Large-scale single-neuron recording in the human cortex using an ultra-flexible electrode array

Domain: neuroscience
Published: 2026-04-13
Editorial outcome: major_revision

Abstract
Monitoring neural population activity at single-cell resolution is essential for fundamental research and clinical innovation. However, translating advanced recording techniques from animal models to humans remains a challenge. Flexible neural electrodes have emerged as powerful tools for large-scale single-unit recordings due to their biocompatibility and high recording density. Here, we demonstrate reliable, high-density single-unit recordings during intraoperative procedures in human patients using ultra-Flexible Implantable Neural Electrode (uFINE) arrays. The uFINE array exhibited sufficient mechanical robustness to maintain structural integrity throughout surgical operations. We successfully recorded 719 single units from 11 patients, with up to 135 single units simultaneously recorded. The flexibility of uFINE array minimized signal disturbances from brain pulsations, enabling stable and continuous single-unit detection. Stimulus and response tuning were observed at the level of individual neurons in awake patients.

Introduction
Single-unit recordings have long been a cornerstone of neuroscience research, offering direct observation of individual neuron activity with exceptional temporal resolution. In the human brain, single-unit recordings provide crucial insights into the neural mechanisms underlying sensory, motor, and particularly human-specific cognitive processes, with broad applications in both basic neuroscience and clinical practice1,2. Traditional approaches to intraoperative single-unit recordings in the human brain, however, are often limited by the number of neurons that can be recorded simultaneously. Commonly used electrode types, such as metal microelectrodes3,4 and microwire bundles5–7, typically yield a small number of single units per electrode (8 microwires)8–11. Although these methods have been instrumental in characterizing the properties of individual neurons, they fall short of capturing the dynamics of neuronal populations. The 96-channel Utah array, which allows for simultaneous recordings of a larger number of neurons, has been widely used in brain-machine interfaces12–17. However, its implantation often leads to significant acute tissue damage 18, making it unsuitable for intraoperative settings that require minimal tissue disruption (for an exception, see Ref19.).

Recently, the intraoperative use of Neuropixels probes has significantly expanded the scale of single-unit recordings in the human brain, providing deeper insights into the neural mechanisms underlying complex functions such as language processing20–24. However, this silicon-based probe still faces challenges from substantial brain movements during surgery, which can compromise recording stability18,20,21,25. Additionally, large lateral brain movements increase the risk of probe breakage, raising concerns about safety and reliability of Neuropixels probes for intraoperative use, as well as their potential for long-term implantation. Thus, there remains a critical need for safer, more effective tools for large-scale single-unit recordings in the human brain.

Ultra-flexible microelectrodes have emerged as a promising solution to these challenges, owing to their excellent biocompatibility and minimal tissue damage26–33. These electrodes have demonstrated success in achieving large-scale, long-term stable single-unit recordings in rodents and non-human primates29–31,34–38. In this study, we present a comprehensive, highly adaptable and user-friendly clinical system that utilizes an ultra-Flexible Implantable Neural Electrode (uFINE) array to record single-unit activity cross cortical depths in the human brain during intraoperative procedures. The flexibility of the uFINE array allows for stable, high-quality recordings even in the presence of rapid brain movements. Using this approach, we also reveal the heterogeneous responses of individual neurons to stimulus and the response period in the dorsolateral prefrontal cortex (dlPFC). Taken together, these results demonstrate the potential of the uFINE array as a powerful and clinically viable neurotechnology with significant basic and translational potential for intraoperative neurophysiological investigations of the human brain, with applications in both healthy and diseased states.

Results
Implantation of the uFINE array in human cortex

Intraoperative single-unit recordings using the uFINE array were attempted in 16 awake patients undergoing Deep Brain Stimulation (DBS) lead implantation or brain tumor resection (Table S1). Successful recordings of single-unit activity were achieved in 11 patients (Figure 1A), while failures in the remaining 5 patients, during the initial troubleshooting stage at each clinical center, were mainly due to excessive electrical noise in the operating room environment and damaged tips of shuttle needles that led to insertion failure. We found that the root mean square (RMS) value of electrical noise could be reduced to 5-10 µV by (1) separating ground and reference (e.g., one placed in the scalp and the other in the cerebrospinal fluid); and (2) turning off the surgical lights and navigator during recordings (Methods). The assembled uFINE array (Figure S2A and methods) and the headstage were connected and encapsulated within a waterproof protective case, forming an enclosed uFINE module (Figure 1B). Furthermore, to prevent damage to shuttle needles tips, we designed a removable protective cap for array tips (Figure S2A) and confirmed the insertion site using a dummy uFINE module before formal implantation (Methods). The 5 patients without single-unit detected were excluded from further analyses in this paper.

To accommodate various neurosurgical procedures, we developed different implantation setups for the uFINE array (Figure 1C and Figure S2C-D). For the cases of DBS implantation, we designed an adaptor that could be mounted onto the Leksell stereotactic frame and coupled with commercial micromanipulators for precise array insertion (Figure 1C and Figure S2C). This setup allowed for insertion through the burr hole, minimizing unnecessary adjustments of the stereotactic frame and preventing additional tissue damage. During surgery, the pre-assembled and sterilized uFINE array and headstage were securely attached to the adaptor by the surgeon. In brain tumor resection surgeries, a custom-designed micromanipulator, mounted on a commercial multi-articulated arm, was used for array insertion (Figure S2D). In all patients, a small superficial incision in the pia was done at the insertion site. During implantation, the uFINE array was guided by coupled tungsten shuttle needles (75 µm in diameter) and inserted to a depth of 5-6 mm. Once the target depth was reached, the shuttle needles were removed, leaving only the flexible array shanks embedded in brain tissue. Therefore, although the back end of the uFINE array was fixed to the holding apparatus, the flexible shanks could follow brain pulsations, which effectively mitigated relative motion between the brain tissue and the recording sites (Figure 1D).

The uFINE array used in intraoperative recordings contained 128 recording sites of 50 μm circle in diameter with 65-µm spacing distributed over two shanks (Figure S1A) for first ten participants. Notably, an upgraded 256-channels uFINE array with 90-µm spacing on four shanks was developed for Pt.11 to enhance the recording coverage and scale. Each shank measured 20 mm in length (35 mm for 256-channels uFINE array), 80 to 245 µm in width with a tapered structure, and 2 µm in thickness, with a typical spacing of 1 mm between the shanks. The 64 recording sites on each shank covered a 4.10-mm distance (5.67-mm distance for 256-channels uFINE array with 90-µm spacing), enabling dense sampling of neural activity at single-cell resolution across cortical depths in the human brain. Compared to penetrating neural electrodes that have been used in human (e.g., microwire bundles and Neuropixels probes), the uFINE array features micron-level dimensions (Figure 1E), superior flexibility (Figure 1F), and mechanical robustness (Figure 1 G&H), making it well-suited for intraoperative use. Especially, for mechanical robustness of uFINE array, the pulling loading until tensile fracture of the array shank was up to 0.33 ± 0.03 N (mean ± std, n = 5 array shank, Figure 1H). The impedance of the uFINE recording sites at 1000 Hz was 52.99 ± 0.49 kΩ (mean ± std, n = 6 recording site, Figure 1I). This performance stems from the optimized recipe of the sputtered porous IrO film, which increases the effective surface area, thereby facilitating interfacial contact and substantially reducing the impedance.

Single-unit recording of neuronal populations across cortical depths

The linear arrangement of uFINE recording sites allowed for sampling of neural activity across cortical depths in the human brain. As shown in Figure 2A-B, recordings from Pt.05 exemplify both raw signals and high-pass filtered (250 Hz) spiking activity captured by the recording sites distributed along the array shanks. Power spectral density (PSD) and laminar profiles of LFP power were analyzed as definitive measures of LFP signal quality. LFP power distribution exhibited clear depth-dependent characteristics (Figure S3A–B). These observations are consistent with previous reports39. Multiple single units with distinctive waveforms were isolated using Kilosort 440 followed by manual curation (Methods). A substantial fraction of single units was detected by more than one recording site, facilitating accurate spike sorting41 (Figure 2C).

In total, 719 single units were isolated from 1302 valid channels (impedance < 4 MΩ) across 11 patients during intraoperative recording, yielding an average of 0.55 ± 0.31 single units per channel (Figure 2D). Additionally, in Pt.11, we performed two recordings both before and after removing tungsten needle. The 103 single units shown in Figure 2D were isolated exclusively from the recordings where tungsten needles were removed from the brain, matching the recording consistency of Pt.01 to Pt.10 where no needle was present during electrophysiological recording. We also observed considerable inter-patient variability in single-unit yield per channel, with a trend of decreased yield with aging (p = 0.09, R² = 0.28, Spearman correlation analysis, Figure S3C), consistent with previously reported age-related declines in neuronal density42. The quality of single units was evaluated by a set of metrics (Figure 2E; see Figure S3D for individual patient metrics). The median and interquartile range of each quality metric were as follows: amplitude: 54.34 μV (46.10 to 66.00 μV); firing rate: 2.27 Hz (0.94 to 5.14 Hz); signal-to-noise ratio (SNR): 3.66 (3.28 to 4.34); autocorrelogram (ACG) rise time: 2.70 ms (0.64 to 9.79 ms); trough-to-peak duration: 0.62 ms (0.51 to 1.01 ms); and peak-trough ratio: 0.47 (0.26 to 0.59). These values are comparable to those reported in previous studies20,30,43.

Although spike waveforms depend on both neuronal type and the location of the recording site relative to the soma, many neuroelectrophysiological studies still infer putative neuronal types from waveform characteristics to better understand neural mechanisms21,44. Based on the polarity and peak-trough ratio of the largest waveform of each single unit (Methods), we classified these units into three categories (Figure 2F-H): positive spikes (PS, n = 412 single units), regular spikes (RS, n = 134), and triphasic spikes (TS, n = 173), which putatively correspond to axonal action potentials, excitatory neurons, and inhibitory neurons, respectively. The spatial distribution of these three classes across penetration depths showed no significant difference (p = 0.57, two-tailed Kruskal-Wallis test). TS units exhibited slightly larger amplitudes (62.08 ± 21.16 μV, mean ± std) compared to RS units (55.00 ± 20.30μV, mean ± std, p = 3.20 × 10-3, two-tailed Welch t test) and PS units (60.44 ± 23.41 μV, mean ± std, p = 0.41, two-tailed Welch t test) (Figure 2I). Taken together, these results demonstrate that the uFINE array is capable of achieving high-quality recordings of single units at scales in the human brain.

Stable intraoperative recording of single-unit activity

A major challenge for intraoperative recordings using rigid electrodes is the substantial human brain pulsations caused by respiratory and cardiac cycles20,21. These rapid brain movements, usually fluctuating around respiratory frequency, pose difficulties for automatic spike-sorting approaches45,46 and have been shown to negatively correlate with single-unit yield20. However, the flexibility of the uFINE array could address this issue by allowing its shanks to follow brain movements, effectively minimizing relative motion between the brain tissue and recording sites and thereby enabling stable single-unit recordings (Figure 1D).

To further estimate the effects of brain movements on recording stability, figure 3A compared spike position drifts over time before (left) and after (right) tungsten needle removal acquired by the 256-channel uFINE array recordings at Pt.11, and without motion correction of kilosort4 when spike-sorting. When constrained by the tungsten needle, spike positions exhibited periodic fluctuations (left panel). These fluctuations synchronized across depth, replicating the drift pattern characteristic of rigid probes. Following needle extraction, spikes showed continuous, stable firing patterns (right panel), demonstrating the uFINE array's native ability to accommodate brain pulsation. Figure 3B quantified spike position drift distributions using kernel density estimation. Needle-constrained recordings exhibited broad drift distributions (median: 88.75μm, range: 0-380 μm), while post-removal recordings showed tightly localized drifts (median: 29.42 μm, predominantly < 200 μm). In addition, for all single units, we also estimated the distance between spikes of each single unit and its nearest neighbouring site (the recording site with the largest waveform for this unit identified from spike sorting) over the course of recordings (Methods). Most of the spikes (60.73%) remained within 32.5 µm of their nearest neighbouring site (Figure 3C), further supporting the minimized relative motion between the recording sites and the brain tissue. In a subset of patients (n = 7) with concurrent video recordings of the brain surface during array insertion (Supplementary Movie 1), we further confirmed that the amplitude of rapid brain movements (Figure S4A) had no significant impact on either the single-unit yield (R² = 0.36, p = 0.15) or the single-unit yield per channel (R² = 0.14, p = 0.41) (Figure S4B&C). To further quantify signal stability under distinct surgical conditions, we evaluated spike position drift across two cohorts with differing craniotomy sizes: approximately 1.5 cm for DBS and 4–10 cm for tumor resection. Participant Pt. 11 was excluded from this analysis due to non-standard 90 μm site spacing, which precluded consistent drift quantification. Given the substantial imbalance in sample sizes between the DBS group (n = 8) and tumor resection group (n = 2), we present descriptive statistics rather than formal hypothesis testing (Figure S4D). The median spike drift was 17.39 μm in the DBS group and 36.89 μm in the tumor resection group.

The mitigated relative motion between the brain tissue and the recording sites should facilitate stable capturing of neuronal activity over longer timescales (e.g., minutes). As shown in Figure 3D, the waveforms of individual single units maintained high similarity over the course of a 36.5-minute recording from Pt.10. To evaluate the recording stability throughout the intraoperative recording period, we divided each recording into 10 time windows and focused on single units with sufficient spike events (termed “active unit”; firing rate > 0.1 Hz and fired at least one spike within each window, Methods). On average, 83.45 ± 16.02 % of single units across 11 patients met this criterion (totaling 600 units, Figure 3E and Table S1). The waveforms of each active unit occupied nearly consistent positions in a low-dimensional PC space, indicating that waveform shape of individual units remained stable over the course of recording (Figure 3F). Throughout the recording, active units exhibited high waveform similarity within each unit (0.93 ± 0.14, mean ± std, Figure 3G), along with excellent stability in spike amplitudes, trough-to-peak duration, peak-trough ratios, and firing rates (Figure 3H-J; see statistics in Table S2). Taken together, these results demonstrate the capability of the uFINE array to achieve stable single-unit recordings over both fast and prolonged timescales in the intraoperative environment.

Stimulus and response tuning at single-cell level in human dlPFC

The capability to record from populations of neurons with single-cell resolution provides a valuable opportunity to investigate sensory, motor, and cognitive processes in human brain. In patients undergoing DBS lead implantation, the uFINE array was implanted in the dlPFC, an area that has extensive connections with sensory and motor cortices and has been implicated in working memory, cognitive flexibility, and planning47–50. We began by characterizing the activity modulation of individual neurons in human dlPFC using a simple “Word-Repeating” task (Figure 4A, 4B). During each trial, the patient (Pt.03, Pt.04 and Pt.08) listened to a word (also displayed on a screen) for 0.5-1.0 s and was then required to orally repeat the word after a 0.6-s delay. On average, each patient completed 79.67 ± 21.01 (mean ± std) trials. We found that the activity of some single units was significantly modulated during the stimulus and response period (Figure 4C-N and Methods), with a slightly higher fraction of units modulated during the response period than the stimulus period (39.10 ± 24.68% vs 22.83 ± 12.64%, mean ± std, Figure 4F, 4J and 4N). Overall, more dlPFC single units exhibited suppression rather than activation (19.65 ± 11.64% vs 3.17 ± 5.50% for the stimulus period, 34.00 ± 28.49% vs 5.10 ± 5.27% for the response period, mean ± std, Figure 4F, 4J and 4N). Our intraoperative recordings highlighted heterogeneous single-unit responses during task performance, showing the potential to facilitate detailed explorations of the neural mechanisms underlying various human cognitive functions at the cellular level.

Methods
Experimental model and study participant details and ethics
The protocols were approved by the Medical Ethics Committee of the First Affiliated Hospital of the Air Force Medical University (KY20232366-F-1) and the Huashan Hospital Institutional Review Board of Fudan University (HIRB, KY2024-103).
Sixteen participants were included in the study across two hospitals (Table S1), all of whom were already scheduled for intraoperative neurophysiological procedures during either DBS implantation surgery under local anesthesia or tumor resection with awake functional mapping as part of normal clinical routine.
Each participant gave informed written consent and was free to withdraw from the study at any time, including during surgery, without any impact on their clinical care. According to the approved ethical protocol and the signed informed consent forms, patients' identities and personal information will remain strictly confidential and will never be disclosed under any circumstances. Therefore, specific consent to publish identifiable information is not applicable.
In strict adherence to your editorial policy, we have limited the disclosure of patient demographic data to only age and sex, which are provided in Table S1. Regarding participant compensation, patients recruited from the First Affiliated Hospital of the Air Force Medical University received no direct financial compensation, whereas participants recruited from Huashan Hospital received a financial compensation of 200 RMB.

Design and fabrication of uFINE electrode array
The uFINE array used in this study consisted of 128 channels arranged in two shanks with shank spacing of 1000 μm. The shank thickness was 2 μm, and the length of a single shank was 20 mm (35 mm for 256-channels uFINE array). Each shank featured 64 recording sites, vertically aligned along its front end, as the recording site segment. The total length of this segment was approximately 4.10 mm (5.67 mm for 256-channels uFINE array), with each circular site having a diameter of 50 μm and a center-to-center distance of 65 μm (90 µm for 256-channels uFINE array). The recording site segment had a tapered structure, with a maximum width of 245 μm at the implantable portion and a width of 80 μm at the tip. Besides the recording site segment, the rest of the shank maintained sufficient redundancy to ensured that it remains in a slack state during brain movement, preventing the portion of the shank inside of the brain from being pulled out.
The uFINE was fabricated using standard planar microfabrication techniques (see Figure S1B). Detailed descriptions of the fabrication and assembly methods were provided in previous publications30. This study implemented several improvements based on prior work, achieving higher uFINE array tensile strength through fabrication and utilizing medical-grade materials throughout the assembly process. The main fabrication procedures of uFINE electrode array were as follows:
(A) The insulating framework was constructed from Polyimide (PI, U-Varnish S, UBE Corporation), prioritized for its high tensile strength, processing flexibility, and long-term biostability in physiological environments. To form the base insulation, PI was spin-coated onto a commercially available silicon (Si) substrate (300 nm thermal oxide, Szjxtech Co., Ltd) at 6000 rpm for 40 s, followed by a graduated thermal curing protocol at 65 °C for 10 min, 95 °C for 2 min, and a final imidization at 230 °C for 3 h.
(B) For the initial metallization, LOR 5 A (KAYAKU Advanced Materials, Inc.) was deposited at 2000 rpm for 35 s and baked at 180 °C for 3 min, then topped with AZ 5214 (Merck) spin-coated at 2000 rpm for 45 s and baked at 95 °C for 2 min. This bilayer resist was patterned using a UV mask aligner (SUSS MA6, SUSS MicroTec) and developed in AZ 300MIF (DuPont Electronic Materials) for 70 s. A Ti/Ni/Au (50/800/200 nm) stack was then evaporated (Ohmiker-60BL, Cello) to form the bottom I/O layer, finalized via lift-off.
(C) The fabrication of metal interconnects utilized a photolithography procedure identical to the aforementioned step. A Ti/Au/Ti (5/100/5 nm) trilayer was deposited through E-beam evaporation and resolved using a standard lift-off technique.
(D) The top encapsulation layer was applied by replicating the PI coating and pre-baking parameters described previously. Subsequently, AZ 5214 was spin-coated at 3000 rpm for 30 s and baked at 95 °C for 2 min to prepare for further patterning.
(E) An image reversal process converted the AZ 5214 to a negative resist via a 40-s bake at 120 °C, followed by a 6-s flood UV exposure and 60 s of development. An 80-nm thick Aluminum (Al) mask was then deposited and lifted off to serve as a hard etch mask for the uFINE-I structures. The exposed PI was removed via reactive ion etching (SI591, Sentech) in a 50% oxygen plasma (60 W, 110 min), after which the Al layer was stripped with hydrofluoric acid (Titanium Etchant TFT, Transene) for 10 s and thoroughly rinsed with deionized water.
(F) Post-etching, the devices underwent a rigorous hard bake at 320 °C for 2 h. This extended thermal treatment was essential to enhance the structural robustness of the array while preserving the mechanical compliance necessary for intravascular navigation.
(G) The photolithographic sequence used for the I/O layer was repeated to define the 80 μm platinum (Pt) electrode regions, which were then established through E-beam evaporation and lift-off.
(H) Individual electrode sites (50 μm diameter) were defined using the same lithographic protocol to deposit a Ti/Au (5/100 nm) seed layer. To minimize interfacial impedance, Iridium Oxide (IrOx) was subsequently grown via reactive sputtering (PRO Line PVD 75, Kurt J. Lesker) at 130 W for 1200 s under an Ar/O2 atmosphere, followed by a final lift-off step.

Assembly of uFINE electrode array
The main assembly procedures of uFINE electrode array were as follows:
(A) Multiple electrodes were fabricated on a single silicon substrate and subsequently separated into individual units via a dicing process.
(B) A flexible printed circuit (FPC) was soldered to the diced uFINE array to establish a connection with the headstage. Specifically, ball grid array (BGA) soldering was employed to connect the FPC pads to the electrode pads in a one-to-one manner using individual solder balls.
(C) The implantable section of the uFINE array probe was immersed in nickel etchant (TFB, Transene Co. Inc.) for 2 to 4 hours at 25°C. This process released the free-standing portion of the probe, while the contact region between the probe and the FPC remained attached to the substrate. The substrate was then cleaved to the desired length prior to implantation.
(D) uFINE array was anchored to tungsten shuttle needles manually. The diameter of the tungsten shuttle needles used for auxiliary electrode implantation was increased from 50 μm to 75 μm. We utilized commercially available 75μm diameter tungsten shuttle wires as the raw material. The sharp tips were fabricated using an electrochemical etching process to achieve the desired tapered morphology.
During device assembly, the unattached proximal section of the flexible filaments, which rests on the silicon substrate, was temporarily immobilized using a water-soluble adhesive (salt crystals) to prevent entanglement.
In this study, the "assembled uFINE array" (Figure S2A) referred to the state in which the uFINE array was anchored to tungsten shuttle needles and soldered to a flexible printed circuit (FPC) for connection between headstage and uFINE array.
(E) A bio-compatible surgical adhesive (Kwik-sil, World Precision Instrument Co.) was used to secure the shuttle needles to the silicon substrate. (In contrast to previous methods, this project did not require complete detachment of the shuttle needles during surgery.)
(F) A waterproof protective case was specifically designed for complete sterilization, encapsulating the assembled uFINE array and headstage (128 channels, Shanghai Stairmed Technology Co.) together, to form an enclosed uFINE module (Figure 1B, Figure S2A). This case was made from a common medical material, polyether ether ketone (PEEK), with reference and ground wires made from needle electrodes (Xian Fude Co.) connected to the headstage. The case consisted of four parts, including a removable protective cap to prevent potential damage to the uFINE array tip before implantation (see Figure S2A). The top of the case featured a port for connecting power and data lines. Prior to implantation, the enclosed uFINE module could be connected to a NeuroFortis (AlphaOmega Co.) micromanipulator fixed to a stereotactic frame (Leksell Co.) or to a custom-designed micromanipulator mounted on a commercial multi-articulated arm (Brainlab Co.).
(G) We also prepared a 1-shank array to avoid complications related to vascular avoidance during the implantation of the 2-shank array. The assembly process for the 1-shank array was similar to that of the 2-shank array; however, the 1-shank array utilized only one tungsten shuttle needles to secure one of the uFINE shanks, resulting in a total of 64 recording sites. Pt.03 and Pt.04 in this study were implanted with the 1-shank array. An upgraded 256-channels, 4-shank uFINE array (inter-shank spacing of 0.75 mm) was developed for Pt.11 to enhance the recording coverage and scale.

Inspection and Biological Safety Testing of Implantable Components
The uFINE arrays have undergone third-party certified type inspection report. The report includes:
(1) EO sterilization verification performed according to GB/T 14233 standard (https://std.samr.gov.cn/gb/search/gbDetailed?id=EB58F4DA904EDB2A2E05397BE0A0A7D33) and https://std.samr.gov.cn/gb/search/gbDetailed?id=028828961R72A250CE06397BE0A0A9F76).
(2) Biocompatibility and toxicity testing of the flexible electrode and the tungsten needle conducted in accordance with the GB/T 16886 standard (equivalent to ISO 10993 standard, https://www.iso.org/standard/10993-1) standards.
For the components implanted into brain tissue (the uFINE array and the tungsten guiding needle), the inspection report includes the corresponding biocompatibility and toxicity tests as detailed below:
(1) uFINE array
• Chemical tests (5 items: appearance/color, reducing substances, residue on evaporation, total heavy metals, UV absorbance); standards: GB/T 14233.1‑2008, GB/T 16886.5‑2017, GB/T 16886.10‑2017.
• Cytotoxicity (GB/T 16886.5‑2017).
• Intracutaneous (intracutaneous/skin) reactivity (GB/T 16886.10‑2017).
• Pyrogenicity (GB/T 16886.11‑2021).
• Acute systemic toxicity (GB/T 16886.11‑2011).
• Skin sensitization (GB/T 16886.10‑2017).
(2) Tungsten guiding needle
• Chemical tests (6 items: appearance/color, pH, reducing substances, residue on evaporation, total heavy metals, UV absorbance); standard: GB/T 14233.1‑2008.
• Cytotoxicity (GB/T 16886.5‑2017).
• Intracutaneous reactivity (GB/T 16886.10‑2017).
• Pyrogenicity (GB/T 16886.11‑2021).
• Acute systemic toxicity (GB/T 16886.11‑2011).
• Skin sensitization (GB/T 16886.10‑2017).

In vitro characterization of the uFINE array
(1) Mechanical
Tensile test of the uFINE array was conducted using an electronic balance (BSM-220.3S, Shanghai ZhuoJing Co.), a 50g weight, and a Digital Stereotaxic Instrument (68025, RWD).
One shank of the uFINE array was released and cut from the wafer. Both ends of the shank were bonded separately to the weight and the holder of the stereotactic device using adhesive. The middle section of the shank was maintained at a length of 1.4±0.1 cm in a relaxed state. The weight was placed on the balance.
The stereotactic device was gradually raised with constant speed monitored by digital display module, stretching the electrode shank and causing slight displacement of the weight, which was reflected in changes to the balance readings. These readings were recorded until the electrode shank broke. The maximum force the shank could withstand was calculated based on the change in balance readings and the electrode’s cross-sectional area, yielding the maximum tensile stress the electrode could endure. This experiment was repeated five times. Results are shown in Figure 1H.
(2) Electrochemical Impedance Spectroscopy
To assess the electrode's ability to maintain good recording performance across a broad frequency range (i.e., capable of recording neural activity across most frequency bands), a Gamry Reference 620 potentiostat (Gamry Instruments) was used to measure the impedance and phase angle of the electrode over a frequency range from 10 Hz to 104 Hz. Measurements were performed in 1 × PBS with an Ag/AgCl reference electrode. The results are shown in Figure 1I.

Preoperative preparation
(1) Sterilization and scenario setup
The items requiring sterilization before surgery included the enclosed uFINE module (containing the assembled uFINE array, FPC, and headstage), data communication cable (Shanghai Stairmed Technology Co., Ltd.), micromanipulators, and a multi-articulated arm (for tumor resection surgery). These items were sterilized in the hospital using ethylene oxide (EO) sterilization following standard protocols.
As shown in Figure S2B-D, other items that needed to be prepared included the StairPlex recording system (Shanghai Stairmed Technology Co., Ltd.), laptop (Windows 11, IE9), battery, and equipment required for behavioral tasks (speakers, microphone, and NI Data Acquisition Card (National Instruments, USB-6009)). These components were transported into the operating room using a custom-assembled cart and connected prior to the experiment. Specifically, the StairPlex, speakers, microphone, and NI card were connected to the computer.
(2) Denoise
Denoising of intraoperative acute recording included two key operations after careful noise signal mitigation tests:
1. Set the ground of headstage connected into the scalp.
2. Eliminate the power line interference from recording devices.

uFINE array positioning and insertion
The positioning and insertion of flexible arrays in DBS surgeries and brain tumor resection surgeries utilized different micromanipulators, but the operational protocols were similar.
For DBS surgeries, after a burr hole for DBS lead implantation was created and the dura mater was incised, the implantation of the uFINE array occurred prior to the DBS lead placement. The specific procedure was as follows. The NeuroFortis micromanipulator was installed in the Leksell stereotactic frame. The implantation trajectory of the uFINE closely aligned with the planned trajectory of the DBS lead, though slight adjustments were made to avoid blood vessels or sulci of the cerebral surface. To safely execute this, a 'dummy module' was first employed to verify the target coordinates and ensure the trajectory was unobstructed by the skull edge. This dummy module was designed with identical dimensions and geometry to the functional uFINE module but lacked the recording headstage. Once the trajectory was confirmed using the dummy, a small superficial incision in the pia was done using an arachnoid surgical knife. Subsequently, the uFINE array replaced the dummy module on the micromanipulator. Sterile ground and reference needle electrodes were placed in the scalp. A sterile data cable connected the headstage to the StairPlex system.
During insertion, the uFINE array was guided into place by the shuttle needles, with an insertion depth of 5-6 mm. Once the target depth was reached, the redundant part of the array (previously fixed to the substrate) was immediately released as the water-soluble salt layer dissolved rapidly upon contact with physiological saline, enabling the electrode filaments to move synchronously with brain pulsation.
The shuttle needles were removed and left suspended approximately 3-5 mm above the brain surface. This ensured that the sharp tips would not scratch the cortex while leaving the flexible array shanks embedded in the tissue. Crucially, there was no need to fully detach the tungsten needles from the insertion holder. The flexible electrode provided redundant length to accommodate brain surface movements, preventing the array from being dislodged. The released redundant part ensured that the uFINE array had sufficient distance when moving with the brain surface, preventing it from being pulled out.
For brain tumor resection surgeries, a custom-designed micromanipulator mounted on a commercial multi-articulated arm (Brainlab Co.) replaced the Leksell stereotactic frame and NeuroFortis for uFINE array implantation. All patients undergoing resection surgery were awakened from anesthesia after the skull and dura mater were opened. Prior to implantation, the target site was selected by identifying a region that can avoid the major surface vasculature and can approach to the apex of the gyrus as close as possible. The multi-articulated arm was then manually adjusted to ensure the uFINE array target in this region and be as perpendicular to the cortical surface as possible, thereby maximizing the likelihood of spanning the full extent of the cortical layers.
Regarding localization of the electrode insertion trajectory, the width of electrode shank was only 200 μm. Direct visualization via standard intraoperative imaging resolutions such as X-ray/CT/MRI are sufficient to visualize the electrode body clearly. Consequently, we employ the following strategies for implantation site localization:
DBS procedures: The uFINE array is implanted according to DBS surgical trajectory planning. the DBS electrode insertion trajectory is determined from preoperative imaging and stereotactic planning. This trajectory defines the uFINE array implantation position, typically without the need for additional imaging-based localization.
Brain tumor resection surgeries: After electrode implantation, we use intraoperative navigation systems (CurveNavigation 17700, BrainlabAG Co.) to mark the electrode entry point (navigation coordinates) on a three-dimensional brain model built from preoperative MRI, so as to document the spatial position of the electrode on the cortex or cortical surface.
The insertion site information for the 11 subjects annotated in Figure 1A was obtained using the two methods described above.

Electrophysiological recordings
In this study, the headstage used (Shanghai Stairmed Technology Co.) had a linear dynamic range of 10 mVpp and a 10-bit sampling resolution. The recording sampling rate was 25 kHz, and impedance measurements were conducted prior to electrophysiological recordings.

uFINE array removal
For both DBS surgeries and brain tumor resection surgeries, after completing electrophysiological recordings, the uFINE array was removed by slowly raising the micromanipulator along the original vertical insertion axis, thereby pulling the array out of the brain. During the removal, the micromanipulator would not be changed position to avoid lateral shear that could cause additional damage. After uFINE array removed, we inspected the electrode shank under the surgical microscope to confirm structural integrity of the implanted segment and to check for breakage or deformation.
In all cases, the uFINE array was completely removed without any left-behind parts. Post-removal microscopic inspection consistently confirmed the structural integrity of the retrieved shank segment.

“Word-Repeating” task
In this study, three patients performed a "Word-repeating" task. The "Word-repeating" task in this study consisted of two types: Pt.03 was assigned Type I, while Pt.04 and Pt.08 were assigned Type II. The paradigms for the two types of tasks were the same (Figure 4A), but the libraries were different. In each trial, patients needed to listen to a specific word, which was synchronously displayed on the screen, as the "stimulus". After a fixed delay of 0.6 s, patients were asked to orally repeat the word as the "response" following a 0.1-s white noise prompt. The library for Type I included "Ji Li (吉利)," "Ji Li (极力)," "Ying Wu (英武)," and "Ying Wu (鹦鹉)." The library for Type II included "Xin Nian Kuai Le (新年快乐)," "Zhu Ni Ping An (祝你平安)," "Zao Ri Kang Fu (早日康复)," and "Qin Lao Yong Gan (勤劳勇敢)." A word was randomly selected from the library for each trial.
Pt.03 had an average stimulus period of 0.51 s (std = 0.01 s) and an average response period of 0.65 s (std = 0.09 s). Pt.04 had an average stimulus period of 0.98 s (std = 0.05 s) and an average response period of 1.04 s (std = 0.09 s). Pt.08 had an average stimulus period of 0.98 s (std = 0.03 s). Due to Pt.08's slower speaking tempo, the average response period was 3.12-s (std = 1.06 s). The three patients completed 101, 79, and 59 valid trials, respectively. It was confirmed before the surgery that all three patients were familiar with the pronunciation and meaning of all the words in the library.
A set of speakers and a screen were used to provide stimuli, and responses were recorded using a microphone. TTL signals from the task laptop were sent to the digital input channels of the StairPlex system via a multi-function I/O module (NI USB-6009, National Instruments) to synchronize the electrophysiological recordings with behavioral data for each trial. TTL triggers were generated during the task using custom code in Python on the task computer. The starting position of each trial was aligned with the starting position of the triggers. The microphone was turned on at the start of the trial and turned off when the trial terminated. Each trial lasted 6 s.

Spike sorting
Before automatic spike-sorting, the raw signals underwent the following preprocessing steps:
1. High-pass filtering (250 Hz);
2. Removal of high-impedance channels higher than 4 MΩ;
3. Removal of channels with RMS noise higher than 2 σ. Based on empirical information, the SNR for such channels is very low; In total, 7.53% of channels (106/1,408) were excluded based on these two factors (mean ± std: 6.50 ± 6.01%).
4. Removal of global channel synchronization artifacts: When all channels recorded a signal greater than 100 μV simultaneously, the portions of the signal surrounding the maximum absolute value point that exceeded 100 μV were set to zero for 10 ms before and after that point.
This study utilized Kilosort 4.040 (https://github.com/MouseLand/Kilosort) for spike sorting. The specific parameter settings were as follows:
1. Nblocks = 0. The spike drift of the flexible electrode array was minimal, so there was no need for a drift correction algorithm to improve spike sorting accuracy during spike sorting. Statistical analysis of spike position drift (Figure 3A-C) confirmed that drift correction was unnecessary.
2. In spike detection, both "Th_universal" and "Th_learned" thresholds were set to 8.
Subsequently, experienced electrophysiologists (SW and CK) performed manual curation of the automatically isolated clusters using Phy (https://github.com/cortex-lab/phy), with the following specific steps:
1. Clusters of abnormal waveforms were labeled as noise, primarily including sinusoidal oscillations and spikes with durations less than 0.1 ms.
2. When a cluster could be clearly observed to have separation in principal component (PC) space, it was split into two or more clusters. If the spatial spread of waveforms, amplitude, and Inter-spike-interval (ISI) distribution were highly similar, and merging did not result in a higher ISI violation, the clusters were merged in Phy. The ISI violations refer to the rate of refractory period violations55.
3. If the ISI violation was less than 0.2 and the FR was above 0.05 Hz, the cluster was labeled as a good unit. Otherwise, the cluster was labeled as multi-units (MUA).
Among the “good units”, those with an average amplitude exceeding 30 μV were considered single units and included in subsequent statistical analyses.

Assessment of recording quality
First, we calculated the single-unit yield per channel to evaluate the efficiency of electrode recordings in capturing neuronal signals. The yield for each patient was defined as the number of sorted single units divided by the number of usable channels, excluding channels with impedance >4 MΩ or abnormal RMS values (as previously described).
Next, to assess signal quality, we analyzed the amplitude, firing rate, SNR, ACG rise time, trough-to-peak duration, and peak-trough ratio of all sorted neurons. For each metric, the 25th, 50th, and 75th percentiles were calculated to summarize variability and central tendency, as shown in Figure 2E. The specific calculation methods for these metrics are as follows:
1. SNR: The signal-to-noise ratio (SNR) was calculated as the peak-to-trough amplitude of the mean waveform divided by twice the standard deviation of the data stream.
2. ACG rise time: Computed using custom scripts based on the method described in Wu et.al56. The score quantifies temporal characteristics of single-unit activity distributions by fitting a triple-exponential equation to the ACG.
3. Peak-trough ratio: Defined as the ratio between the absolute amplitudes of the minimum trough and the following peak44, as illustrated in Figure 2F.
4. Trough-to-peak duration: Defined as the time interval (ms) between the waveform trough and the subsequent peak in the mean waveform, as illustrated in Figure 2F.
Then, for each unit, we analyzed the mean waveform (averaged from 500 randomly sampled spikes) on the primary channel and its 10 nearest neighbors (using a sliding window of 11 channels). A channel was included in the spread calculation if the peak-to-peak amplitude of the unit's mean waveform on that channel exceeded 2 times the baseline RMS noise (SNR > 2).

Waveform classification
Waveforms of single units were extracted and manually curated using custom code, followed by classification using a standardized approach to categorize the units into regular-spiking (RS), triphasic-spiking (TS), and positive-spiking (PS) types, based on the spike waveform recorded at the channel exhibiting the largest amplitude for each unit44,57,58.
As illustrated in Figure 2F, PS (positive-spiking) waveforms were characterized by positive peaks whose magnitude exceeds that of the negative troughs. TS (triphasic-spiking) waveforms display a prominent initial positive peak, followed by a larger negative trough (with a first peak-trough ratio >0.1), and a subsequent positive peak occurring within 1 ms after the initial peak. In contrast, RS (regular-spiking) waveforms typically exhibit dominant negative troughs.

Assessment of recording stability
(1) Spike positions calculation
The spike positions for each spike were obtained from the "spike_positions.npy" file automatically generated by Kilosort4. Since the uFINE array's recording sites are arranged in a vertical linear configuration, the file contains only longitudinal position data.
(2) Waveform similarity
We assessed recording stability by analyzing the waveform similarity of single units across different recording periods59. For each single unit, the mean waveform was calculated within each recording segment. Then, the Pearson correlation coefficients were computed for pairs of average waveform vectors across segments to evaluate waveform similarity.
(3) Waveform shape stability in PC space
We assessed waveform shape stability using PCA. We projected all spikes from each single unit into a three-dimensional coordinate system. The waveform length for each spike was set to 2.5 ms. The x-axis and y-axis represented the first and second dimensions of the PCA of all waveforms, while the z-axis indicated the recording length in seconds.
(4) The definition of active units
A unit was defined as an “active unit” when both conditions met:
1. Its firing rate was higher than 0.1 Hz;
2. Fired at least one spike within each window.
We standardized the recording time for each patient by dividing their recordings into 10 equal time windows, irrespective of the varying recording lengths.
(5) Recording quality of active units in 10 time windows
For each time window, the mean and standard deviation of spike amplitude, trough-to-peak duration, and peak-trough ratio of all active units were calculated. These metrics were used to assess changes in unit waveform characteristics over time, with smaller waveform variations indicating more stable recording quality. The mean firing rate within each time window was used to characterize neuronal activity over time.
To further assess recording stability, we performed two-tailed ANOVA to compare each parameter across time windows. The p-value for the comparison between each time window with respect to the rest of the windows was calculated, with p > 0.05 indicating no significant difference. The results are shown in Table S2.
(6) The estimation of brain pulsation movement
We used custom MATLAB code to extract markers from videos recorded with a surgical microscope including brain pulsation trajectories to estimate the patients' brain pulsation movement. All microscope videos were recorded at 30 frames per second. A total of seven patients had microscope videos that effectively captured brain pulsation. The estimation process was as follows:
1. A segment of video of at least 30 s was extracted, ensuring that both the length calibration marker (typically the cross-section of the silicon substrate holding the uFINE array, with a constant length of 4 mm) and the brain movement marker (usually a clearly visible reflection point corresponding to the pulsation trajectory at the surface of the brain) were unobstructed, and that the shooting angle and distance remained unchanged.
2. The number of pixel points for the length calibration marker was measured, and the length-to-pixel ratio was calculated based on its actual length.
3. Each frame of the video was extracted and converted to grayscale. Regions greater than the grayscale threshold (with a threshold value of 220) were extracted from a fixed area in each frame as the Region of Interest (ROI), with the center of the ROI designated as the brain movement marker.
4. The trajectory of the brain movement marker within the selected video segment in step (1) was extracted and converted into actual distance using the length-to-pixel ratio.
5. The trajectory curve of the brain movement marker was plotted, and the peak-to-peak motion for each pulsation was calculated, yielding the mean peak-to-peak motion as the average brain pulsation movement distance for each patient.
The limitations of the estimation method used in this study were as follows:
1. The videos lacked markers for depth-of-field calibration;
2. Brain movement described a trajectory in three-dimensional space (primarily represented as vertical movement relative to the uFINE array, but also included some lateral movement), while the video only depicted this three-dimensional motion projected onto a two-dimensional plane at the shooting angle.

Stimulus and response tuning of single units
The FR tuning curve for each single unit was calculated through the following steps:
1. A commonly used speech analysis tool for phonetics, Praat (Phonetic Science, Amsterdam), was utilized to mark the onsets and offsets of the stimulus and response for all trials.
2. The duration from stimulus onset to stimulus offset for each trial was defined as the stimulus duration, and the trial-averaged stimulus duration was calculated. Similarly, the trial-averaged response duration was obtained.
3. One stimulus period was defined as 0.5 s before the stimulus onset to 0.5 s after the stimulus offset. The response period was defined similarly. Each period was divided into time bins with a length of 50 ms. The number of spikes fired during each time bin was counted and converted into spikes per second (Hz) to obtain the FR for that time bin.
4. If a single unit had fewer than 20 trials and a trial-averaged FR below 5 Hz, it was excluded from the tuning analysis. The remaining single units were used in the following analysis. In Figures 4D, 4H and 4I, the raster plot for each single unit displayed only 15 randomly selected trials.
5. The trial-averaged firing rate (FR) and the standard error of the mean (SEM) of FR for each time bin were calculated for each valid tuning-analysis unit.
6. The FR tuning curve was plotted using the trial-averaged FRs from the time bins. To reflect the overall tuning trend, a smoothing sliding window of 50 ms with an overlap of 25 ms was applied.
7. The average FR in the 0.5 to 0.25 s before the stimulus onset was defined as the FR baseline. Each time bin after the stimulus onset and response onset was subjected to a significance test (two-tailed Wilcoxon signed-rank test, p<0.05) against the FR baseline to determine whether that time bin had significance. Time bins with a significant increase in FR relative to the baseline FR were defined as significant activation periods, while time bins with a significant decrease were defined as significant suppression periods.
8. When a single unit's significance period lasted continuously over 150 ms24, it was defined as a significant activation or suppression unit.
9. The proportions of significant activation units and significant suppression units were calculated for each patient during the stimulus and response periods.
10. The difference between the baseline FR and the FR for each time bin was defined as △FR.
A heatmap displayed a matrix of △FR values, with each row corresponding to a valid tuning-analysis unit and each column corresponding to the △FR for each time bin of that valid tuning-analysis unit. This heatmap was used to compare the fluctuations of △FR between units during the stimulus period or response period. The heatmaps for the response and stimulus were sorted in descending order based on the △FR values during the response period.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The authors present their new high-density electrode uFINE and demonstrate its successful use in humans to record high-quality single neurons acutely during brain surgery. The recording quality, yield, resistance to brain motion, and quality of characterization is very impressive throughout.\nThis new electrode addresses a very significant problem – while high density acute recordings in humans during surgery have been relatively common with the Neuropixel electrode, the rigid electrode poses significant challenges in terms of recording stability and breakage risk. The present technology addresses this significant limitation.\nThis first-in-human test of this new type of electrode is novel and interesting. The paper is well written, and the example case study (Fig 4) of word responses in dlPFC is an interesting use case. I recommend publication after addressing the following relatively minor issues.\nMajor issues:\n1.The electrode-to-electrode spacing was 65um, with 50um diameter electrode pads. A useful way to characterize units is to examine on how many nearby electrodes a given unit is visible. Thus the question: what was the number of electrode pads that a given unit was visible? If it is several, that provides additional resolution to estimate brain motion.\n2.Material verification and Sterilization. The methods mention that ‘standard EO sterilization’ was used. What testing was done to verify that the resulting product was sterile (sterilization verification)? Second, I didn’t see consideration of the materials and their toxicity/biocompatibility. What testing was done to assure that the materials used in the parts of the electrode that enter the brain have acceptable toxicity profiles?\n3.Electrode removal. I didn’t see description of electrode removal steps and experience. What are the considerations for successful removal and was testing done to assure that removal would always be possible/successful? Was removal without left-behind parts successful in all cases?\n4.Tungsten guide wire removal. I couldn’t decipher how the tungsten wire removal is initiated – how is the tungsten wire attached to the electrode and how is it detached so that I can be removed?\n5.LFP. Did the electrode allow reliable recording of LFPs and if so what were the characteristics of the LFPs recorded?\n6.Localization. How did the authors localize their recordings, both in terms of exact position inserted (is the electrode visible on X-Ray or intra-op CT?) and for assigning cortical layers to different depths on the electrode?\nMinor issues:\n1. ECG. Have the authors recording simultaneous ECG? That way effects of brain motion can be quantified rigorously (Mosher et al 2020, Cell Reports). If this data is not available, sufficient to mention as future possibility.\n2.The stated ‘tensile strengths’ of 831 MPa – what does this number look like for other commonly used electrodes, such as Neuropixel or tungsten microwires ? Would be helpful to have this comparison to put this number into perspective (it is hard for me to say if this is good or bad).", "reviewer_2": "Wu et al describe the application of a novel flexible electrode array for single neuron recording in human cortex. They characterize mechanical properties of the electrode array and describe the setup for intraoperative recordings in human patients undergoing DBS or brain tumor surgeries. In 11 cases where they successfully recorded neurons, they describe the yield and electrophysiological properties of the cells, including waveform shapes. In one case, they directly compare recordings performed with the tungsten stiffener attached to after the stiffener was removed, showing that neuron tracking is superior when the array is able to move with brain pulsation. Finally, they show neurons with task-evoked activity from dLPFC, where most neurons were suppressed during the speaking portion of a verbal repetition task.\nIn general, this is a nice paper that describes a timely advance in neurophysiological array technology for humans. The paper is simple and clearly written, and I think will be of interest to the field, especially if these arrays can be made available either commercially or through partnerships.\nI have several questions and comments that I think would help strengthen the paper:\n1) It would help to know if the patient numbering scheme used reflects the sequential order of recording. Specifically, were there no issues after the first 2 DBS cases, and was this more of a challenge for the tumor cases? Similarly, is there something about the tumor cases that made these recordings more likely to suffer from noise issues? I assume that the tumor cases involved larger craniotomies, though this was not stated explicitly in the paper.\n2) Other than avoiding vasculature, how was the probe insertion targeted on the cortical surface? Was there an attempt to place the array perpendicularly to the surface in the middle of a gyrus to maximize the chance of spanning the cortical layers? Furthermore, once the tungsten needle was removed, can they be certain that the probe maintained this perpendicular orientation? This is important to understand for the results that discuss effects relative to depth, cell type, etc.\n3) In general, it would help to provide more context and comparison to other thin-film arrays that have been developed. Some of these have been used in human recordings (for example, work from Shadi Dayeh as well Neuralink), and while some papers are cited, it is not explained how the uFINE approach differs.\n4) The comparison between recording with and without the tungsten needle in a single participant is impressive and highly valuable for validating the importance of a flexible array design. To fully test the flexible vs stiff electrode comparison, it would be helpful to perform motion correction on the recording with the needle and quantify the yield and unit quality metrics. If the flexible configuration outperforms the stiff recording with motion correction, this is a valuable quantification for the field.\n5) The claim that the flexible array helps preserve neuronal health during recordings is important, but I do not think it is fully supported here. Without a direct comparison of the waveform similarity metrics to a recording that is done with the needle or to other arrays like Neuropixels, I do not think that the analysis in the paragraph on lines 252-266 can be interpreted beyond saying that they are able to hold cells over time (which is also true for other arrays).\n6) While I appreciate the inclusion of the verbal task for the dLPFC recordings, the description of the results is rather sparse. In particular, it would be helpful to have some basic characterization of whether the neurons recorded during this task carry any task-related information. For example, can word identity be decoded from activity? If so, does this tell us anything about the fact that the firing seems to be primarily suppressed? I don’t think they need to drastically expand the scope of the paper, but it would be helpful to provide a bit more about this task to give more context for what kinds of activity can be recorded with these arrays.\n7) The discussion makes some statements that I don’t think are actually supported by the data. For example, “This reduction in cross-sectional area results in a more tissue-friendly implantation, allowing for faster recovery of neural activity after insertion”, while likely true, was not tested so we can’t be certain. Similarly, “The single-unit yield of the uFINE array is comparable to that of microelectrodes with higher channel counts (e.g., Neuropixels probe)19–23”. I suppose one could look through those references and count yields, but the paper itself doesn’t report this comparison, and the high variability in yield across recordings – both for uFINE and Neuropixels – makes this a somewhat more complicated question.", "reviewer_3": "In this manuscript, Tian et al. demonstrate a polyimide-based flexible neural probe (uFINE) capable of capturing individual neuronal spiking activity in the human brain intraoperatively. Building on their previous work in non-human primates, they fabricated a multi-channel human probe using Ti/IrOx electrode material. Intraoperative recordings were conducted in 11 patients, and the authors report acquiring spikes with high yield and consistency. Overall, this represents a significant technical achievement; however, several issues and missing technical details need to be addressed.\nThe key justification for their approach is the flexibility of the polymeric substrate. The combination of mechanical flexibility and ductility provides major safety and SNR advantages over rigid, brittle Si-based probes such as Neuropixels. However, very little quantification is provided regarding the buckling force or required flexibility. For instance, what differences in neural recording stability were observed between stand-alone flexible probes and shuttle-attached conditions?\nThe detachment/attachment of the probe from its shuttle is unclear. It appears that the probe was glued to the shuttle using KWIK-SIL. Is there any mechanical anchoring feature on the probe? Figure S1 shows a perforation at the tip—was this used for anchoring? If the adhesion is robust enough for insertion, how does the shuttle decouple from the probe during recording? How do the authors ensure that no glue particles remain in the brain during detachment? It should also be noted that KWIK-SIL is not approved for human use.\nThe “Methods/Design, Fabrication, and Assembly of the uFINE Electrode Array” section should be revised to include all necessary information without reliance on previous publications. Steps C–E are critical for understanding the implantation procedure; in its current form, steps C and E may appear contradictory.\nNo information is provided regarding the acquisition system used or how the probe is connected/bonded. Details such as yield, electrode size, and other relevant parameters are missing.\nThe electrodes are 50 µm diameter circles with an impedance of approximately 50 kΩ at 1 kHz. This is a remarkably low impedance, potentially a record for Ir/IrOx electrodes. It is important to clearly explain how such low impedance values were achieved.", "reviewer_4": "The authors present the development of a penetrating, flexible, high-density electrode array designed for human single-neuron recordings. The device, fabricated from medical-grade polyimide using standard microfabrication techniques, was characterized in vitro and subsequently implanted in patients undergoing DBS lead implantation or brain tumor resection surgery.\nThe flexible probes consisted of 1, 2 or 4 shanks, each containing 64 microelectrodes arranged linearly with an electrode pitch of 65 µm or 90 µm. The device was capable of simultaneously recording the activity of up to 135 single units from the human neurocortex, achieving signal quality comparable to recordings obtained with the variant of Neuropixels silicon probes developed for human intraoperative settings. The main advantage of the proposed device over conventional silicon probes lies in its improved recording stability and and reduced susceptibility to brain pulsations, an issue commonly affecting rigid probes used in human neuronal recordings.\nThe authors also demonstrated a research application of the system by examining the heterogenity of individual neurononal responses during a word-repeating task in awake patients.\nOverall, the manuscript is well written and presents a notable technological advancement with relevance for both human neuroscience research and potential clinical applications. However, a more detailed description of the methodology is needed, and several aspects of the manuscript require clarification. My specific comments and questions are listed below.\nMajor concerns:\n1. The implantation procedure is not described in sufficient detail. Please provide more information on how the shuttle was driven into the tissue with the flexible probe, specifically, at what speed and by what mechanism, and how the shuttle was removed after implantation. Is the curved segment of the shank (e.g., Fig. 1D) a design element? Or is this folded part achieved during implatation? Please also describe how the flexible probes were removed from the brain tissue after recordings were completed.\n2. Probes with multiple, closely placed shanks can cause significant brain dimpling, potentially hindering proper implantation (e.g., resulting in only a subset of electrodes reaching the cortex). Was any dimpling observed during or after the implantation of the 2- or 4-shank probes? Were any implantation attempts unsuccessful or resulted only in partial insertion?\n3. The amplitude of brain pulsations likely depends on the size of the craniotomy: larger cranial windows typically cause greater brain motion. Please report the approximate sizes of the craniotomy prepared during DBS and tumor resection surgeries. The latter is presumably larger (~2 cm vs. ~4-8 cm), potentially resulting in greater brain movements. Anesthesia might also affect brain pulsations. Therefore, I suggest comparing signal stability between the two surgical conditions (DBS vs. tumor resection).\n4. The authors classified the recorded single units into three categories: regular spiking, triphasic, and positive spikes. However, in the studies cited for this analysis (Paulk et al. 2022, Nat Neurosci; Sun et al. 2021, J Physiol), two additional categories were identified: compound/broad spikes and fast-spiking units, the latter typically corresponding to interneurons, similar to triphasic spikes. Did the authors observe any units that would fall into these two categories? While compound spikes appear to be rare, fast-spiking units were reported to be relatively abundant in Paulk et al (2022). Please clarify whether such units were detected and, if not, discuss possible reasons.\n5. While the focus of the study is on single-unit recordings, the authors also note that the system can record local field potentials (LFPs). It would be helpful to include basic quality measures for LFPs.\n6. What is the potential for scaling up the developed device? How many channels and what electrode density could be achieved with this technology?\n7. I recommend comparing the present device with other flexible probes intended for human brain-machine interface or clinical applications (e.g. Neuralink probes, Axoft electrode arrays, or the probe demonstrated in Lee et al., 2024. Nat Comm) and with other laminar probes designed for human intracortical recordings (e.g., the thumbtack probe used in Cash et al. 2009, Science; Csercsa et al. 2010, Brain). A brief comparison in the Discussion section would strengthen the manuscript.\nMinor comments:\nLines (L)48-49: “However, its implantation often leads to significant acute tissue damage, …”. Please provide studies supporting the claim that Utah arrays cause significant acute damage.\nL59-60: “owing to their excellent biocompatibility and minimal tissue damage25-27”. The cited studies do not primarily assess biocompatibility. I recommend including additional publications that present comprehensive histological analyses of brain tissue after chronic implantation of flexible probes.\nL86-87: “and confirmed the insertion site using a dummy uFINE module before formal implantation (Methods).” Please provide more details on how the dummy module was used to confirm the insertion site.\nL109-110 and L395-396: “The 64 recording sites on each shank covered a 4-mm distance (6-mm distance for 256-channels uFINE array with 90-μm spacing)” and “The total length of this segment was approximately 4.15 mm (5.76 mm for 256-channels uFINE array)” In the second sentence, the authors give an estimation for the shank length covered with electrodes which seem closer to exact value than provided in the first sentence which states exact lenghts (but are in reality only estimations). Please clarify the inconsistency.\nL115: Specify whether the sample number (n = 5) refers to shanks or probes.\nL117: Similarly, clarify whether (n = 6) refers to probes or recording sites. Please also report the average number or percentage of nonfunctional sites per probe.\nL137-138: “both low raw signals” Please clarify what is meant by “low raw signals.” Do you refer to low-frequency signals (LFPs, <250 Hz) or raw wideband signals?\nL144: “we performed twice recordings”. I suggest replacing “twice recordings” with “two recordings.”\nL146: “exclusively from the recordings that tungsten needles”. I suggest replacing “that tungsten needles” with “where tungsten needles.”\nL152-154: I recommend adding missing units (µV, Hz, ms) for the median values.\nL155-156: The phrase “previous primate studies” suggests multiple studies, but only one is cited. Please extend the reference list.\nL219-220: The text refers to “a subset of patients (n = 7) with concurrent video recordings,” but the Methods mentions only six (L614–615). Please clarify.\nL267-268: I suggest replacing “an area has extensive connections” with “an area that has extensive connections.”\nL277-278: I think the percentages for the stimulus and response periods appear swapped based on Figure 4. Please revise.\nL334-335: “This reduction in cross-sectional area results in a more tissue-friendly implantation, allowing for faster recovery of neural activity after insertion.” Please add supporting references for the claim that reduced cross-sectional area leads to faster recovery of neural activity.\nL355-356: The authors state that by “using finer tungsten wires could … reduce the time required to obtain stable neural signals”. Could the authors please explain why using finer wires would reduce the time required to obtain stable neural signals?\nL410: I would suggest providing more details on the design and fabrication of the tungsten shuttle wires.\nL413-414: The statement “this project did not require a needle withdrawal design during surgery” is unclear. The manuscript indicates that tungsten needles were withdrawn after implantation (L488–489: “The shuttle wires were withdrawn…”). Please clarify.\nL413: Based on the description in the Methods, the silicon substrate is a part of the uFINE module, but it is not shown in the figures. Please clarify what part of the uFINE module is the silicon substrate, why it is needed and how the polymer shanks are mounted to it.\nL415: “the uFINE array was anchored to tungsten shuttle wires” Please describe in more detail how the shuttles were assembled with the uFINE arrays.\nL456: Specify the fluid (e.g., PBS, saline) and reference electrode used during EIS measurements.\nL460: Please define “FPC”.\nL484: Please describe how the dummy module differs from the uFINE module.\nL488: “the redundant part of the array prefixed at the substrate was released immediately.” Please clarify the meaning of “the redundant part” of the array? How was it prefixed?\nL499: Did the authors record wideband recordings (LFP and unit activity at the same time)? Please report the frequency range of recordings.\nL501: Please report the total number of trials used in the word-repeating task.\nL527-528: Please provide the approximate percentage of channels excluded due to high impedance and high RMS noise.\nL539: “Subsequently, experienced electrophysiologists (SW)” Please confirm whether manual curation was performed by a single electrophysiologist.\nL547: Please report the method used to calculate the ISI violation.\nL565: “Defined as the maximum value among the first and second peak-to-trough ratios” The definition is not clear for the reviewer. Please clarify why or how peak-to-trough ratios are used to calculate the peak–trough ratio.\nFigure 1D: In the left side, some of the labels appear misplaced (e.g., “Skull” is positioned on the scalp, while “Dura” indicates the skull). Please clarify.\nFigure 1E: The uFINE probe seems not to scale. Based on the 100 µm scale bar and the 65 µm electrode pitch, the schematic of the uFINE array appears undersized. Please revise.\nFigure 1F: Please specify whether this is the array with the 65 µm or the 90 µm electrode pitch.\nFigure 2A: Spikes on high-passed channels are barely visible, possibly due to PDF compression. Also, while the caption states that traces are 2-sec long, the scale bar suggests 3-4 sec. Please clarify.\nFurther, consider marking or noting nonfunctional channels explicitly. I noticed that there are missing channels (Chn18, Chn107) and also channels with no or very low activity (Chn1-2, Chn8, Chn10). What is the difference between these two channel groups?\nFigure 2B: The figure caption mentions asterisks, but only arrows are visible. Please revise. The mean spike waveform of Unit#72 shown on the right should also be mentioned in the caption.\nFigure 2C: Please briefly explain how the spread of channels was calculated.\nFigure 2I: For the four-shank probe with a larger electrode pitch, electrodes span >4 mm (5.67 mm), yet the y-axis only shows 4 mm. Were single units outside this range excluded?\nFigure 3D: The caption refers to three units, but four are shown. Please revise.\nFigure 3F: Are the numbers in the panel single unit IDs? Please clarify.\nFigure 4M: The two columns representing stimulus (listen) and response (speak) appear swapped since unit #2 and #3 (speak onset) are activated in panel I; however, in panel M unit #2 and #3 appear with red color (activation) in the stimulus column not in the response column. Please revise.\nFigure 4C, G, K: How do unit numbers correspond to the number of active units presented for patients in Table S1. While Pt. 03 has 9 active units in Table S1, in panel C the number of units is higher (12). In contrast, Pt. 04 has 21 active units in Table S1, while fewer (19 units) in panel G. Please clarify.\nFigure 4D: The caption mentions asterisks marking units in panel C, but they are not visible. Please revise.\nTable S1: I suggest replacing “(2) at least one spike each time window” with “(2) fired at least one spike each time window”?\nTable S1: I would suggest adding a footnote indicating that a four-shank probe was used in Pt.11.\nFigure S2B: What is function of the rod-like structure on the left side of the uFINE module?\nFiure S3B: The unit in the y-axis should be µV for the “Amplitude” instead of µm. Also, please clarify the meaning of “notch” in the caption."}, "author_response": "1 REVIEWER #1:\n3 in humans to record high-quality single neurons acutely during brain surgery. The recording\n4 quality, yield, resistance to brain motion, and quality of characterization is very impressive\n5 throughout.\n7 in humans during surgery have been relatively common with the Neuropixel electrode, the rigid\n8 electrode poses significant challenges in terms of recording stability and breakage risk. The\n9 present technology addresses this significant limitation.\n11 written, and the example case study (Fig 4) of word responses in dlPFC is an interesting use\n12 case.\n14 1. The electrode-to-electrode spacing was 65um, with 50um diameter electrode pads. A useful\n15 way to characterize units is to examine on how many nearby electrodes a given unit is\n16 visible. Thus the question: what was the number of electrode pads that a given unit was\n17 visible? If it is several, that provides additional resolution to estimate brain motion.\n19 characterizing unit visibility across the electrode array for assessing motion resolution.\n21 channels. The number of single units whose waveforms were visible on only single electrode site\n22 was 40. The remaining single units can be recorded simultaneously on two or more channels,\n23 with nearly half of the units from Pt.05 detected simultaneously on two sites. This method could\n24 provide additional brain resolution for estimating brain motion by employing a spline-based fast-\n25 motion correction (Windolf et al., Nature Methods, 2025, Figure 2b) that tracks high-amplitude\n26 units across channels and correlates with LFP-estimated movement.\n27\n28 2. Material verification and Sterilization. The methods mention that ‘standard EO sterilization’\n29 was used. What testing was done to verify that the resulting product was sterile (sterilization\n30 verification)? Second, I didn’t see consideration of the materials and their\n31 toxicity/biocompatibility. What testing was done to assure that the materials used in the parts\n32 of the electrode that enter the brain have acceptable toxicity profiles?\n34 which are indeed critical for clinical experiments. We fully agree that ensuring safety in these\n35 areas is paramount.\n36  \"The methods mention that ‘standard EO sterilization’ was used. What testing was done to\n37 verify that the resulting product was sterile (sterilization verification)? \"\n39 party type-test report is an official safety and performance certificate issued by an accredited\n40 independent laboratory. It provides verifiable evidence of device performance and safety and was\n41 used here as a prerequisite for Ethics Committee approval of this Investigator-Initiated Trial (IIT).\n43 (1) EO sterilization verification performed according to GB/T 14233 standard\n44 (https://std.samr.gov.cn/gb/search/gbDetailed?id=EB58F4DA904DB2A2E05397BE0A0A7D33)\n45 and\n46 https://std.samr.gov.cn/gb/search/gbDetailed?id=02882896172A250CE06397BE0A0A9F76);\n47 and\n48 (2) Biocompatibility and toxicity testing of the flexible electrode and the tungsten needle conducted\n49 in accordance with the GB/T 16886 standard (equivalent to ISO 10993 standard,\n50 https://www.iso.org/standard/10993-1) standards.\n51  “ Second, I didn’t see consideration of the materials and their toxicity/biocompatibility. What\n52 testing was done to assure that the materials used in the parts of the electrode that enter the\n53 brain have acceptable toxicity profiles?”\n55 inspection report includes biocompatibility and toxicity testing for both components as follows:\n56 (1) uFINE array\n57  Chemical tests (5 items: appearance/color, reducing substances, residue on evaporation, total\n58 heavy metals, UV absorbance); standards: GB/T 14233.1‑2008, GB/T 16886.5‑2017, GB/T\n59 16886.10‑2017.\n60  Cytotoxicity (GB/T 16886.5‑2017).\n61  Intracutaneous (intracutaneous/skin) reactivity (GB/T 16886.10‑2017).\n62  Pyrogenicity (GB/T 16886.11‑2021).\n63  Acute systemic toxicity (GB/T 16886.11‑2011).\n64  Skin sensitization (GB/T 16886.10‑2017).\n65 (2) Tungsten guiding needle\n66  Chemical tests (6 items: appearance/color, pH, reducing substances, residue on evaporation,\n67 total heavy metals, UV absorbance); standard: GB/T 14233.1‑2008.\n68  Cytotoxicity (GB/T 16886.5‑2017).\n69  Intracutaneous reactivity (GB/T 16886.10‑2017).\n70  Pyrogenicity (GB/T 16886.11‑2021).\n71  Acute systemic toxicity (GB/T 16886.11‑2011).\n72  Skin sensitization (GB/T 16886.10‑2017).\n73 3. Electrode removal. I didn’t see description of electrode removal steps and experience. What\n74 are the considerations for successful removal and was testing done to assure that removal\n75 would always be possible/successful? Was removal without left-behind parts successful in\n76 all cases?\n78  \"What are the considerations for successful removal and was testing done to assure that\n79 removal would always be possible/successful?\"\n81 the mechanical characteristics of the uFINE array.\n82 (1) Procedure: After completing electrophysiological recordings, the uFINE array was removed\n83 by slowly raising the micromanipulator along the original vertical insertion axis, thereby pulling the\n84 array out of the brain. When uFINE array removed, we inspected the electrode shank under the\n85 surgical microscope to confirm structural integrity of the implanted segment and to check for\n86 breakage or deformation. We will add this electrode removal procedure to the Methods (uFINE\n87 array removal; Revised manuscript, Line 573-582).\n88 (2) Mechanical characteristics: Although highly flexible, the uFINE array has sufficient mechanical\n89 strength to withstand peak tensile forces measured in mechanical testing (>10 g or 0.1 N, Figure\n90 1G). The uFINE array can withstand a pulling force of up to 10 g (Figure 1G). In contrast, the\n91 frictional force between flexible electrodes and brain tissue during insertion and removal is\n92 typically in the micronewton (μN) range, which is negligible (Tian et al., Micromachines, 2025).\n94 structural integrity without breakage during the pull-out process.\n95  \"Was removal without left-behind parts successful in all cases?\"\n97 microscopic inspection consistently confirmed the structural integrity of the retrieved shank\n98 segment. After multiple insertion and removal tests in pigs, our results showed that all uFINE\n99 array could be removed completely without left-behind parts.\n100 4. Tungsten guide wire removal. I couldn’t decipher how the tungsten wire removal is initiated –\n101 how is the tungsten wire attached to the electrode and how is it detached so that I can be\n102 removed?\n104  “How is the tungsten wire attached to the electrode”:\n106 shank’s silicon substrate using a biocompatible surgical adhesive (Kwik-sil). The tip of each uFINE\n107 array features a small circular aperture. The aperture is anchored to the tungsten shuttle needle,\n108 thereby completing the attachment of the electrode to the tungsten needle. See the enlarged\n109 images of the electrode tip for illustration (left panel for implantable segment of uFINE array and\n111\n112  “How is it detached so that the electrode can be left in place and the tungsten removed?”\n114 redundant uFINE array filament stacked on the surface of silicon substrate was flashed by saline.\n116 vertical movement of the needle) to retract the tungsten guide needle. Once the tungsten guide\n117 needle is withdrawn from the brain, the uFINE array remains implanted. After removing the guide\n118 needle, we keep the tungsten tip ~3–5 mm above the cortical surface to avoid contact or\n119 scratching of the cortex. Subsequently, the recording can proceed.\n120 5. LFP. Did the electrode allow reliable recording of LFPs and if so what were the\n121 characteristics of the LFPs recorded?\n123 data within a bandwidth of 0.1–10,000 Hz. Following low-pass filtering (cutoff frequency <250 Hz),\n124 robust LFP signals were extracted. The primary characteristics of the acquired LFPs are as\n125 follows:\n126 (1) Spatial consistency and resolution: LFP signals recorded on the same shank exhibit a high\n127 degree of correlation (as illustrated in the “raw data” column of Figure 2A), while simultaneously\n128 demonstrating local variations between specific recording sites. For example, in the “raw data” of\n130 on shank 1. Similarly, Chn#116 and Chn#17 differ from other channels on shank 2.\n131 (2) Energy distribution:\n133 beta and gamma frequency bands, specifically below approximately 50 Hz (as illustrated in the\n134 low-pass filtered panels of Figure 2B).\n136 layers (left panel), whereas gamma oscillations are more prominent in superficial layers (middle\n137 panel). The combined depth distribution of theta/beta/gamma oscillations is summarized in the\n138 right panel, which aligns with findings from prior research (Buzsáki et al., Nature Reviews\n140\n141 6. Localization. How did the authors localize their recordings, both in terms of exact position\n142 inserted (is the electrode visible on X-Ray or intra-op CT?) and for assigning cortical layers\n143 to different depths on the electrode?\n145 cortical layers to varying depths. The following details our current methodology, its limitations,\n146 and ongoing efforts to improve accuracy.\n147 (1) Localization of the electrode insertion trajectory:\n149 200 μm. Direct visualization via standard intraoperative imaging resolutions such as X-\n150 ray/CT/MRI are insufficient to visualize the electrode body clearly. Consequently, we employ the\n151 following strategies for implantation site localization:\n152  DBS procedures: The uFINE array is implanted according to DBS surgical trajectory planning.\n153 the DBS electrode insertion trajectory is determined from preoperative imaging and\n154 stereotactic planning. This trajectory defines the uFINE array implantation position, typically\n155 without the need for additional imaging-based localization.\n156  Brain tumor resection surgeries: After electrode implantation, we use intraoperative navigation\n157 systems (CurveNavigation 17700, BrainlabAG Co.) to mark the electrode entry point\n158 (navigation coordinates) on a three-dimensional brain model built from preoperative MRI, so\n159 as to document the spatial position of the electrode on the cortex or cortical surface. The\n160 navigation accuracy of this device is difficult to quantify, as it primarily depends on the\n161 resolution of the MRI images and the precision of the 3D brain modeling derived from those\n162 images.\n164 two methods described above. We added the above description to the Methods subsection\n165 “uFINE array positioning and insertion” to clarify how the exact insertion locations were\n166 determined (revised manuscript, Line 555-568).\n167 (2) Cortical layers assignment to different depths on the electrode:\n169 process. Initially, a tungsten needles was approached to the cortical surface using a\n170 micromanipulator until proximity was achieved without physical contact. The vertical position of\n171 the tungsten needle was continuously monitored. Subsequently, the vertical fine-adjustment\n172 control was carefully manipulated to lower the tungsten needle tip perpendicularly until initial\n173 contact with the cortical surface was detected, and the corresponding position was recorded.\n175 axis to the designated implantation depth. This methodology was implemented to a consistent,\n176 relatively orthogonal trajectory of electrode insertion relative to the local cortical surface.\n178 and achieved implant depth can be inevitable. This deviation is attributable to brain pulsation and\n179 cortical surface indentation during the insertion process. Brain pulsation is modulated by\n180 intracranial pressure, cardiac activity and respiration. The cortical surface dimpling is influenced\n181 by pulsation, insertion velocity, the remaining thickness of the arachnoid and pia mater, and the\n182 angle of insertion. Consequently, the approach used to control implantation depth in this study\n183 may make deviation from the true depth of the electrode by approximately a few millimeters, which\n184 can make the accurate assignment of cortical layers difficult.\n186 precision, suitable for routine clinical use that can directly visualize the depth of uFINE array at\n187 an order of hundreds-of-micrometers scale for cortical layer assignment. Therefore, precise\n188 cortical layer assignment to different depths on the electrode remains a challenge.\n191 studies using uFINE array, we are attempting to observe the relative depth of the uFINE with\n192 respect to the cortical surface using intraoperative ultrasound or optical coherence tomography,\n193 but resolution and workable depth range remain limited.\n195 electrophysiological features (e.g., depth‑dependent LFP spectral distributions, amplitude/phase\n196 gradients) combined with current source density analysis (iCSD). The LFP depth distributions in\n197 the theta/beta and gamma bands described above support preliminary electrophysiology‑based\n198 cortical layer assignment.\n200 layer in the “Limitations and future directions” section. (Revised manuscript, Line 354-365).\n202 1. ECG. Have the authors recording simultaneous ECG? That way effects of brain motion can\n203 be quantified rigorously (Mosher et al 2020, Cell Reports). If this data is not available,\n204 sufficient to mention as future possibility.\n206 study. We acknowledge that brain pulsation is modulated by both respiration and cardiac activity.\n208 with neurophysiological signals, thereby enabling better quantification and subsequent mitigation\n209 of motion artifacts in LFP analysis. In our ongoing clinical neuroscience research, we have tried\n210 recording synchronized ECG and electrophysiological data to specifically address and eliminate\n211 the impact of brain motion on LFP signals.\n214 2. The stated ‘tensile strengths’ of 831 MPa – what does this number look like for other\n215 commonly used electrodes, such as Neuropixel or tungsten microwires? Would be helpful to\n216 have this comparison to put this number into perspective (it is hard for me to say if this is\n217 good or bad).\n219 during procedure of electrode implantation and removal was difficult to understand. Consequently,\n220 we have revised Figure 1H, the corresponding descriptions (Line 114-115) and the figure legend\n221 to represent the strength of the uFINE array by pulling force.\n223 0.33 N. Since the frictional forces between flexible electrodes and brain tissue during implantation\n224 and removal were reported to be in the micro-newton (μN) level, our findings confirm that the\n225 mechanical strength of the uFINE array provides a sufficient safety margin for the removal\n226 process.\n229 in human cortex. They characterize mechanical properties of the electrode array and describe\n230 the setup for intraoperative recordings in human patients undergoing DBS or brain tumor\n231 surgeries. In 11 cases where they successfully recorded neurons, they describe the yield and\n232 electrophysiological properties of the cells, including waveform shapes. In one case, they\n233 directly compare recordings performed with the tungsten stiffener attached to after the stiffener\n234 was removed, showing that neuron tracking is superior when the array is able to move with\n235 brain pulsation. Finally, they show neurons with task-evoked activity from dLPFC, where most\n236 neurons were suppressed during the speaking portion of a verbal repetition task.\n238 technology for humans. The paper is simple and clearly written, and I think will be of interest to\n239 the field, especially if these arrays can be made available either commercially or through\n240 partnerships.\n241 1. It would help to know if the patient numbering scheme used reflects the sequential order of\n242 recording. Specifically, were there no issues after the first 2 DBS cases, and was this more\n243 of a challenge for the tumor cases? Similarly, is there something about the tumor cases that\n244 made these recordings more likely to suffer from noise issues? I assume that the tumor\n245 cases involved larger craniotomies, though this was not stated explicitly in the paper.\n247  “It would help to know if the patient numbering scheme used reflects the sequential order of\n248 recording.”\n250 chronological order of the recordings.\n251  “Specifically, were there no issues after the first 2 DBS cases, and was this more of a\n252 challenge for the tumor cases? Similarly, is there something about the tumor cases that made\n253 these recordings more likely to suffer from noise issues? I assume that the tumor cases\n254 involved larger craniotomies, though this was not stated explicitly in the paper.\n257 integration of advanced auxiliary equipment such as neuronavigation systems and\n258 somatosensory evoked potential (SSEP) monitoring. This makes the tumor cases encounter more\n259 technical challenges, especially more noise issues, during the tumor cases compared to the initial\n260 two DBS cases.\n262 intraoperative equipment. Specifically, tumor resections typically utilize surgical navigation\n263 systems, which were not used in the DBS cases. We discovered that the radiofrequency (RF)\n264 components of the navigation system introduced significant electromagnetic interference into the\n265 intraoperative electrophysiological recordings. It took three cases for our team to systematically\n266 troubleshoot, and finally identified the navigation system as the specific source of this noise.\n267 2. Other than avoiding vasculature, how was the probe insertion targeted on the cortical\n268 surface? Was there an attempt to place the array perpendicularly to the surface in the\n269 middle of a gyrus to maximize the chance of spanning the cortical layers? Furthermore,\n270 once the tungsten needle was removed, can they be certain that the probe maintained this\n271 perpendicular orientation? This is important to understand for the results that discuss effects\n272 relative to depth, cell type, etc.\n274 maintenance of implanted shank posture. Our point-by-point clarifications are provided below.\n275  “Other than avoiding vasculature, how was the probe insertion targeted on the cortical surface?\n277 to maximize the chance of spanning the cortical layers?”\n279 targeting site on the cortical surface, our protocol integrates both avoiding vasculature and\n280 anatomical landmarks.\n282 (1) Vascular Avoidance: We first identify a region on the target gyrus that is less of major surface\n283 vasculature, as a safe zone.\n284 (2) Anatomical landmark: Within this 'safe zone,' we prioritize the apex or the area near the midline\n285 of the gyrus as close as possible.\n286 (3) Perpendicular to brain surface: The multi-articulated arm and the micromanipulator are\n287 adjusted to be perpendicular to brain surface of the targeted gyrus center before the tungsten\n288 needle is advanced.\n290 “Prior to implantation, the target site was selected by identifying a region that can avoid the major\n291 surface vasculature and can approach to the apex of the gyrus as close as possible. The multi-\n292 articulated arm was then manually adjusted to ensure the uFINE array target in this region and\n293 be as perpendicular to the cortical surface as possible, thereby maximizing the likelihood of\n294 spanning the full extent of the cortical layers.”\n295  “ Furthermore, once the tungsten needle was removed, can they be certain that the probe\n296 maintained this perpendicular orientation? This is important to understand for the results that\n297 discuss effects relative to depth, cell type, etc.”\n299 upon insertion. For instance, Zhao et al., Nature Biomedical Engineering, 2023 showcased the\n300 volumetric distribution of a 64-shank NET array (the predecessor of the uFINE array) within the\n301 mouse visual cortex via Micro-CT imaging (Figure 1e).\n302\n304 post-implantation. This structural integrity was further supported in an earlier work (Zhao et al.,\n306 the filaments both in vitro and in vivo. Figure 7B and 7C demonstrate that the shank retains their\n307 vertical alignment in in vitro tests following the retraction of the guiding needle.\n308\n309 3. In general, it would help to provide more context and comparison to other thin-film arrays\n310 that have been developed. Some of these have been used in human recordings (for\n311 example, work from Shadi Dayeh as well Neuralink), and while some papers are cited, it is\n312 not explained how the uFINE approach differs.\n314 comparison with current state-of-the-art neural probes is essential to properly position our work,\n315 and we have added a dedicated section to revised manuscript (Line 334-336) to address this.\n317 there is currently no published work that provides robust statistical data on unit recording\n318 performance using flexible depth electrodes in the human brain. Our work addresses this critical\n319 gap.\n321 human settings:\n322  Shadi Dayeh’s work (Lee et al., Nature Communications, 2024): This work did not disclose\n323 the recording of single-unit activity in the human brain.\n324  Neuralink:1024-channel flexible threads, wireless BMI system. No disclosure. To our\n325 knowledge, no statistical data regarding SUA yield in human subjects has been publicly\n326 disclosed.\n327  Axoft Electrode Arrays (Lee et al., medRxiv, 2025): Limited disclosure, single case. During\n328 intraoperative recording, the diagrams showed that 22 out of 32 electrodes were able to record\n329 31 spikes. The calculated single-unit yield per channel is 0.969, with a sample size of n=1.\n331 relevant study to demonstrate single-unit activity recording using thin-film electrodes in the human\n332 brain. We have successfully provided systemic statistical data on the recording yield which was\n333 previously missing in literature.\n335 systematically compare our findings with theirs.\n336 4. The comparison between recording with and without the tungsten needle in a single\n337 participant is impressive and highly valuable for validating the importance of a flexible array\n338 design. To fully test the flexible vs stiff electrode comparison, it would be helpful to perform\n339 motion correction on the recording with the needle and quantify the yield and unit quality\n340 metrics. If the flexible configuration outperforms the stiff recording with motion correction,\n341 this is a valuable quantification for the field.\n343 have indeed performed spike sorting and attempted motion correction on the recording with\n344 tungsten needle. However, we have ultimately decided not to include these results as quantitative\n345 baseline evidence and have removed the statement suggesting that brain pulsation significantly\n346 impacts yield to ensure the rigor of our conclusions.\n348 needle, we found that the results were not meaningful. The site pitch of our electrode used to\n349 make comparison between recording with and without the tungsten needle is 90 μm, whereas the\n351 designed for Neuropixels, for example \"DREDge\" algorithm (Windolf et al., Nature Methods, 2025),\n352 could not be effectively implemented with our spacing. Additionally, considering the subjective\n353 interference during the manual curation process, we think this comparison lacks significant and\n354 rigorous meaning.\n355 5. The claim that the flexible array helps preserve neuronal health during recordings is\n356 important, but I do not think it is fully supported here.\n358 recordings\" may overstate what our current data can support. After careful consideration, we do\n359 agree with this comment.\n361 similarity metrics, primarily demonstrates the uFINE array's ability to mitigate the impact of brain\n362 pulsations during intraoperative procedures. This ensures stable recordings and the consistent\n363 identification of single units. However, these results should not be directly interpreted as superior\n364 “preservation of neuronal health” compared to rigid probes like Neuropixels, as direct comparative\n365 biological or histological evidence is currently lacking. Hence, we delete the claim of “preserve\n366 neuronal health during recordings” in the manuscript.\n368 reflects our findings.\n370 should facilitate stable capturing of neuronal activity over longer timescales (e.g., minutes).\n372 between the electrodes and the brain tissue, minimizing spike drifting during prolonged recordings,\n373 and reducing loss of spiking information when doing spike-sorting.\n374 6. While I appreciate the inclusion of the verbal task for the dLPFC recordings, the description\n375 of the results is rather sparse. In particular, it would be helpful to have some basic\n376 characterization of whether the neurons recorded during this task carry any task-related\n377 information. For example, can word identity be decoded from activity? If so, does this tell us\n378 anything about the fact that the firing seems to be primarily suppressed? I don’t think they\n379 need to drastically expand the scope of the paper, but it would be helpful to provide a bit\n380 more about this task to give more context for what kinds of activity can be recorded with\n381 these arrays.\n383 fully agree that assessing whether neurons carry task-relevant information (e.g., word decodability)\n384 is crucial for understanding the functional significance of the observed inhibitory-dominant neural\n385 activity.\n387 were not included in this manuscript:\n388 (1) The primary objective of this study was to establish and validate the complete toolbox for\n390 stage clinical trials, where the word-repeat task was designed for maximum simplicity to minimize\n391 patient burden and prioritize the evaluation of surgical implantation procedures and signal quality.\n393 analysis (e.g., in terms of trial counts, randomized stimulus sequences, or control conditions).\n394 (2) The data in Figure 4 are mainly intended to demonstrate that the uFINE array can record\n395 tuned single-unit activity during auditory stimulation or speech production, with no direct intention\n396 to provide analysis of neural mechanisms.\n398 data can support. In our ongoing clinical studies, we have partnered with neuroscience teams to\n399 implement specialized paradigms for working memory and language processing. We plan to\n400 perform systematic decoding and mechanistic explorations in these refined datasets, with a\n401 subsequent manuscript currently in preparation.\n402 7. The discussion makes some statements that I don’t think are actually supported by the data.\n404 implantation, allowing for faster recovery of neural activity after insertion”, while likely true,\n405 was not tested so we can’t be certain. Similarly, “The single-unit yield of the uFINE array is\n406 comparable to that of microelectrodes with higher channel counts (e.g., Neuropixels\n407 probe)19–23”. I suppose one could look through those references and count yields, but the\n408 paper itself doesn’t report this comparison, and the high variability in yield across recordings\n409 – both for uFINE and Neuropixels – makes this a somewhat more complicated question.\n411 direct quantitative support within the context of this specific study.\n413 friendly implantation, allowing for faster recovery of neural activity after insertion”, and “The single-\n414 unit yield of the uFINE array is comparable to that of microelectrodes with higher channel counts\n415 (e.g., Neuropixels probe)19–23”.\n418 capable of capturing individual neuronal spiking activity in the human brain intraoperatively.\n420 probe using Ti/IrOx electrode material. Intraoperative recordings were conducted in 11 patients,\n421 and the authors report acquiring spikes with high yield and consistency. Overall, this represents\n422 a significant technical achievement; however, several issues and missing technical details need\n423 to be addressed.\n424 1. The key justification for their approach is the flexibility of the polymeric substrate. The\n425 combination of mechanical flexibility and ductility provides major safety and SNR\n426 advantages over rigid, brittle Si-based probes such as Neuropixels. However, very little\n427 quantification is provided regarding the buckling force or required flexibility. For instance,\n428 what differences in neural recording stability were observed between stand-alone flexible\n429 probes and shuttle-attached conditions?\n431 provide quantitative evidence for the mechanical flexibility.\n432  “The key justification for their approach is the flexibility of the polymeric substrate. The\n433 combination of mechanical flexibility and ductility provides major safety and SNR advantages\n434 over rigid, brittle Si-based probes such as Neuropixels. However, very little quantification is\n435 provided regarding the buckling force or required flexibility.”\n437 properties of our flexible electrodes.\n439 2020) that systematically compares the mechanical characteristics of Si-based probes, metal\n440 microwires, and various flexible materials (such as SU-8, Polyimide, and Parylene-C). Based on\n441 these properties, we calculated the bending stiffness for our uFINE electrode (dimensions: 200\n442 μm×1). The results are summarized as follows:\n443  The bending stiffness of the Polyimide (PI)-based uFINE electrode is approximately 0.04\n444 pNm2.\n445  For comparison, similarly sized probes made of SU-8 and Parylene-C yield values of\n446 approximately 0.004 pNm2 and 0.05 pNm2, respectively.\n447  In contrast, a Silicon-based probe (transformed to Neuropixels dimensions) has a bending\n448 stiffness of approximately 1.05×107 pNm2.\n450 magnitude more flexible than traditional Si-based probes. This extreme flexibility is key to\n451 reducing the mechanical mismatch between the implant and the brain tissue, thereby enhancing\n452 long-term safety and signal stability.\n453  “For instance, what differences in neural recording stability were observed between stand-\n454 alone flexible probes and shuttle-attached conditions?”\n456 our manuscript, with the key findings presented in Figure 3A and 3B (Line 205-217):\n458 tungsten needles, we observed significant periodic fluctuations in spike positions (median drift:\n459 88.75 μm). This periodic fluctuation mirrors the motion artifacts commonly observed in\n461 (Paulk et al., Nature Neuroscience, 2022; Chung et al., Neuron, 2022)\n463 array demonstrated its native ability to accommodate brain movements (Figure 3A). This resulted\n464 in more stable and continuous firing patterns, with the median drift reducing to 29.42 μm (mostly\n465 below 50 μm, as quantified in Figure 3B).\n466 2. The detachment/attachment of the probe from its shuttle is unclear. It appears that the probe\n467 was glued to the shuttle using KWIK-SIL. Is there any mechanical anchoring feature on the\n468 probe? Figure S1 shows a perforation at the tip—was this used for anchoring? If the\n469 adhesion is robust enough for insertion, how does the shuttle decouple from the probe\n470 during recording? How do the authors ensure that no glue particles remain in the brain\n471 during detachment? It should also be noted that KWIK-SIL is not approved for human use.\n473 clarity of our anchoring mechanism.\n475 enters the brain tissue. As specified in the Methods (Line 466-468), KWIK-SIL was used\n476 exclusively outside the skull to bond the proximal end of the shuttle needles to the silicon carrier\n477 substrate. Therefore, there is no risk of glue particles remaining in the brain.\n479 mechanical \"threading\" design rather than chemical adhesion. As shown in the enlarged view of\n480 the probe tip below, each shank features an 18 μm-diameter aperture. During assembly, the\n481 conical tips of the shuttle needles were threaded through these apertures, providing the necessary\n482 mechanical anchoring for insertion without the need for glue on the shanks themselves. (Please\n483 refer to the enlarged view of the probe tip provided below.)\n485 reaching the target depth, the \"redundant portion\" of the array—temporarily secured to the carrier\n486 substrate by salt crystallization—was released by applying sterile saline to dissolve the crystals.\n488 arrays were only mechanically threaded through the apertures, they easily slid out, allowing the\n489 rigid shuttles to be completely withdrawn while leaving only the flexible uFINE shanks embedded\n490 in brain tissue.\n491\n492 3. The “Methods/Design, Fabrication, and Assembly of the uFINE Electrode Array” section\n493 should be revised to include all necessary information without reliance on previous\n494 publications. Steps C–E are critical for understanding the implantation procedure; in its\n495 current form, steps C and E may appear contradictory.\n498 section to ensure we provide all necessary technical details without requiring to refer to previous\n499 publications. Furthermore, to resolve potential confusion regarding the procedure, we relocated\n502 eliminating any ambiguity. The revised descriptions can be found in Line 421-484.\n503 4. No information is provided regarding the acquisition system used or how the probe is\n504 connected/bonded. Details such as yield, electrode size, and other relevant parameters are\n505 missing.\n507 characterizing unit visibility across the electrode array for assessing motion resolution.\n509 system, probe connection, and physical parameters were included in the manuscript. We have\n510 summarized these details below and ensured they are easily locatable:\n512 (Shanghai Stairmed Technology Co., Ltd.), which supports up to 1,024 channels (Methods, Line\n513 508-509). The system setup, including the enclosed uFINE module (comprising the array, FPC,\n514 and headstage) and the data communication cables, is described in the Methods, Line 512-517.\n516 mechanism (Q1), the probe is connected to the shuttle needles via mechanical threading rather\n517 than chemical bonding. The conical tips of the tungsten shuttle needles were threaded through\n518 18 μm-diameter apertures at the distal tips of the flexible shanks, allowing for stable insertion\n519 without the use of adhesives on the implantable portion.\n521 channel flexible printed circuit (FPC), which was connected to the headstage via a Zero Insertion\n523 using a custom-designed Type-C cable.\n525 circuit (FPC) was soldered for connection between headstage and uFINE array.\n528 units from 1,302 valid channels (impedance < 4 MΩ), resulting in an average yield of 0.55 ± 0.31\n529 single units per channel.\n531 are provided in the Methods (Line 407-416) and Figure S1. Key parameters include:\n532  Shank dimensions: 2 μm thickness; 20 mm length (35 mm for 256-channel version).\n533  Recording sites: Each circular site has a diameter of 50 μm, with a center-to-center pitch of\n534 65 μm (90 μm for 256-channel version).\n535  Shank geometry: A tapered structure with a tip width of 80 μm and a maximum width of 245\n536 μm at the implantable portion.\n537\n538 5. The electrodes are 50 µm diameter circles with an impedance of approximately 50 kΩ at\n539 1 kHz. This is a remarkably low impedance, potentially a record for Ir/IrOx electrodes. It is\n540 important to clearly explain how such low impedance values were achieved.\n542 clarify the mechanism behind this performance.\nx\n545 visually resembling the texture of a soda cracker cross-section. This porous morphology\n546 drastically increases the effective surface area relative to the geometric surface area. By\n547 maximizing the contact interface between the IrO layer and the fluid, we effectively minimized\nx\n548 the interfacial impedance.\n550 the film's porosity is highly sensitive to deposition conditions. By precisely fine-tuning critical\n551 parameters—specifically the oxygen (O ) partial pressure, chamber pressure, and sputtering\n2\n552 duration—we were able to engineer the film growth to transition from a dense structure to the\n553 high-surface-area porous structure presented in this work.\n555 difference between a standard coating (general example, left panel) and our optimized low-\n556 impedance coating (good example, right panel). The \"Good\" sample clearly demonstrates the\n557 porous, high-surface-area architecture that facilitates such low impedance values.\nx\n560 increases the effective surface area, thereby facilitating interfacial contact and substantially\n561 reducing the impedance.\n563 of uFINE electrode array\" in the revised manuscript (Line 450-453).\n565 deposit a Ti/Au (5/100 nm) seed layer. To minimize interfacial impedance, Iridium Oxide (IrO )\nx\n566 was subsequently grown via reactive sputtering (PRO Line PVD 75, Kurt J. Lesker) at 130 W for\n567 1200 s under an Ar/O atmosphere, followed by a final lift-off step.\n2\n568\n571 designed for human single-neuron recordings. The device, fabricated from medical-grade\n572 polyimide using standard microfabrication techniques, was characterized in vitro and\n573 subsequently implanted in patients undergoing DBS lead implantation or brain tumor resection\n574 surgery.\n576 linearly with an electrode pitch of 65 µm or 90 µm. The device was capable of simultaneously\n577 recording the activity of up to 135 single units from the human neurocortex, achieving signal\n578 quality comparable to recordings obtained with the variant of Neuropixels silicon probes\n579 developed for human intraoperative settings. The main advantage of the proposed device over\n580 conventional silicon probes lies in its improved recording stability and and reduced susceptibility\n581 to brain pulsations, an issue commonly affecting rigid probes used in human neuronal\n582 recordings.\n584 heterogenity of individual neurononal responses during a word-repeating task in awake patients.\n586 relevance for both human neuroscience research and potential clinical applications. However, a\n587 more detailed description of the methodology is needed, and several aspects of the manuscript\n588 require clarification. My specific comments and questions are listed below.\n590 1. The implantation procedure is not described in sufficient detail. Please provide more\n591 information on how the shuttle was driven into the tissue with the flexible probe, specifically,\n592 at what speed and by what mechanism, and how the shuttle was removed after\n593 implantation. Is the curved segment of the shank (e.g., Fig. 1D) a design element? Or is this\n594 folded part achieved during implatation? Please also describe how the flexible probes were\n595 removed from the brain tissue after recordings were completed.\n597 (1) Mechanism and speed of uFINE array implantation\n599 the micro-drive mechanism. As illustrated in Figure S2, we employed two distinct systems\n600 depending on the surgical context:\n601  For DBS procedures, the commercial NeuroFortis (AlphaOmega Co.) micromanipulator was\n602 employed.\n603  For tumor resection surgeries, a custom-designed micromanipulator was used.\n605 surgeon manually. The surgeon achieves this movement by hand-turning the knob of\n606 micromanipulator, which directly drives the electrode shank and the tungsten needle shuttle into\n607 the tissue. The implantation speed is therefore surgeon-controlled, maintained at an estimated\n608 slow rate of approximately 1 mm/s.\n609 (2) Removal of the shuttle after implantation\n610  Redundant Segment Release: Upon reaching the desired depth, the redundant portion of the\n611 array which had been temporarily held to the substrate by salt crystallization, and was rapidly\n612 released once the crystals were dissolved with saline.\n613  Shuttle removal: Following this release, the micromanipulator operation was reversed to\n614 carefully retract the shuttle needles and remove the rigid tungsten needle shuttle from the\n615 brain tissue. The needles were then suspended 3–5 mm above the brain surface, leaving only\n616 the uFINE shanks embedded.\n617 (3) Curved segment of the shank\n619 manufactured in a bent shape). Instead, this morphology is illustrated to highlight the ultraflexible\n620 nature of the uFINE array, which allows it to bend and conform to brain tissue dynamics freely.\n621 (4) Removal of uFINE Arrays After Recordings\n623 the micromanipulator along the original vertical insertion axis, thereby pulling the array out of the\n624 brain. After uFINE array removed, we inspected the electrode shank under the surgical\n625 microscope to confirm structural integrity of the implanted segment and to check for breakage or\n626 deformation.\n628 manuscript, Line 573-582).\n629 2. Probes with multiple, closely placed shanks can cause significant brain dimpling, potentially\n630 hindering proper implantation (e.g., resulting in only a subset of electrodes reaching the\n631 cortex). Was any dimpling observed during or after the implantation of the 2- or 4-shank\n632 probes? Were any implantation attempts unsuccessful or resulted only in partial insertion?\n634 for multi-shank probes.\n636 the initial contact, which resolved immediately upon penetration. Consequently, this did not hinder\n637 the implantation process.\n639 probes (both 2- and 4-shank) were successfully and completely implanted. We believe this high\n640 success rate was largely due to two factors:\n641 (1) Pia Incision: As described in the Methods (Line 534-535), before inserting the probe, a small\n642 superficial incision in the pia was performed using an arachnoid surgical knife.\n643 (2) Simultaneous Contact: We carefully adjusted the electrode position to ensure all the shanks\n644 made simultaneous contact with the brain surface.\n646 shank spacing).\n647 3. The amplitude of brain pulsations likely depends on the size of the craniotomy: larger cranial\n648 windows typically cause greater brain motion. Please report the approximate sizes of the\n649 craniotomy prepared during DBS and tumor resection surgeries. The latter is presumably\n650 larger (~2 cm vs. ~4-8 cm), potentially resulting in greater brain movements. Anesthesia\n651 might also affect brain pulsations. Therefore, I suggest comparing signal stability between\n652 the two surgical conditions (DBS vs. tumor resection).\n654 conditions. We agree that the surgical paradigm significantly influences the recording environment.\n655 (1) Difference in Craniotomy Size: We confirm that the craniotomy sizes differ significantly:\n656 approximately 1.5 cm for DBS and 4–10 cm for tumor resection.\n657 (2) Comparison of Signal Stability: To address the reviewer's request for a stability comparison,\n658 we performed the following analyses:\n659  Physical Motion (Video Analysis):\n661 brain motion visually as detailed in (Figure S3, D and E). Unfortunately, a direct statistical\n662 comparison of physical brain pulsation amplitude (video-based) between the two surgical groups\n663 remains unfeasible due to the limited number of tumor resection cases with high-quality video\n664 recordings (n=1).\n665\nNO. Surgical conditions Anesthesia Yield_per_chn\non_distance\n667  Functional Stability (Spike Position Drift):\n669 the spike position drift — a direct functional measure of signal stability.\n671 which prevents consistent drift calculation. Consequently, due to the disparity in sample sizes\n672 between the DBS group (n=8) and the Tumor Resection group (n=2), we relied on descriptive\n673 statistics rather than formal hypothesis testing.\n674  DBS Group: Median spike drift = 17.39 μm\n675  Tumor Resection Group: Median spike drift =36.89 μm\n676\n677 4. The authors classified the recorded single units into three categories: regular spiking,\n678 triphasic, and positive spikes. However, in the studies cited for this analysis (Paulk et al.\n679 2022, Nat Neurosci; Sun et al. 2021, J Physiol), two additional categories were identified:\n680 compound/broad spikes and fast-spiking units, the latter typically corresponding to\n681 interneurons, similar to triphasic spikes. Did the authors observe any units that would fall\n682 into these two categories? While compound spikes appear to be rare, fast-spiking units were\n683 reported to be relatively abundant in Paulk et al (2022). Please clarify whether such units\n684 were detected and, if not, discuss possible reasons.\n686 compound spike units.\n688 units described in Paulk et al., Nature Neuroscience, 2022, and the \"compound spikes\" described in\n692 waveforms into three types: Positive, Regular, and Triphasic. After separating the positive spikes,\n693 we distinguished Triphasic spikes from Regular spikes based on the criteria: [1st peak–trough\n694 ratio > 0.1 and peak-to-peak time < 1 ms]. This aligns with the first stage of the classification\n695 system in Sun et al. (2021). We acknowledge that Sun et al. (2021) further subdivides the non-\n696 triphasic units by identifying \"Compound spikes\" (peak-to-peak time > 1 ms) and clustering the\n697 remainder into Regular and Fast types. Therefore, under our current classification, the \"Regular\"\n698 category effectively encompasses both potential Compound spikes and some Fast spikes that\n699 lack a prominent pre-trough peak.\n701 groups units into RS, FS, and PS based strictly on the spike waveform duration (valley-to-peak)\n702 of the largest peak (FS < 0.3 ms; RS > 0.3 ms), we re-analyzed our data using this definition.\nExample unit\n1st peak–trough ratio 0.33 0.08\npeak-to-peak time (ms) 0.40 0.64\nvalley-to-peak time (ms) 0.20 0.16\nClassification in our study Triphasic spike Regular spike\nClassification in Paulk's study Fast spike Fast spike\nPatient ID\nDetected (V2P < 0.3ms) (TS) in our study (RS) in our study\nPt.01 12 10 2\nPt.02 5 4 1\nPt.03 0 0 0\nPt.04 1 1 0\nPt.05 15 15 0\nPt.06 4 2 2\nPt.07 1 1 0\nPt.08 0 0 0\nPt.09 19 9 10\nPt.10 2 1 1\nPt.11 19 14 5\nSum 78 57 5\n707\n708 5. While the focus of the study is on single-unit recordings, the authors also note that the\n709 system can record local field potentials (LFPs). It would be helpful to include basic quality\n710 measures for LFPs.\n712 a crucial component of neural activity and likely contain important information beyond spiking\n713 activity.\n715 sampling rate with a broad raw bandwidth of 0.1–10,000 Hz. To visually demonstrate the raw\n716 signal quality, we have provided a representative 20-second segment of LFP traces across\n717 multiple channels in the figure below (data from same patients in Figure 2 A). As observed, the\n718 system captures stable, high-fidelity local field potentials with clear oscillatory patterns. Reliable\n720 in Figure 2B of the revised manuscript.\n722 of LFP traces across multiple channels in the figure below.\n723\n725 and laminar profiles.\n727 in deeper layers (left panel), while gamma oscillations are more prominent in superficial layers\n728 (middle panel). The overall depth-dependent distribution of theta/beta/gamma bands is\n729 summarized in the right panel. These depth-dependent features are consistent with previous\n730 reports (e.g., Buzsáki et al., Nature Reviews Neuroscience, 2012).\n731 6. What is the potential for scaling up the developed device? How many channels and what\n732 electrode density could be achieved with this technology?\n734 indeed a critical advantage of our technology.\n735 (1) Current Achievement: In this study, we have successfully demonstrated scalability by doubling\n736 the channel count from 128 to 256 channels (as shown in Pt. 11).\n737 (2) Near-Term Scaling Plan: Considering the constraints of the intraoperative surgical window\n738 and utilizing our current standard photolithography capability, we anticipate achieving a system\n739 capacity approaching ~1,000 channels in the near term. This scale will be realized by\n740 implementing a modular array consisting of 8–12 shanks, with the site count per shank ranging\n741 from 16 to 64 depending on the target region.\n742 (3) Strategies to Address Scaling Challenges: We acknowledge that the primary challenges for\n743 such scaling are the efficiency of the implantation process and the physical dimensions of the\n744 headstage packaging. We propose the following strategies to address these scaling issues:\n745  Automated Implantation: To achieve the implantation of multiple shanks within a limited\n746 surgical window, we aim to utilize automated insertion robots. This will ensure high precision\n747 and consistency as the shank count increases.\n748  Miniaturized Headstage Design: We are working on developing more compact, high-density\n749 headstage modules to reduce the overall weight and volume of the system, which is essential\n750 for scaling up the channel count in practical applications.\n753 miniaturization, this platform will be capable of supporting high-density recordings of 500–1,000\n754 channels during surgery in near term.\n755 7. I recommend comparing the present device with other flexible probes intended for human\n756 brain-machine interface or clinical applications (e.g. Neuralink probes, Axoft electrode\n757 arrays, or the probe demonstrated in Lee et al., 2024. Nat Comm) and with other laminar\n758 probes designed for human intracortical recordings (e.g., the thumbtack probe used in Cash\n759 et al. 2009, Science; Csercsa et al. 2010, Brain). A brief comparison in the Discussion\n760 section would strengthen the manuscript.\n762 comparison with current state-of-the-art neural probes is essential to properly position our work,\n763 and we have added a dedicated section to the Discussion to address this (Line 334-336).\n765 there is currently no published work that provides robust statistical data on unit recording\n766 performance using flexible electrodes in the human brain. Our work addresses this critical gap.\n768 human SUA recording statistics:\n769  Neuralink Probes, 1024-channel flexible threads, wireless BMI system. No disclosure. To our\n770 knowledge, no statistical data regarding SUA yield in human subjects has been publicly\n771 disclosed.\n772  Axoft Electrode Arrays (Lee et al., medRxiv, 2025): Limited disclosure, single case. During\n773 intraoperative recording, the diagrams showed that 22 out of 32 electrodes were able to record\n774 31 spikes. The calculated single-unit yield per channel is 0.969, with a sample size of n=1.\n775  Lee et al., 2024. Nat Comm: This work did not disclose the recording of single-unit activity in\n776 the human brain.\n777  The Thumbtack Probe: We are unsure if you are referring to a rigid short laminar probe\n778 (Neuronelectrod Kft., Budaörs, Hungary). This is a rigid electrode.\n779  We have not found SUA recording details in Cash et al., Science, 2009\n780  Csercsa et al., Brain, 2010: Statistically disclosed. The study recorded 33 SUA across\n781 5 electrodes (120 total channels) in 5 cases of 5~7day recording each. Single units\n782 yield per channel = 0.458 ± 0.072（if contain all 5 patients, 0.275 ± 0.256）\nPt No. of SUA Yield per channel\n1 9 37.5%\n2 0 0\n3 0 0\n4 12 50%\n5 12 50%\n783\n785 1. Lines (L)48-49: “However, its implantation often leads to significant acute tissue\n786 damage, …”. Please provide studies supporting the claim that Utah arrays cause significant\n787 acute damage.\n790 neurons and glial cells are killed or injured during insertion, blood vessels are disrupted, and the\n791 blood brain barrier is compromised (Figure 2A, B). Furthermore, although the insertion of the UEA\n792 did not result in clinically relevant hemorrhages, petechial hemorrhages were sometimes found\n793 located below the tips of the electrodes (Figure 2C, D).\n794\n795 2. L59-60: “owing to their excellent biocompatibility and minimal tissue damage25-27”. The\n796 cited studies do not primarily assess biocompatibility. I recommend including additional\n797 publications that present comprehensive histological analyses of brain tissue after chronic\n798 implantation of flexible probes.\n800 substantiate the biocompatibility claim and we have now revised the manuscript to cite additional\n801 studies in Line 59-60.\n803 histological analyses of high-density electrode implantation in mice, and Ref 29 (Tian et al.,\n805 support this claim. We also added Ref 30 (Zhao et al., Nature Neuroscience, 2023), which\n806 demonstrates glial-scar-free integration and stable tracking of neural activity throughout the entire\n807 adult life of mice. Additionally, we included Ref 31 (He et al., iScience, 2020) and Ref 32 (Tang et\n808 al., Nature Electronics, 2023) to further support the minimal tissue damage and long-term stability\n809 of flexible interfaces.\n810 3. L86-87: “and confirmed the insertion site using a dummy uFINE module before formal\n811 implantation (Methods).” Please provide more details on how the dummy module was used\n812 to confirm the insertion site.\n814 provided additional details regarding the confirmation process in Line 530-536.\n816 ensure the trajectory was unobstructed by the skull edge. This dummy module was designed with\n817 identical dimensions and geometry to the functional uFINE module but lacked the recording\n818 headstage. Once the trajectory was confirmed using the dummy, a small superficial incision in\n819 the pia was done using an arachnoid surgical knife. Subsequently, the uFINE array replaced the\n820 dummy module on the micromanipulator.\n821 4. L109-110 and L395-396: “The 64 recording sites on each shank covered a 4-mm distance\n822 (6-mm distance for 256-channels uFINE array with 90-μm spacing)” and “The total length of\n823 this segment was approximately 4.15 mm (5.76 mm for 256-channels uFINE array)” In the\n824 second sentence, the authors give an estimation for the shank length covered with\n825 electrodes which seem closer to exact value than provided in the first sentence which states\n826 exact lenghts (but are in reality only estimations). Please clarify the inconsistency.\n828 were intended as approximations of electrode coverage. The values of 4.15 mm and 5.76 mm in\n829 the second sentence (Line 410-411) are the precise design values based on the calculation of\n830 electrode count and spacing. We have revised the values in Line 109 from 4 mm and 6 mm to\n831 4.15 mm and 5.76 mm to ensure consistency.\n832 5. L115: Specify whether the sample number (n = 5) refers to shanks or probes.\n834 6. L117: Similarly, clarify whether (n = 6) refers to probes or recording sites. Please also report\n835 the average number or percentage of nonfunctional sites per probe.\n837 average, the percentage of nonfunctional sites per probe was 6.50±6.01%.\n838 7. L137-138: “both low raw signals” Please clarify what is meant by “low raw signals.” Do you\n839 refer to low-frequency signals (LFPs, <250 Hz) or raw wideband signals?\n841 this in the revised manuscript (Line 138).\n842 8. L144: “we performed twice recordings”. I suggest replacing “twice recordings” with “two\n843 recordings.”\n845 145.\n846 9. L146: “exclusively from the recordings that tungsten needles”. I suggest replacing “that\n847 tungsten needles” with “where tungsten needles.”\n849 needles” in Line 147.\n850 10. L152-154: I recommend adding missing units (µV, Hz, ms) for the median values.\n852 associated with its unit, we have revised the sentence (Line 152-156) as follows:\n854 (46.10 to 66.00 μV); firing rate: 2.27 Hz (0.94 to 5.14 Hz); signal-to-noise ratio (SNR): 3.66 (3.28\n855 to 4.34); autocorrelogram (ACG) rise time: 2.70 ms (0.64 to 9.79 ms); trough-to-peak duration:\n856 0.62 ms (0.51 to 1.01 ms); and peak-trough ratio: 0.47(0.26 to 0.59)\n857 11. L155-156: The phrase “previous primate studies” suggests multiple studies, but only one is\n858 cited. Please extend the reference list.\n860 studies on flexible electrode recordings in primates, adding Ref 29 (Tian et al., Advanced Science,\n861 2023), and Ref 41 (Wang et al., Microsystems & Nanoengineering, 2023) to support the statement.\n862 (Line 157)\n863 12. L219-220: The text refers to “a subset of patients (n = 7) with concurrent video recordings,”\n864 but the Methods mentions only six (L614–615). Please clarify.\n866 recordings is 7. This was a typo in the Methods section (Line 702), and we have corrected it to\n867 \"seven\" to match the main text.\n868 13. L267-268: I suggest replacing “an area has extensive connections” with “an area that has\n869 extensive connections.”\n871 connections\" as recommended. (Line 267-268)\n872 14. L277-278: I think the percentages for the stimulus and response periods appear swapped\n873 based on Figure 4. Please revise.\n875 with Figures 4F, 4J, and 4N. We would like to clarify that our statement in Line 277-278 is correct:\n876  The data 19.65% vs 3.17% refers to Suppression vs. Activation during the stimulus period.\n877  The data 34.00 vs 5.10 % refers to Suppression vs. Activation during the response period.\n878 15. L334-335: “This reduction in cross-sectional area results in a more tissue-friendly\n879 implantation, allowing for faster recovery of neural activity after insertion.” Please add\n880 supporting references for the claim that reduced cross-sectional area leads to faster\n881 recovery of neural activity.\n883 avoid any potential overstatement, we have decided to remove this sentence from the revised\n884 text.\n885 16. L355-356: The authors state that by “using finer tungsten wires could … reduce the time\n886 required to obtain stable neural signals”. Could the authors please explain why using finer\n887 wires would reduce the time required to obtain stable neural signals?\n889 and signal stability. To maintain the scientific rigor of the manuscript and avoid any potential\n890 overstatement, we have decided to remove this sentence from the revised text.\n892 stable neural signals due to the following mechanisms:\n893  Reduced Mechanical Stiffness and Acute Injury: Finer shuttle needles significantly lower the\n894 overall structural stiffness of the implant during insertion. This reduction in stiffness helps\n895 minimize the \"plowing effect\" and acute tissue damage as the probe penetrates the brain\n896 parenchyma.\n897  Minimized Tissue Void and Faster Recovery: Smaller shuttle needles leave behind a smaller\n898 footprint or \"void\" in the tissue upon removal. A reduced void size facilitates faster tissue\n899 collapse and accelerated recovery of the local microenvironment.\n901 in the field of neural interfaces to be directly correlated with a shorter period required to reach a\n902 stable state for neural recordings.\n903 17. L410: I would suggest providing more details on the design and fabrication of the tungsten\n904 shuttle wires.\n906 fabrication of the tungsten shuttles in the revised manuscript (Methods, Line 459-462).\n908 tips were fabricated using an electrochemical etching process to achieve the desired tapered\n909 morphology.\n911 gradual taper (approx. 30°) to a 10 μm ultra-fine tip. Standard tungsten needles (75 μm) were\n912 obtained and processed using a custom-built fabrication setup. The shaping process relied on\n913 electrochemical etching, where a motorized clamping system was employed to precisely control\n914 the immersion depth and duration. throughout the process, the needle profile was monitored in\n915 real-time using a CCM (Compact Camera Module) to ensure the final geometry matched our\n916 design specifications.\n917 18. L413-414: The statement “this project did not require a needle withdrawal design during\n918 surgery” is unclear. The manuscript indicates that tungsten needles were withdrawn after\n919 implantation (L488–489: “The shuttle wires were withdrawn…”). Please clarify.\n921 tungsten shuttle needles are indeed retracted from the brain tissue to release the flexible\n922 electrodes, they are not completely detached or removed from the insertion assembly. They are\n923 simply pulled up and remain suspended above the brain surface.\n925 (1) In the section “Design, fabrication and assembly of uFINE electrode array” (Line 467-468):\n927 needles during surgery.\n928 (2) In the section “uFINE array positioning and insertion” (Line 540-542):\n930 above the brain surface. This ensured that the sharp tips would not scratch the cortex while\n931 leaving the flexible array shanks embedded in the tissue.\n933 flexible electrode provided redundant length to accommodate brain surface movements,\n934 preventing the array from being dislodged.\n935 19. L413: Based on the description in the Methods, the silicon substrate is a part of the uFINE\n936 module, but it is not shown in the figures. Please clarify what part of the uFINE module is the\n937 silicon substrate, why it is needed and how the polymer shanks are mounted to it.\n940 serves as a temporary carrier primarily used during the fabrication and assembly processes. It\n941 provides a rigid, planar surface necessary for achieving micron-level precision in alignment,\n942 processing, and bonding during manufacturing.\n944 beneath the flexible section. This layer anchored the device during fabrication and was\n945 subsequently chemically etched to release the arrays.\n948 are attached to the silicon plate.\n949\n950 20. L415: “the uFINE array was anchored to tungsten shuttle wires” Please describe in more\n951 detail how the shuttles were assembled with the uFINE arrays.\n953 aperture. The aperture is anchored to the tungsten shuttle needle, thereby completing the\n954 attachment of the electrode to the tungsten needle. See the enlarged images of the electrode tip\n955 for illustration (left panel for implantable segment of uFINE array and Tungsten needle, and right\n956 panels for zoom-in of electrode tip).\n957\n958 21. L456: Specify the fluid (e.g., PBS, saline) and reference electrode used during EIS\n959 measurements.\n961 experimental setup in the Methods section (Line 503-504).\n963 22. L460: Please define “FPC”.\n965 23. L484: Please describe how the dummy module differs from the uFINE module.\n967 revised text reads: This dummy module was designed with identical dimensions and geometry to\n968 the functional uFINE module but lacked the recording headstage.\n969 24. L488: “the redundant part of the array prefixed at the substrate was released immediately.”\n973 filaments that rests on the silicon substrate, as illustrated in figure below.\n975 using a water-soluble adhesive (salt crystals) to prevent entanglement. Upon contact with saline\n976 during surgery, the salt layer dissolved immediately, allowing the electrode filaments to move\n977 synchronously with brain pulsation.\n978\n979 25. L499: Did the authors record wideband recordings (LFP and unit activity at the same time)?\n982 raw data is 0.1 Hz to 10,000 Hz. Reliable LFP signals (0.1–250 Hz) were isolated through low-\n983 pass filtering, as shown in Figure 2B.\n984 26. L501: Please report the total number of trials used in the word-repeating task.\n987 trials were used in the word-repeating task.\n988 27. L527-528: Please provide the approximate percentage of channels excluded due to high\n989 impedance and high RMS noise.\n993 of channels excluded based on these two factors was 7.53% (106 out of 1,408 channels).\n994 28. L539: “Subsequently, experienced electrophysiologists (SW)” Please confirm whether\n995 manual curation was performed by a single electrophysiologist.\n997 experienced electrophysiologists, Shun Wu and Cen Kong. We have updated the manuscript in\n999 29. L547: Please report the method used to calculate the ISI violation.\n1001 processing pipeline in Kilosort4, which we believe the default parameter is 2 ms based on\n1003 30. L565: “Defined as the maximum value among the first and second peak-to-trough ratios”\n1005 are used to calculate the peak–trough ratio.\n1008 definition was not sufficiently precise. We have now revised the definition and calculation of the\n1009 peak-to-trough ratio in the manuscript to align with these established standards.\n1011 definitions are:\n1012  Peak-trough ratio: The ratio between the absolute amplitudes of the minimum trough and the\n1013 following peak.\n1014  First peak-trough ratio: The ratio between the absolute amplitudes of the minimum trough and\n1015 the previous peak (if it exists).\n1016  Duration is the time between the minimum trough and the subsequent peak.\n1018 the absolute amplitudes of the minimum trough and the following peak, as illustrated in Figure 2F.\n1020 manuscript to support this calculation method. Accordingly, we have updated the peak-to-trough\n1021 ratio and duration used in our analysis.\n1023 relationship between peaks and troughs. This occasionally allowed for erroneous cases where\n1024 both peaks were identified either preceding or succeeding the main trough. Consequently, we\n1025 have refined the logic for both waveform classification and feature extraction\n1026  Baseline Correction: The average value of the first 10 data points and the last 10 data points\n1027 of the waveform is calculated and set as the zero potential.\n1028  Polarity Detection and Feature Extraction\n1029  First, compare the absolute values of the global maximum (Max) and the global\n1030 minimum (Min):\n1031  Case A: Positive Dominant (Max > |Min|)\n1032  Case B: Negative Dominant (|Min| > Max)\n1033  Classification Criteria: Features are calculated based on the detected points, and waveforms\n1034 are classified sequentially according to the following rules:\n1035  PS (Positive Spiking): Applied to any waveform that is Positive Dominant (Max > |Min|).\n1036  TS (Triphasic Spiking):\n1037  Must be Negative Dominant (Max < |Min|);\n1038  AND exhibit a significant 1st Peak (Height of 1st Peak > 10% of Trough Depth);\n1039  AND have a short duration (Time interval between the two peaks < 1 ms).\n1040  RS (Regular Spiking):\n1041  All other Negative Dominant waveforms that do not meet TS criteria.\n1044 peak, and trough definition); and Figure 2I (waveform classification).\n1045 31. Figure 1D: In the left side, some of the labels appear misplaced (e.g., “Skull” is positioned on\n1046 the scalp, while “Dura” indicates the skull). Please clarify.\n1048 \"Skull,\" \"Dura,\" and \"Scalp\" in the revised Figure 1D.\n1049 32. Figure 1E: The uFINE probe seems not to scale. Based on the 100 µm scale bar and the 65\n1050 µm electrode pitch, the schematic of the uFINE array appears undersized. Please revise.\n1052 array is accurately scaled relative to the 100 µm scale bar and the 65 µm electrode pitch.\n1053 33. Figure 1F: Please specify whether this is the array with the 65 µm or the 90 µm electrode\n1054 pitch.\n1056 added this specification to the figure caption.\n1057 34. Figure 2A: Spikes on high-passed channels are barely visible, possibly due to PDF\n1058 compression. Also, while the caption states that traces are 2-sec long, the scale bar\n1059 suggests 3-4 sec. Please clarify.\n1061 ensure the spikes are clearly visible, we have provided a high-resolution version of Figure 2A as\n1062 a supplementary file.\n1064 and high-pass traces are 4 seconds long, and we have updated the figure caption (Line 172).\n1065 35. Further, consider marking or noting nonfunctional channels explicitly. I noticed that there are\n1066 missing channels (Chn18, Chn107) and also channels with no or very low activity (Chn1-2,\n1069 (1) Missing channels (e.g., Chn18, Chn107, Chn108): These are the nonfunctional channels that\n1070 were identified and excluded based on our screening criteria for impedance and/or RMS noise.\n1071 (2) Low-activity channels (e.g., Chn1-2, Chn8, Chn10): These are channels that were not filtered\n1072 out during our screening of impedance and RMS noise, but they showed low or no activity.\n1073 36. Figure 2B: The figure caption mentions asterisks, but only arrows are visible. Please revise.\n1075 caption.\n1077 (Line 179).\n1078 (1) We have revised the figure caption to correctly refer to arrows instead of asterisks.\n1079 (2) We have also added a description of the mean spike waveform of Unit#72 shown on the right\n1080 side of the panel.\n1082 shows the waveform of individual spikes (marked by arrows) and the mean spike waveform of\n1084 37. Figure 2C: Please briefly explain how the spread of channels was calculated.\n1086 in the \"Assessment of recording quality\" section of the Methods (Line 654-657).\n1088 showing significant neural activity. Specifically, for each unit, we analyzed the mean waveform\n1089 (averaged from 500 randomly sampled spikes) on the primary channel and its 10 nearest\n1090 neighbors (using a sliding window of 11 channels). A channel was included in the spread\n1091 calculation if the peak-to-peak amplitude of the unit's mean waveform on that channel exceeded\n1092 2 times the baseline RMS noise (SNR > 2).\n1093 38. Figure 2I: For the four-shank probe with a larger electrode pitch, electrodes span >4 mm\n1094 (5.67 mm), yet the y-axis only shows 4 mm. Were single units outside this range excluded?\n1096 shank probe. In our initial analysis, the depth information for Pt.11 was incorrectly calculated using\n1097 a 65 μm pitch instead of the actual 90μm pitch. We have now revised Figure 2I and corrected the\n1098 calculation. Consequently, single units falling outside the intended analysis range (4100μm) have\n1099 been excluded (23 units in total). We have also explicitly noted this calculation method and the\n1100 unit count in the figure caption.\n1102 the mean amplitude (x axis) and the distance from the array tip (y axis, calculated by multiplying\n1103 the channel number by site spacing) for each single unit within this range (n = 696).\"\n1105 39. Figure 3D: The caption refers to three units, but four are shown. Please revise.\n1107 state that four units are shown in the panel (Line 251).\n1108 40. Figure 3F: Are the numbers in the panel single unit IDs? Please clarify.\n1110 41. Figure 4M: The two columns representing stimulus (listen) and response (speak) appear\n1111 swapped since unit #2 and #3 (speak onset) are activated in panel I; however, in panel M\n1112 unit #2 and #3 appear with red color (activation) in the stimulus column not in the response\n1113 column. Please revise.\n1115 indeed inadvertently swapped in Figure 4M. We have corrected the figure in the revised\n1116 manuscript to align with the raster plots in Panel I.\n1117 42. Figure 4C, G, K: How do unit numbers correspond to the number of active units presented\n1118 for patients in Table S1. While Pt. 03 has 9 active units in Table S1, in panel C the number\n1119 of units is higher (12). In contrast, Pt. 04 has 21 active units in Table S1, while fewer (19\n1120 units) in panel G. Please clarify.\n1122 the distinct temporal windows and inclusion criteria applied for the general summary versus the\n1123 specific task analysis:\n1124 (1) Table S1: Summarizes \"Active Units\" across the entire recording session (whole process).\n1126 each time window.\n1127 (2) Figure 4 (Task-Specific Analysis): Focuses strictly on the task paradigm period. To ensure\n1128 statistical reliability for tuning curves, we applied a stricter criterion: FR > 5 Hz specifically during\n1129 the task trials (as detailed in Methods).\n1131 from that in Figure 4 due to these distinct selection criteria and temporal windows.\n1132 43. Figure 4D: The caption mentions asterisks marking units in panel C, but they are not visible.\n1136 described.\n1137 44. Table S1: I suggest replacing “(2) at least one spike each time window” with “(2) fired at\n1138 least one spike each time window”?\n1140 replaced “(2) at least one spike each time window” with “(2) fired at least one spike each time\n1141 window” in Table S1 and revised manuscript Line 228-229.\n1142 45. Table S1: I would suggest adding a footnote indicating that a four-shank probe was used in\n1145 have added a footnote to Table S1 to explicitly indicate that a four-shank probe was used in\n1146 participant Pt. 11.\n1147 46. Figure S2B: What is function of the rod-like structure on the left side of the uFINE module?\n1149 tightened screw, as indicated by the arrow in the picture below (Figure 1C).\n1150\n1151 47. Figure S3B: The unit in the y-axis should be µV for the “Amplitude” instead of µm. Also,\n1152 please clarify the meaning of “notch” in the caption.\n1154 in the revised Figure S3B.\n1156 represents the 95% confidence interval of the median. non-overlapping notches indicate a\n1157 statistically significant difference between medians (p < 0.05).\n3 all the major and minor issues I had raised conclusively. I am supportive of publication of the\n4 present version.\n6 regarding how sterility and biocompatibility was assured and what standards were satisfied\n7 somewhere into the manuscript (methods or supplement). This is very helpful information.\n11 of Implantable Components” within the Methods (Line 428-454) to address the sterility and\n12 biocompatibility standards.\n13\n18 process, which has helped to improve the quality of our manuscript.\n19\n24 versions of the manuscript.\n25\n28 are still recommended to further improve the manuscript.\n29\n31 3. The amplitude of brain pulsations likely depends on the size of the craniotomy: larger cranial\n32 windows typically cause greater brain motion. Please report the approximate sizes of the\n33 craniotomy prepared during DBS and tumor resection surgeries. The latter is presumably larger\n34 (~2 cm vs. ~4-8 cm), potentially resulting in greater brain movements. Anesthesia might also\n35 affect brain pulsations. Therefore, I suggest comparing signal stability between the two surgical\n36 conditions (DBS vs. tumor resection).\n39 adding the figure prepared by the authors (“Functional Stability - Spike Position Drift”) to the\n41 craniotomy size between the two types of surgeries.\n45 surgeries and cited the related figure (“Functional Stability - Spike Position Drift”, Figure S4D) in\n46 the Main Text (Line 182-189). The figure and its caption are located in the Supplementary\n48\n50 5. While the focus of the study is on single-unit recordings, the authors also note that the system\n51 can record local field potentials (LFPs). It would be helpful to include basic quality measures for\n58 power distribution”) in the Main Text (Line 122-124). The figure and its caption are located in the\n60\n62 1. Lines (L)48-49: “However, its implantation often leads to significant acute tissue\n63 damage, …”. Please provide studies supporting the claim that Utah arrays cause significant\n64 acute damage.\n68 neurons and glial cells are killed or injured during insertion, blood vessels are disrupted, and the\n69 blood brain barrier is compromised (Figure 2A, B). Furthermore, although the insertion of the\n71 found located below the tips of the electrodes (Figure 2C, D).\n74 (2014). Eisenkolb et al. (2023) may be an appropriate reference to show an exception, but it\n75 would be helpful to also cite Fernández et al. as an example supporting the claim that Utah\n76 array implantation often leads to significant acute tissue damage.\n78 challenges and future prospects.\" Frontiers in neuroengineering 7 (2014): 24.\n82\n84 6. L117: Similarly, clarify whether (n = 6) refers to probes or recording sites. Please also report\n85 the average number or percentage of nonfunctional sites per probe.\n88 average, the percentage of nonfunctional sites per probe was 6.50±6.01%.\n91 than recording sites, as this is ambiguous in its current form. In addition, I recommend reporting\n92 the total number of sites measured and including the percentage of nonfunctional sites provided\n93 in the authors’ response.\n96 examining our data, we confirmed that for the EIS measurements, n = 6 refers to specific\n97 recording sites, not probes. We have now clarified this in the revised manuscript (Line 113-114).\n99 exclusion statistics in Line 583-584: “In total, 7.53% of channels (106/1,408) were excluded\n100 based on these two factors (mean ± std: 6.50 ± 6.01%).”\n101\n103 14. L277-278: I think the percentages for the stimulus and response periods appear swapped\n104 based on Figure 4. Please revise.\n107 them with Figures 4F, 4J, and 4N. We would like to clarify that our statement in Line 277-278 is\n108 correct:\n109 • The data 19.65% vs 3.17% refers to Suppression vs. Activation during the stimulus period.\n110 • The data 34.00 vs 5.10 % refers to Suppression vs. Activation during the response period.\n113 indicating that units corresponding to the stimulus period are shown in the outer circle and those\n114 corresponding to the response period in the inner circle, I derive the following percentages:\n115\n121\n123 percentages seem to correspond to the response period, and vice versa (or the figure caption\n124 should be revised).\n126 activated/suppressed units shown in Fig. 4M. For example, Fig. 4M indicates no activated units\n127 during the stimulation period, whereas Fig. 4N reports that 4.76% of units were activated during\n128 listening. Please clarify these discrepancies.\n131 identify critical inconsistencies in our visualization. We have carefully re-examined the data and\n133 1. We confirmed that there was an inconsistency between the sub-figures. In the original Figure\n134 4F&J, the outer circle represented the response period, whereas in Figure 4N (and the original\n135 caption), the outer circle represented the stimulus period. To resolve this confusion, we have\n136 standardized the layout across all figures (Figure 4F&J&N) and the captions. In the revised\n137 manuscript, the outer circle uniformly denotes the response period, and the inner circle\n138 uniformly denotes the stimulus period. Figure 4N has been updated to match this standard.\n139 2. Correction of Figure 4M (Unit 9): Regarding the discrepancy in Pt. 08, the reviewer is correct.\n141 response” in the original Figure 4M. We have corrected this labeling error in the revised Figure\n142 4M, which now matches the statistics presented in Figure 4N.\n143\n145 24. L488: “the redundant part of the array prefixed at the substrate was released\n146 immediately.” Please clarify the meaning of “the redundant part” of the array? How was it\n147 prefixed?\n151 filaments that rests on the silicon substrate, as illustrated in figure below.\n153 using a water-soluble adhesive (salt crystals) to prevent entanglement. Upon contact with saline\n154 during surgery, the salt layer dissolved immediately, allowing the electrode filaments to move\n155 synchronously with brain pulsation.\n158 authors’ response into the Methods section.\n161 the manuscript. We have added the detailed description to the Methods section at Line 403-405\n162 and Line 509-511.\n164 filaments, which rests on the silicon substrate, was temporarily immobilized using a water-\n165 soluble adhesive (salt crystals) to prevent entanglement.\"\n167 fixed to the substrate) was immediately released as the water-soluble salt layer dissolved\n168 rapidly upon contact with physiological saline, enabling the electrode filaments to move\n169 synchronously with brain pulsation.\"\n170\n173 particular, it is not clear how the silicon substrate component is fabricated—whether it is\n174 produced during probe fabrication or manufactured separately. Additionally, clarification is\n175 needed regarding how the silicon substrate is attached to the flexible printed circuit (FPC)\n176 assembled with the uFINE array, as well as the overall order of assembly.\n178 substrate to the FPC? Likewise, is the uFINE array initially anchored to the tungsten needle,\n179 with the tungsten needles subsequently secured to the silicon substrate using Kwik-Sil (as\n180 currently described in the manuscript), or does this occur in the reverse order? How is the\n181 redundart part of the probe achieved? Most of my confusion arises from the timing and method\n182 by which the silicon substrate is attached to the FPC and whether the uFINE array is originally\n183 part of this silicon piece.\n185 complete assembly (uFINE array, tungsten needle, silicon substrate, and FPC). Currently, the\n186 figures show only photos of individual uFINE arrays or schematics of the FPC and silicon\n187 substrate with tungsten needles, which makes it difficult to fully understand the assembly\n188 process.\n191 assembly. We agree that the distinction between the fabrication and assembly steps needed\n192 better definition. To better describe these procedures, we have revised the relevant descriptions\n193 in the Methods and separated the assembly process into an independent section for greater\n194 clarity.\n196 1. Silicon Substrate Origin: The silicon substrate was a commercially available finished product\n197 (300 nm thermal oxide wafer, Szjxtech Co., Ltd). We utilized this substrate as the starting\n198 material to initiate the probe fabrication process, as described in Line 357-358.\n199 2. Fabrication Steps: For the detailed fabrication steps, please refer to Fabrication Procedures\n201 3. Assembly Sequence: We have clarified the step-by-step workflow in the revised text (Line\n202 387-397):\n203  Fabrication & Dicing: Multiple electrode units are fabricated on a single silicon wafer, which\n204 is then diced into individual chips (Assembly Procedure A).\n205  FPC Connection: The diced silicon chips (containing the electrodes) are bonded to the\n207 electrode pads to the FPC pads (Assembly Procedure B).\n208  Release & Cleaving: The device is submerged in nickel etchant (TFB, Transene Co. Inc.) to\n209 release the implantable portion of the probe from the silicon substrate. Crucially, the contact\n210 region remains anchored to the silicon substrate for stability. The substrate is then cleaved\n211 to the desired length (Assembly Procedure C).\n212  Tungsten Needle Assembly: Finally, the released uFINE array is manually anchored to the\n213 tungsten shuttle needles (Assembly Procedure D).\n214  Visualization: As requested, we have added a photograph of the fully assembled device to"}]

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