# Chip-Based Cytometry Illuminated by a Blade-Shape Continuous Light for Multispectral Detection
## Materials and Methods
### Instrumental Setup and Flow Cytometry  
Figure 2 presents a schematic representation of the proposed multi-spectral micro-flow cytometry system. The bulk of the experimental system is built on an inverted microscope (Eclipse TE2000U, Nikon, Tokyo, Japan) equipped with a high numerical aperture (N.A.) objective lens (CFI\({}_{60}\) 20\(\times\)/0.75, Nikon, Tokyo, Japan) and a light-stop film as the excited unit. In accordance with the proof-of-concept study performed in [36], the spatial resolution of the cytometer was further enhanced by passing the light emitted from the dark-field condenser through a pinhole prior to entering the high N.A. objective lens. Additionally, a small interrogation region is beneficial to particle/cell detection and counting, and the experiments were performed using a circular pinhole with a diameter of 1 mm for forming the interrogation region with a diameter of 100 \(\mu\)m.  
Figure 1: Working principle of the proposed diascopic illumination technique in multispectral cytometry analyses.  
Figure 2: Schematic for the experimental setup of the proposed multi-parameter cytometry detection system. N.A., numerical aperture; UV-Vis-NIR, ultraviolet-visible light-near infrared.  
The light emitted from the interrogation region of the cytometer was collected by a low N.A. objective lens (CFI\({}_{60}\) 20\(\times\)/0.45, Nikon, Tokyo, Japan) and was then coupled into a UV-Vis-NIR spectrometer (HR4000, Ocean Optics, Dunedin, FL, USA) via a multi-mode fused silica fiber (P400-2-UV-Vis, Ocean Optic, Dunedin, FL, USA). Note that an objective lens with a lower N.A. than that of the excitation objective lens was deliberately chosen on the detection side of the cytometer in order to restrict the collected light to just the scattered light and fluorescence signals emitted from the particles. The detection signal was processed by the spectrometer and then collected using a personal computer via a USB interface and software (OOIBase32, Ocean Optics, Dunedin, FL, USA) at a sampling rate of 20 acquisitions per second. Finally, the spectra were analyzed and post-processed by a proprietary program written in Matlab(r) (R2012, The MathWorks, Natick, MA, USA). In order to meet the optical detection scheme of this study, the microfluidic chip was fabricated in low-cost microscope slides with a rapid fabrication process [37]. The details regarding the chip design and the reagents for the experiments are provided in the supplementary information.  
To confirm the focusing capability of the two sheath flows, an initial experiment was performed using Rhodamine B as the sample solution. Figure 3 presents the visualization results obtained using a general fluorescence microscope after the vacuum pump was removed, and the sheath and sample moved under the effects of inertia and hydrostatic forces alone. The results presented in Figure 3a confirm that the sheath flows confine the sample flow to a narrow stream within the center of the microchannel. The width of the focused stream is dependent on the sample and sheath flow rates [38], and in the present case, was found to have a value of 20 \(\upmu\)m. However, a slight diffusion of the focused stream was observed in the downstream region of the chip due to the low flow rate.  
Having confirmed the focusing capability of the two sheath flows, a second experiment was performed using a sample containing red fluorescent-labeled particles with a size of 15 \(\upmu\)m. The corresponding visualization results are presented in Figure 3b. It can be seen that the focusing effect causes the particles to flow through the interrogation region of the chip in a single-line fashion, thereby permitting their individual characterization and counting. The linear velocity of the focused sample stream was about 500 \(\upmu\)m/s for the particle movement, and the velocity was necessary to provide sufficient exposure time for the spectral measurement using the UV-Vis-NIR spectrometer with an integrated time of 20 ms.