Quantitative assessment of smartphone fluorescence microscope designs for enhanced imaging of micro/nanoparticles and leukocytes
Creators: name: Sami, Muhammad Ahsan
Publisher: name: No Publisher Supplied
Year: 2023

description: Smartphone fluorescence microscopes (SFM) offer some of the major functionalities offered by their benchtop counterparts at a fraction of the cost. Though precise and accurate, these devices still have a lot of limitations, such as being limited to only one fluorophore, and the unavailability of multiple magnifications to name a few. To overcome these limitations, the design of a new SFM prototype that offers the user ability to work with multiple fluorophores and smartphones at multiple magnification levels is presented. A 1951 USAF resolution test target was used to quantify the maximum resolution of the microscope which was found to be 3.9 µm. The performance of the designed SFM was compared with a regular benchtop microscope by counting the imaged fluorescent microbeads. SFM prototype demonstrated a high correlation coefficient (R2 = 0.99). Further, to automate the process of detecting and counting fluorescent particles imaged using the SFM, multiple artificial neural networks (ANNs) were trained using various algorithms, and their performances were compared to the control (ImageJ) and a high correlation (R2 = 0.99) was found. Imaging and quantification of bioparticles in the sub-micron and nanometer range remain challenging as it requires aggressive robustness and high-performance metrics of the building blocks of SFM. To address this, SFMs equipped with multiple excitation modalities were designed and their imaging capabilities were analyzed. Employing spatial positional variations of the excitation source with respect to the imaging sample plane (i.e., parallel, perpendicular, oblique), three distinct SFM variants were developed. These SFM variants were tested using green-fluorescent beads of four different sizes (8.3, 2, 1, 0.8 µm). Optimal excitation voltage regions for the SFM variants were determined by imaging these beads at a range of voltages to check for the data loss region, optimal excitation region, and high noise region. The relative performances of these SFM variants were also quantified by calculating signal difference to noise ratio (SDNR) and contrast to noise ratio (CNR) from the captured images. SFM with oblique excitation generated the highest SDNR and CNR values but required the most power as well. Afterward, an automated algorithm (AQAFI) for the quantitative assessment of the quality of images captured using SFMs was developed. Green-fluorescent beads of four different sizes (8.3, 2, 1, 0.8 µm) were imaged using our custom-designed SFM employing an oblique excitation scheme. Mean bead intensity, bead vicinity noise, and image background noise were picked as the parameters of interest. Manual calculation of these parameters is done using ImageJ to work as control values for validating the performance of the designed algorithm. R2 values of greater than 0.95 were obtained for the parameters of interest calculated using the automated algorithm and control (ImageJ). Finally, SDNR and CNR values were calculated for the captured images using the parameters of interest obtained from both the algorithm and ImageJ, and a good accord was found. Next, the utility of signal averaging techniques was investigated for improving the quality of fluorescent images captured using the SFM and to further reduce its operational complexity by obviating the need to tune excitation source intensity depending on the specimen size. Averaging and gaussian filters of five kernel sizes (3 x 3, 7 x 7, 11 x 11, 15 x 15, and 21 x 21) and three levels of standard deviations σ (1,3,5) (for gaussian only) were applied on the images of different sizes of green-fluorescent beads of (8.3, 2, 1, 0.8 µm) imaged using the SFM at excitation voltages ranging from 4.3 to 4.5 V. Application of the filters significantly improved the visual quality of the captured images. Averaging and gaussian filters (σ = 5) with a kernel size of 21 x 21 produced the best results for all four beads. AQAFI was then used to validate and quantify this improvement in image quality by calculating the signal difference to noise ratio (SDNR) and contrast to noise ratio (CNR) of filtered and unfiltered original images. We then imaged fluorescently tagged peripheral blood leukocytes using the SFM (4.3 – 4.5 V) and applied averaging and gaussian filters with the previously identified optimal parameters. Similar to fluorescent beads, significant improvement in visual image quality was observed for the leukocytes. This visual improvement in image quality was then validated and quantified by using AQAFI which calculated the SDNR and CNR values of the leukocyte images before and after the application of the digital filters. Going forward, we plan on employing the developed SFM for monitoring single-cell level neutrophil phagocytic activity for early diagnosis of sepsis. Using our setup, as a first step, we have already imaged the submicron IgG-coated fluorescent beads that are used to incite the phagocytic activity in human peripheral blood neutrophils. Furthermore, we are also working on coming up with a generalized tool that can work with a range of microscopes to compute their parameters of interest and key performance indicators (SDNR and CNR). Based on the obtained results, necessary image enhancement techniques would be applied to the captured images to further improve their quality.