Fig 1.
Optical schematic and image of the UV fluorescence microscope system.
(a) Optical schematic of the designed UV microscope and (b) image of the fully assembled system. Samples were mounted on an xyz stage to enable focus adjustment and lateral scanning.
Table 1.
Summary of optical design parameters for the UV fluorescence microscope.
Fig 2.
Performance of the assembled fluorescence microscope.
(a) MTF plot calculated for the image plane in both the tangential and sagittal directions showing the nominal performance of the system. (b) Image taken with a 455 nm LED of a high resolution 1951 USAF target presenting the measured performance of the system. The smallest resolvable feature on the target was group 8 element 1 (shown enlarged and outlined in white), demonstrated by the intensity profiles through the vertical and horizontal elements indicated by the red lines and arrows. Resolution target image has been contrast enhanced for visualization purposes.
Fig 3.
Fluorescence image of WBCs stained with AO.
(a) Image taken using the 280 nm excitation source with an exposure time of 500 ms and detector gain of 0 dB without excitation or emission filters. The large, central white box represents the 1 mm x 1 mm FOV where WBCs are in focus. Insert image shows a pair of arbitrarily selected WBCs. (b) Image of the same FOV with an inexpensive absorption foil, which served as an emission filter, that only allowed the fluorescence signal from the WBCs. Color images were reconstructed from raw intensity values given by the Bayer mask of the camera.
Fig 4.
(a) Image of AO stained whole blood taken under UV excitation in the 1 mm x 1 mm FOV, (b) WBCs after applying a Sobel filter, and (c) the segmented WBCs, displayed as the red circles, used for analysis based on the size criteria. Scale bars represents 100 μm.
Fig 5.
Example histogram of the R/G ratios in a single FOV.
The red line shows the bimodal shape of the histogram, delineating what appears to be two distinct populations of WBCs (agranulocytes in the left population and granulocytes in the right population).
Fig 6.
Comparison of calculated WBC values to commercial hematology analyzer reported values.
Bar graphs show the (a) total WBC/μl, (b) percent agranulocytes, and (c) percent granulocytes for five separate venous draws. Purple bars on the left show the mean value of 12 FOVs as calculated by the algorithm (with error bars indicating ± one standard deviation) while turquoise bars on the right show the gold standard value as reported by the commercial hematology analyzer (with error bars indicating ± 5% error, given in the hematology analyzer specifications on accuracy [16]).