Development of a universal, tunable, miniature fluorescence microscope for use at the point of care

Abstract

Fluorescence microscopy can be a powerful tool for cell-based diagnostic assays; however, imaging can be time consuming and labor intensive to perform. Tunable systems give the ability to electronically focus at user selected depths inside an object volume and may simplify the opto-mechanical design of the imaging system. We present a prototype of a universal, tunable, miniature fluorescence microscope built from poly(methyl methacrylate) singlets that incorporates miniature, electrowetted lenses for electronic focusing. We demonstrate the ability of this system to perform clinically relevant differential white blood cell counts using single use custom cartridges pre-loaded with the fluorescent dye acridine orange.

Keywords: (170.1470) Blood or tissue constituent monitoring; (170.2520) Fluorescence microscopy; (170.3880) Medical and biological imaging.

Fig. 1

Optical schematic of the infinity-corrected tunable microscope. The red arrows indicate the tunable Arctic 316 lenses. The first three singlets plus the two tunable lenses comprise the objective while the last two singlets comprise the tube lens. The total length of the system is 46 mm.

Fig. 2

Typical response of an Arctic 316 tunable lens in response to driving voltage [11] (a). Plot depicting the power of each of the two tunable lenses with respect to the working distance is represented by black dots and circles (b). The dashed blue line shows the estimated power of the electrowetted optics calculated as the sum of the powers of the two tunable lenses. The red squares depict the total power of the tunable sub-system as calculated by a ray tracing algorithm. The Strehl ratio of the system as a function of working distance is plotted with the solid orange line. Calculations were performed at a wavelength of 590 nm. Horizontal dotted lines in both plots depict the linear, hysteresis free region for driving the tunable lenses.

Fig. 3

Performance metrics of the miniature tunable fluorescence microscope for nominal working conditions, calculated for the image plane in both the tangential (T) and sagittal (S) directions. The MTF is shown for (a) 530 nm, (b) 590 nm, (c) 660 nm, and (d) 720 nm configurations. Spots diagrams for the 0.0 mm and 0.60 mm y-field points are presented in (e-h) for all monochromatic configurations. The Airy disk radii for consequtive configurations is: 6.65 μm for 530 nm, 7.37 μm for 590 nm, 8.04 μm for 660 nm and 8.04 μm for 720 nm.

Fig. 4

A cross sectional view of the threaded objective holder, which contains three PMMA singlet aspheric lenses and two Arctic316 electrowetted lenses.

Fig. 5

Components of the miniature tunable microscope: Arctic 316-P tunable lens [11] (a), the 3D printed holder for the tunable objective (b), and the 3D printed tube lens holder with C mount threading for a Flea3 image detector (c).

Fig. 6

Plot of the nominal axial chromatic shift in the image space as a function of the focused wavelength. The location of the plane of best focus is when Δz equals zero.

Fig. 7

Images of a positive high resolution USAF target taken with the miniature tunable microscope. The first row represents the system focused at 450 nm, the second row at 550 nm, the third row at 600 nm, the fourth row at 650 nm, and the fifth row at 700 nm. The insets in the bottom left of the main diagonal images are the enlarged Group 8, elements 4-6 of their respective image. Due to the filter and LED combination, the images taken at 450 nm appeared darker than the images at other wavelengths and were contrast enhanced for visualization purposes.

Fig. 8

Example images of AO-stained blood. Image (a) is the sample with the tunable lenses tuned to emphasize the DNA fluorescence in green. Image (b) is the same field of view with the tunable lenses tuned to emphasize the RNA fluorescence in red. The insets of (a) and (b) show a representative WBC. An intensity plot of a horizontal cross section through the center of the WBC shown in (a) and (b) is given in (c) and (d) respectively. Scale bars represent 100 µm. Images have been contrast enhanced for vizualization purposes.

Fig. 9

Imaging WBCs at different depths in AO stained blood. Image (a) is the sample with the tunable lenses tuned to emphasize the DNA fluorescence in green, while (d) is the sample with the tunable lenses tuned to emphasize the RNA fluorescence in red. The intensity plots in (b) and (e) correspond to the bottom left WBC, while the intensity plots in (c) and (f) correspond to the top right WBC. Intensities were taken as a horizontal cross section through their respective WBC. The arrows indicate the WBCs and their corresponding plots. Scale bar represents 100 µm. Images have been contrast enhanced for vizualization purposes.