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.

© 2018 Optical Society of America under the terms of the OSA Open Access Publishing Agreement

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References

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    [Crossref] [PubMed]
  5. A. Forcucci, M. E. Pawlowski, C. Majors, R. Richards-Kortum, and T. S. Tkaczyk, “All-plastic, miniature, digital fluorescence microscope for three part white blood cell differential measurements at the point of care,” Biomed. Opt. Express 6(11), 4433–4446 (2015).
    [Crossref] [PubMed]
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    [PubMed]
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    [PubMed]
  18. M. Zheng, J. C. Lin, H. Kasdan, and Y. Tai, “Fluorescent labeling, sensing, and differentiation of leukocytes from undiluted whole blood samples,” Sens. Actuators B Chem. 132(2), 558–567 (2008).
    [Crossref]
  19. B. Houwen, “The differential cell count,” Lab. Hematol. 7, 89–100 (2001).
  20. Centers for Disease Control and Prevention, “Antibiotics Aren’t Always the Answer,” https://www.cdc.gov/features/getsmart/index.html . Accessed: 11 July 2017.
  21. C. E. Majors, M. E. Pawlowski, T. Tkaczyk, and R. R. Richards-Kortum, “Low-Cost Disposable Cartridge for Performing a White Blood Cell Count and Partial Differential at the Point-of-Care,” Health Innovation Point of Care Conference, 1–10 ( 2014).
    [Crossref]

2017 (1)

2015 (3)

A. Forcucci, M. E. Pawlowski, C. Majors, R. Richards-Kortum, and T. S. Tkaczyk, “All-plastic, miniature, digital fluorescence microscope for three part white blood cell differential measurements at the point of care,” Biomed. Opt. Express 6(11), 4433–4446 (2015).
[Crossref] [PubMed]

A. Forcucci, M. E. Pawlowski, Z. Crannell, I. Pavlova, R. Richards-Kortum, and T. S. Tkaczyk, “All-plastic miniature fluorescence microscope for point-of-care readout of bead-based bioassays,” J. Biomed. Opt. 20(10), 105010 (2015).
[Crossref] [PubMed]

Y. Nakai, M. Ozeki, T. Hiraiwa, R. Tanimoto, A. Funahashi, N. Hiroi, A. Taniguchi, S. Nonaka, V. Boilot, R. Shrestha, J. Clark, N. Tamura, V. M. Draviam, and H. Oku, “High-speed microscopy with an electrically tunable lens to image the dynamics of in vivo molecular complexes,” Rev. Sci. Instrum. 86(1), 013707 (2015).
[Crossref] [PubMed]

2014 (1)

2011 (2)

2008 (1)

M. Zheng, J. C. Lin, H. Kasdan, and Y. Tai, “Fluorescent labeling, sensing, and differentiation of leukocytes from undiluted whole blood samples,” Sens. Actuators B Chem. 132(2), 558–567 (2008).
[Crossref]

2006 (1)

2001 (1)

B. Houwen, “The differential cell count,” Lab. Hematol. 7, 89–100 (2001).

1971 (1)

L. R. Adams and L. A. Kamentsky, “Machine characterization of human leukocytes by acridine orange fluorescence,” Acta Cytol. 15(3), 289–291 (1971).
[PubMed]

1961 (1)

J. F. Jackson, “Supravital Blood Studies, Using Acridine Orange Fluorescence,” Blood 17, 643–649 (1961).
[PubMed]

Adams, L. R.

L. R. Adams and L. A. Kamentsky, “Machine characterization of human leukocytes by acridine orange fluorescence,” Acta Cytol. 15(3), 289–291 (1971).
[PubMed]

Baggett, B. K.

Banerjee, A.

Boilot, V.

Y. Nakai, M. Ozeki, T. Hiraiwa, R. Tanimoto, A. Funahashi, N. Hiroi, A. Taniguchi, S. Nonaka, V. Boilot, R. Shrestha, J. Clark, N. Tamura, V. M. Draviam, and H. Oku, “High-speed microscopy with an electrically tunable lens to image the dynamics of in vivo molecular complexes,” Rev. Sci. Instrum. 86(1), 013707 (2015).
[Crossref] [PubMed]

Carlson, K. D.

Chidley, M. D.

Clark, J.

Y. Nakai, M. Ozeki, T. Hiraiwa, R. Tanimoto, A. Funahashi, N. Hiroi, A. Taniguchi, S. Nonaka, V. Boilot, R. Shrestha, J. Clark, N. Tamura, V. M. Draviam, and H. Oku, “High-speed microscopy with an electrically tunable lens to image the dynamics of in vivo molecular complexes,” Rev. Sci. Instrum. 86(1), 013707 (2015).
[Crossref] [PubMed]

Crannell, Z.

A. Forcucci, M. E. Pawlowski, Z. Crannell, I. Pavlova, R. Richards-Kortum, and T. S. Tkaczyk, “All-plastic miniature fluorescence microscope for point-of-care readout of bead-based bioassays,” J. Biomed. Opt. 20(10), 105010 (2015).
[Crossref] [PubMed]

Descour, M. R.

Draviam, V. M.

Y. Nakai, M. Ozeki, T. Hiraiwa, R. Tanimoto, A. Funahashi, N. Hiroi, A. Taniguchi, S. Nonaka, V. Boilot, R. Shrestha, J. Clark, N. Tamura, V. M. Draviam, and H. Oku, “High-speed microscopy with an electrically tunable lens to image the dynamics of in vivo molecular complexes,” Rev. Sci. Instrum. 86(1), 013707 (2015).
[Crossref] [PubMed]

Forcucci, A.

A. Forcucci, M. E. Pawlowski, Z. Crannell, I. Pavlova, R. Richards-Kortum, and T. S. Tkaczyk, “All-plastic miniature fluorescence microscope for point-of-care readout of bead-based bioassays,” J. Biomed. Opt. 20(10), 105010 (2015).
[Crossref] [PubMed]

A. Forcucci, M. E. Pawlowski, C. Majors, R. Richards-Kortum, and T. S. Tkaczyk, “All-plastic, miniature, digital fluorescence microscope for three part white blood cell differential measurements at the point of care,” Biomed. Opt. Express 6(11), 4433–4446 (2015).
[Crossref] [PubMed]

Funahashi, A.

Y. Nakai, M. Ozeki, T. Hiraiwa, R. Tanimoto, A. Funahashi, N. Hiroi, A. Taniguchi, S. Nonaka, V. Boilot, R. Shrestha, J. Clark, N. Tamura, V. M. Draviam, and H. Oku, “High-speed microscopy with an electrically tunable lens to image the dynamics of in vivo molecular complexes,” Rev. Sci. Instrum. 86(1), 013707 (2015).
[Crossref] [PubMed]

Grewe, B. F.

Hasan, N.

Helmchen, F.

Hiraiwa, T.

Y. Nakai, M. Ozeki, T. Hiraiwa, R. Tanimoto, A. Funahashi, N. Hiroi, A. Taniguchi, S. Nonaka, V. Boilot, R. Shrestha, J. Clark, N. Tamura, V. M. Draviam, and H. Oku, “High-speed microscopy with an electrically tunable lens to image the dynamics of in vivo molecular complexes,” Rev. Sci. Instrum. 86(1), 013707 (2015).
[Crossref] [PubMed]

Hiroi, N.

Y. Nakai, M. Ozeki, T. Hiraiwa, R. Tanimoto, A. Funahashi, N. Hiroi, A. Taniguchi, S. Nonaka, V. Boilot, R. Shrestha, J. Clark, N. Tamura, V. M. Draviam, and H. Oku, “High-speed microscopy with an electrically tunable lens to image the dynamics of in vivo molecular complexes,” Rev. Sci. Instrum. 86(1), 013707 (2015).
[Crossref] [PubMed]

Houwen, B.

B. Houwen, “The differential cell count,” Lab. Hematol. 7, 89–100 (2001).

Jackson, J. F.

J. F. Jackson, “Supravital Blood Studies, Using Acridine Orange Fluorescence,” Blood 17, 643–649 (1961).
[PubMed]

Kamentsky, L. A.

L. R. Adams and L. A. Kamentsky, “Machine characterization of human leukocytes by acridine orange fluorescence,” Acta Cytol. 15(3), 289–291 (1971).
[PubMed]

Kasdan, H.

M. Zheng, J. C. Lin, H. Kasdan, and Y. Tai, “Fluorescent labeling, sensing, and differentiation of leukocytes from undiluted whole blood samples,” Sens. Actuators B Chem. 132(2), 558–567 (2008).
[Crossref]

Kim, H.

Kyrish, M.

Li, B.

Lin, J. C.

M. Zheng, J. C. Lin, H. Kasdan, and Y. Tai, “Fluorescent labeling, sensing, and differentiation of leukocytes from undiluted whole blood samples,” Sens. Actuators B Chem. 132(2), 558–567 (2008).
[Crossref]

Majors, C.

Mastrangelo, C. H.

Nakai, Y.

Y. Nakai, M. Ozeki, T. Hiraiwa, R. Tanimoto, A. Funahashi, N. Hiroi, A. Taniguchi, S. Nonaka, V. Boilot, R. Shrestha, J. Clark, N. Tamura, V. M. Draviam, and H. Oku, “High-speed microscopy with an electrically tunable lens to image the dynamics of in vivo molecular complexes,” Rev. Sci. Instrum. 86(1), 013707 (2015).
[Crossref] [PubMed]

Nonaka, S.

Y. Nakai, M. Ozeki, T. Hiraiwa, R. Tanimoto, A. Funahashi, N. Hiroi, A. Taniguchi, S. Nonaka, V. Boilot, R. Shrestha, J. Clark, N. Tamura, V. M. Draviam, and H. Oku, “High-speed microscopy with an electrically tunable lens to image the dynamics of in vivo molecular complexes,” Rev. Sci. Instrum. 86(1), 013707 (2015).
[Crossref] [PubMed]

Oku, H.

Y. Nakai, M. Ozeki, T. Hiraiwa, R. Tanimoto, A. Funahashi, N. Hiroi, A. Taniguchi, S. Nonaka, V. Boilot, R. Shrestha, J. Clark, N. Tamura, V. M. Draviam, and H. Oku, “High-speed microscopy with an electrically tunable lens to image the dynamics of in vivo molecular complexes,” Rev. Sci. Instrum. 86(1), 013707 (2015).
[Crossref] [PubMed]

Ozeki, M.

Y. Nakai, M. Ozeki, T. Hiraiwa, R. Tanimoto, A. Funahashi, N. Hiroi, A. Taniguchi, S. Nonaka, V. Boilot, R. Shrestha, J. Clark, N. Tamura, V. M. Draviam, and H. Oku, “High-speed microscopy with an electrically tunable lens to image the dynamics of in vivo molecular complexes,” Rev. Sci. Instrum. 86(1), 013707 (2015).
[Crossref] [PubMed]

Pavlova, I.

A. Forcucci, M. E. Pawlowski, Z. Crannell, I. Pavlova, R. Richards-Kortum, and T. S. Tkaczyk, “All-plastic miniature fluorescence microscope for point-of-care readout of bead-based bioassays,” J. Biomed. Opt. 20(10), 105010 (2015).
[Crossref] [PubMed]

Pawlowski, M. E.

A. Forcucci, M. E. Pawlowski, C. Majors, R. Richards-Kortum, and T. S. Tkaczyk, “All-plastic, miniature, digital fluorescence microscope for three part white blood cell differential measurements at the point of care,” Biomed. Opt. Express 6(11), 4433–4446 (2015).
[Crossref] [PubMed]

A. Forcucci, M. E. Pawlowski, Z. Crannell, I. Pavlova, R. Richards-Kortum, and T. S. Tkaczyk, “All-plastic miniature fluorescence microscope for point-of-care readout of bead-based bioassays,” J. Biomed. Opt. 20(10), 105010 (2015).
[Crossref] [PubMed]

Qin, H.

Richards-Kortum, R.

A. Forcucci, M. E. Pawlowski, C. Majors, R. Richards-Kortum, and T. S. Tkaczyk, “All-plastic, miniature, digital fluorescence microscope for three part white blood cell differential measurements at the point of care,” Biomed. Opt. Express 6(11), 4433–4446 (2015).
[Crossref] [PubMed]

A. Forcucci, M. E. Pawlowski, Z. Crannell, I. Pavlova, R. Richards-Kortum, and T. S. Tkaczyk, “All-plastic miniature fluorescence microscope for point-of-care readout of bead-based bioassays,” J. Biomed. Opt. 20(10), 105010 (2015).
[Crossref] [PubMed]

Richards-Kortum, R. R.

Shrestha, R.

Y. Nakai, M. Ozeki, T. Hiraiwa, R. Tanimoto, A. Funahashi, N. Hiroi, A. Taniguchi, S. Nonaka, V. Boilot, R. Shrestha, J. Clark, N. Tamura, V. M. Draviam, and H. Oku, “High-speed microscopy with an electrically tunable lens to image the dynamics of in vivo molecular complexes,” Rev. Sci. Instrum. 86(1), 013707 (2015).
[Crossref] [PubMed]

Tai, Y.

M. Zheng, J. C. Lin, H. Kasdan, and Y. Tai, “Fluorescent labeling, sensing, and differentiation of leukocytes from undiluted whole blood samples,” Sens. Actuators B Chem. 132(2), 558–567 (2008).
[Crossref]

Tamura, N.

Y. Nakai, M. Ozeki, T. Hiraiwa, R. Tanimoto, A. Funahashi, N. Hiroi, A. Taniguchi, S. Nonaka, V. Boilot, R. Shrestha, J. Clark, N. Tamura, V. M. Draviam, and H. Oku, “High-speed microscopy with an electrically tunable lens to image the dynamics of in vivo molecular complexes,” Rev. Sci. Instrum. 86(1), 013707 (2015).
[Crossref] [PubMed]

Taniguchi, A.

Y. Nakai, M. Ozeki, T. Hiraiwa, R. Tanimoto, A. Funahashi, N. Hiroi, A. Taniguchi, S. Nonaka, V. Boilot, R. Shrestha, J. Clark, N. Tamura, V. M. Draviam, and H. Oku, “High-speed microscopy with an electrically tunable lens to image the dynamics of in vivo molecular complexes,” Rev. Sci. Instrum. 86(1), 013707 (2015).
[Crossref] [PubMed]

Tanimoto, R.

Y. Nakai, M. Ozeki, T. Hiraiwa, R. Tanimoto, A. Funahashi, N. Hiroi, A. Taniguchi, S. Nonaka, V. Boilot, R. Shrestha, J. Clark, N. Tamura, V. M. Draviam, and H. Oku, “High-speed microscopy with an electrically tunable lens to image the dynamics of in vivo molecular complexes,” Rev. Sci. Instrum. 86(1), 013707 (2015).
[Crossref] [PubMed]

Tkaczyk, T. S.

Utzinger, U.

van ’t Hoff, M.

Voigt, F. F.

Xing, D.

Yang, S.

Zheng, M.

M. Zheng, J. C. Lin, H. Kasdan, and Y. Tai, “Fluorescent labeling, sensing, and differentiation of leukocytes from undiluted whole blood samples,” Sens. Actuators B Chem. 132(2), 558–567 (2008).
[Crossref]

Acta Cytol. (1)

L. R. Adams and L. A. Kamentsky, “Machine characterization of human leukocytes by acridine orange fluorescence,” Acta Cytol. 15(3), 289–291 (1971).
[PubMed]

Appl. Opt. (1)

Biomed. Opt. Express (2)

Blood (1)

J. F. Jackson, “Supravital Blood Studies, Using Acridine Orange Fluorescence,” Blood 17, 643–649 (1961).
[PubMed]

J. Biomed. Opt. (1)

A. Forcucci, M. E. Pawlowski, Z. Crannell, I. Pavlova, R. Richards-Kortum, and T. S. Tkaczyk, “All-plastic miniature fluorescence microscope for point-of-care readout of bead-based bioassays,” J. Biomed. Opt. 20(10), 105010 (2015).
[Crossref] [PubMed]

Lab. Hematol. (1)

B. Houwen, “The differential cell count,” Lab. Hematol. 7, 89–100 (2001).

Opt. Express (3)

Rev. Sci. Instrum. (1)

Y. Nakai, M. Ozeki, T. Hiraiwa, R. Tanimoto, A. Funahashi, N. Hiroi, A. Taniguchi, S. Nonaka, V. Boilot, R. Shrestha, J. Clark, N. Tamura, V. M. Draviam, and H. Oku, “High-speed microscopy with an electrically tunable lens to image the dynamics of in vivo molecular complexes,” Rev. Sci. Instrum. 86(1), 013707 (2015).
[Crossref] [PubMed]

Sens. Actuators B Chem. (1)

M. Zheng, J. C. Lin, H. Kasdan, and Y. Tai, “Fluorescent labeling, sensing, and differentiation of leukocytes from undiluted whole blood samples,” Sens. Actuators B Chem. 132(2), 558–567 (2008).
[Crossref]

Other (9)

SCHOTT Nexterion, “Coverslips,” http://www.schott.com/nexterion/english/products/coverslips.html Accessed: 16-May-2017.

World Health Organization, “Diagnostic testing,” http://www.who.int/malaria/areas/diagnosis/en/ . Accessed: 29-Jun-2017.

World Health Organization, “TB detection and diagnosis,” http://www.who.int/tb/areas-of-work/laboratory/en/ . Accessed: 29-Jun-2017.

Nikon MicroscopyU, “Introduction to Fluorescence Microscopy,” https://www.microscopyu.com/techniques/fluorescence/introduction-to-fluorescence-microscopy . Accessed: 30-Jan-2017.

C. L. Grendol, “Apparatus and method for injection molding lenses.” US Patent 450534A. Issued: September 10, 1985.

Varioptic documentation, “Arctic 316 Family,” (Varioptic, 2015).

J. M. Cavagnaro, “Polymer optics: progress in plastic optics follows advances in materials and manufacturing,” (Laser Focus World, 2011), http://www.laserfocusworld.com/articles/print/volume-47/issue-9/features/polymer-optics-progress-in-plastic-optics-follows-advances-in-materials-and-manufacturing.html . Accessed: 16-May-2017.

Centers for Disease Control and Prevention, “Antibiotics Aren’t Always the Answer,” https://www.cdc.gov/features/getsmart/index.html . Accessed: 11 July 2017.

C. E. Majors, M. E. Pawlowski, T. Tkaczyk, and R. R. Richards-Kortum, “Low-Cost Disposable Cartridge for Performing a White Blood Cell Count and Partial Differential at the Point-of-Care,” Health Innovation Point of Care Conference, 1–10 ( 2014).
[Crossref]

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Figures (9)

Fig. 1
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
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
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
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
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
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
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
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
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.

Tables (5)

Tables Icon

Table 1 Summary of optical design parameters of the tunable fluorescence microscope.

Tables Icon

Table 2 Optical prescription data of the tunable microscope for configurations 1-4* (Radii, thickness, semi-diameters (SD), and conic values are given in units of [mm])

Tables Icon

Table 3 Tolerance parameters of the miniature tunable microscope

Tables Icon

Table 4 Summary of the resolution of the tunable system for 450, 550, 600, 650, and 700 nm wavelengths. The theoretical resolution (located in the ‘Calculated Resolution’ column) was found using the Rayleigh criterion, and the corresponding Group and Element number of the 1951 USAF resolution target is given in the column ‘Resolution Limit (theoretical).’ The experimentally measured resolution was reported in column ‘Resolution Limit (actual).’ The columns ‘Image’ and ‘Intensity Plot’ show the magnified image of the smallest resolvable element of the 1951 USAF target together with the intensity cross-section through the pixels marked with the white line.

Tables Icon

Table 5 Comparison of differential WBC counts from the commercial hematology analyser and the tunable microscope.

Equations (3)

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OP=1.48DV56.22
d= 0.61λ NA
d= λn N A 2

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