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Miniaturized integration of a fluorescence microscope

Nature Methods volume 8, pages 871–878 (2011)Cite this article

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Abstract

The light microscope is traditionally an instrument of substantial size and expense. Its miniaturized integration would enable many new applications based on mass-producible, tiny microscopes. Key prospective usages include brain imaging in behaving animals for relating cellular dynamics to animal behavior. Here we introduce a miniature (1.9 g) integrated fluorescence microscope made from mass-producible parts, including a semiconductor light source and sensor. This device enables high-speed cellular imaging across ∼0.5 mm2 areas in active mice. This capability allowed concurrent tracking of Ca2+ spiking in >200 Purkinje neurons across nine cerebellar microzones. During mouse locomotion, individual microzones exhibited large-scale, synchronized Ca2+ spiking. This is a mesoscopic neural dynamic missed by prior techniques for studying the brain at other length scales. Overall, the integrated microscope is a potentially transformative technology that permits distribution to many animals and enables diverse usages, such as portable diagnostics or microscope arrays for large-scale screens.

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Figure 1: Design and fabrication of an integrated fluorescence microscope.
Figure 2: Cerebellar microcirculatory dynamics in freely behaving mice.
Figure 3: Nonuniform regulation of cerebellar capillaries during locomotion.
Figure 4: Purkinje neurons' Ca2+ spiking dynamics during motor behavior.
Figure 5: Cerebellar microzones exhibit large-scale, synchronized Ca2+ spiking during motor behavior.

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Acknowledgements

We thank E.J. Baron, S.S. Gambhir, E.T.W. Ho, R. Luo, E. Mukamel, J. Perlin, L. Sasportas, W. Talbot and members of the Stanford Varian machine shop for technical assistance, and L. Looger (HHMI Janelia Farm Research Campus) for the GCaMP3 plasmid. We acknowledge graduate fellowships from the US National Science Foundation and Stanford University (L.D.B.), postdoctoral fellowships from the Human Frontier Science Program (A.N.) and the Machiah Foundation (Y.Z.), and research funding to M.J.S. from Lawrence Livermore National Laboratory, the Office of Naval Research, the US National Institutes of Health Nanomedicine Development Center for Optical Control of Biological Function, the National Science Foundation Center for Biophotonics, the Packard, the Bill and Melinda Gates and the Paul G. Allen Family Foundations, and the Stanford University CNC program.

Author information

Author notes

  1. Kunal K Ghosh, Laurie D Burns and Eric D Cocker: These authors contributed equally to this work.

Authors and Affiliations

  1. David Packard Electrical Engineering Building, Stanford University, Stanford, California, USA

    Kunal K Ghosh & Abbas El Gamal

  2. James H. Clark Center, Stanford University, Stanford, California, USA

    Kunal K Ghosh, Laurie D Burns, Eric D Cocker, Axel Nimmerjahn, Yaniv Ziv & Mark J Schnitzer

  3. Howard Hughes Medical Institute, Stanford University, Stanford, California, USA

    Mark J Schnitzer

  4. CNC Program, Stanford University, Stanford, California, USA

    Mark J Schnitzer

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Contributions

K.K.G. performed optical analysis, designed electronic circuits, assembled microscopy systems, wrote cell-counting software and performed the zebrafish, tuberculosis and cell-counting experiments. L.D.B. performed optical analysis, designed the optical pathway, assembled microscopy systems, performed cerebellum and hippocampal imaging studies, and analyzed the Ca2+-imaging data. E.D.C. designed the mechanical housing, heat dissipation, focusing mechanisms and illumination control circuitry, assembled microscopy systems, designed and built behavioral enclosures with video acquisition, and analyzed the behavioral and microcirculation data. A.N. developed the cerebellar preparation and performed cerebellar imaging studies. Y.Z. developed and performed the hippocampal imaging methodology. A.E.G. supervised the project. M.J.S. supervised the project and wrote the paper. All authors designed experiments and edited the paper.

Corresponding author

Correspondence to Mark J Schnitzer.

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Competing interests

K.K.G., E.D.C., A.E.G. and M.J.S. have equity in a company (Inscopix) pursuing imaging applications based on the integrated microscope.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–2, Supplementary Table 1 and Supplementary Methods (PDF 3670 kb)

Supplementary Video 1

Mouse behavior and microcirculation in the cerebellar vermis recorded concurrently in a mouse with an integrated microscope mounted on the cranium. This movie presents the simultaneous video clips of mouse behavior and microcirculation in the vermis for two example behaviors. The first example shows the mouse walking about the behavioral arena. The second example shows the mouse running on an exercise wheel. Behavioral data (left) were recorded at 30 Hz with an overhead camera and infrared illumination. Microcirculation (right) was recorded using the integrated microscope at 100 Hz after an intravenous injection of FITC-dextran. This fluorescent dye brightly labels the blood plasma, allowing erythrocytes to be seen in dark relief. Individual erythrocytes are apparent flowing through the capillaries. Brain motion artifacts are so minimal as to be virtually undetectable. Scale bar, 100 μm. (MOV 6923 kb)

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Ghosh, K., Burns, L., Cocker, E. et al. Miniaturized integration of a fluorescence microscope. Nat Methods 8, 871–878 (2011). https://doi.org/10.1038/nmeth.1694

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