Miniaturized integration of a fluorescence microscope

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.

Figure 1. Design and fabrication of an integrated fluorescence microscope

a. Computer-assisted design of an integrated microscope, shown in cross-section. Blue and green arrows mark illumination and emission pathways, respectively. Scale bar is 5 mm. b. Assembled integrated microscope. Insets show, clockwise from bottom left: filter cube holding dichroic mirror and excitation and emission filters; printed circuit board (PCB) holding the complementary metal-oxide-semiconductor (CMOS) camera chip; PCB holding the light emitting diode (LED) illumination source. The wire bundles for LED and CMOS boards are visible. Scale bar is 5 mm and applies to all four photographs. c. Electronics for real-time image acquisition and control. The LED and CMOS sensoreach have their own PCB. These boards are connected to a custom, external PCB via nine fine wires (two to the LED and seven to the camera) encased in a single polyvinyl chloride sheath. The external PCB interfaces with a computer via a USB adaptor board. Labeled components summarize circuitry for providing power, controlling the LED and CMOS camera, and transferring data. Abbreviations: PD, flash programming device; OSC, Quartz crystal oscillator; I²C, Two-wire Inter-Integrated Circuit serial communication interface.

Figure 2. Cerebellar microcirculatory dynamics in freely behaving mice

a. Microvasculature in cerebellar cortex of a freely behaving mouse, after intravascular injection of fluorescein-dextran dye. The image shown (300 × 300 pixels; 440 μW illumination power at the specimen plane) is the standard deviation of a 10 s movie, a computation that highlights vasculature (Online Methods). Colored dots mark the locations of corresponding measurements in (c, d). Scale bar is 50 μm. b. Map of erythrocyte flow speeds averaged over an interval of 30 s for vessels of (a). Scale bar is 50 μm. c, d. Erythrocyte flow speeds, (c), and vessel diameter changes, (d), for the 4 vessels marked in (a). Blue shading marks periods of movement about the cage. Red shading indicates running on an exercise wheel. White shading marks when the mouse rested orbarely moved. Black vertical lines separate different records from the same mouse and specimen field.

Figure 3. Non-uniform regulation of cerebellar capillaries during locomotion

a, b. Erythrocyte speeds, (a), and vessel diameters, (b), compared between rest and locomotion. Each datum represents a vessel location in the vermis. Data above the diagonal indicate up-regulation in speed or diameter during motor activity (blue points, walking within the cage; red points, wheel running). Shaded blue and red areas demarcate 1 s.d. of measurement fluctuations calculated using the data below the diagonal. c. Cumulative histogram of changes in flow speeds during walking about the cage (blue) or wheel running (red), compared to rest. Histogram portions above and to the right of areas enclosed by colored dashed lines represent data for vessel locations lying above the color corresponding shaded areas in (a). (Inset) Mean ± s.e.m. changes compared to rest. (*) Indicates significant difference between walking and running (P = 4 · 10⁻⁵); (†) indicates significant difference from rest (P = 2 · 10⁻⁴), using Wilcoxon signed-rank tests. d. Cumulative histogram of changes in vessel diameters during walking (blue) or running (red), compared to rest. Histogram portions outside areas enclosed by colored dashed lines represent measurements lying above the corresponding shaded areas in (b). (Inset) Mean ± s.e.m. changes. (*) Indicates significant difference between walking and running (P = 6 · 10⁻³); (†) and (††) indicate significant differences from rest of P = 1 · 10⁻³ and P = 1 · 10⁻⁴, respectively, using Wilcoxon signed-rank tests. Data in (a–d) comprise n = 97 vessel locations from 3 mice.

Figure 4. Purkinje neurons' Ca²⁺ spiking dynamics during motor behavior

a. Contours of 206 Purkinje neurons identified in a freely behaving mouse, super imposed on a time-averaged fluorescence image (480 × 480 pixels; 170-250 μW illumination at the specimen plane) of the cerebellar surface after injection of the Ca²⁺-indicatorOregeon-Green-BAPTA-1-AM. Each color indicates one of nine identified microzones. Filled contours mark neurons whose activity is shown in (b). Scale bar is 100 μm. b. Relative changes in fluorescence (ΔF/F) from filled neurons in (a). Black dots mark detected Ca²⁺ spikes. Scale bars: 1 s (horizontal); 3% ΔF/F (vertical). c. Spike train correlation coefficients for pairs of neurons, during resting, grooming, and locomotion. Colored outlines indicate microzones identified by cluster analysis of the correlation coefficients and correspond to those in (a).

Figure 5. Cerebellar microzones exhibit large-scale, synchronized Ca²⁺ spiking during motor behavior

a. Ca²⁺ spike (black dots) raster plots for neurons of Fig. 4a. Colored shading indicates the mouse's behavior (peach, grooming; green, locomotion; gray, resting; other small movements, blue). Microzone rasters (colored dots) show Ca²⁺ spiking by > 35% (open dots) or > 50% (closed dots) of neurons identified in each microzone. Scale bar is 5 s. b. Expanded view of the locomotion and first resting periods of (a). Scale bar is 5 s. c. Mean ± s.e.m rates of individual neuronal spiking (top) and synchronized microzone activation (bottom: > 35% cells synchronized, unfilled bars; > 50% cells, solid bars). d. Spike rates for individual cells (small data points) and synchronized microzonal activation (> 35% cells, large open points; > 50% cells, large solid points) plotted for periods of grooming vs. rest (yellow points) or locomotion vs. rest (green points). e. Cumulative histograms of percentages of cell's spikes occurring during synchronized activation (> 35% activation, open points; > 50% activation, solid points; locomotion, green; grooming, yellow; resting, gray). Data of (c-e) are from n = 3 mice, 336 cells, and 16 microzones.