A few prior studies have explored in vivo
cellular imaging in unrestrained animals but used fiber-optics and benchtop instrumentation not conducive to mass-production3,6,7
. By comparison, our microscope's composition from mass-producible components should also help promote its dissemination. The LED, image sensor, filters, and microlenses are all made via batch fabrication, so their costs per microscope decline rapidly with production volume. The microscope housings were machined, but these could be molded to facilitate greater volumes, with the cost dominated by the initial mold. Overall, we expect costs per microscope to fall substantially with volume manufacturing. This is not the case with conventional high-resolution microscopes, for which multiple parts still resist batch-fabrication.
Other aspects of the integrated microscope that should aid dissemination include the energy efficiency of LED illumination and the microscope's ease of transport without need for re-alignment. We made seven identical copies of the microscope in the initial production round. These devices contain all optical parts but require a computer and are not standalone. Future, standalone versions should be possible and could benefit in-the-field diagnostics, which demand a high degree of portability.
Creation of the integrated microscope capitalized on recent advances in LEDs and image sensors, but the main innovation was in the system design. We created a miniaturized optical pathway, a data acquisition system, three circuit boards, and a housing with fine focusing capability, while keeping the microscope to 1.9 g mass. Prior approaches to fluorescence imaging in miniaturized format have generally required accessory, tabletop optical instrumentation6,11,12,31
. An earlier integrated system for use in mobile rats exhibited a Nyquist resolution of ~20 μm, insufficient for observing individual cells32
. Optofluidic chips have been incompatible with fluorescence contrast and required assumptions about the specimen, such as that it was flowing at certain speeds at zero optical working distance13
. Lensless approaches do not produce direct images but involve deciphering diffraction patterns produced by cells immobilized at a fixed working distance14
. Due to its fluorescence capability, lack of ancillary optical instrumentation, alignment-free portability, and suitability for volume production, the integrated microscope differs from prior miniaturized devices and can address distinct applications, including imaging the dynamics of hundreds of individual neurons in behaving animals.
The integrated microscope will permit Ca2+
imaging in deep brain areas of behaving mice when it is combined with recently developed methods for time-lapse micro-optical imaging33
, genetically encoded Ca2+
-indicators that are non-ratiometric34
, and an accessory microendoscope to access deep tissues6,33
,. To illustrate, we performed Ca2+
-imaging in hippocampal area CA1 of active mice using a GCaMP3 Ca2+
genetically targeted to pyramidal neurons (Supplementary Fig. 2
). These studies revealed prominent Ca2+
transients that likely correspond to action potential bursts, as GCaMP3 is not sufficiently sensitive to report single action potentials in these neurons34
These proof-of-concept data show neuronal Ca2+
-imaging during naturalistic mouse behavior is not restricted to studies of cerebellar cortex. Brain areas where cellular-level epi-fluorescence imaging has worked well in behaving rodents include cerebellum6
, olfactory bulb35
, and neocortex37
-imaging should be similarly feasible in various other brain regions. Many institutions are investing in rodent behavioral facilities, and the integrated microscope appears compatible with widely used assays including fear conditioning, novel object recognition, tests of rodent social interaction, or assays involving olfactory cues or foraging for rewards. This flexibility should help add a brain-imaging component to behavioral research using instrumentation that could be less costly, sizable, and challenging to operate than current optical apparatus.
In comparison to two-photon microscopes used to study alert, head-restrained rodents4,5,8,9
, the integrated microscope provides broader fields of view and decreased susceptibility to motion artifacts due to its greater depth of field and faster frame rates (Supplementary Video 1
). For comparison, two-photon microscopy in behaving mice allowed tracking of ~100 Purkinje neurons' Ca2+
spiking at < 12 Hz frame rates4,5,9
; the integrated microscope monitored 206 Purkinje neurons at 46 Hz (). Two-photon microscopy has other virtues, including optical sectioning, lower background fluorescence, superior resolution, and greater robustness to scattering. These are key advantages for imaging cells multiple scattering lengths from the objective lens. However, the pixel dwell times in two-photon microscopy (~0.1–3 μs) are so brief relative to one-photon imaging frame-rates that in moderate light scattering regimes neuronal Ca2+
-spikes are approximately equally detectable in either modality, since the greater photon counts and less severe photon shot noise in one-photon imaging can compensate for this modality's lesser contrast (B. Wilt, J. Fitzgerald, and M.J Schnitzer, unpublished calculations
The two modalities also appear complementary regarding rodent behavioral assays. The integrated microscope permits free-ranging behavior, whereas head-fixation of an animal for two-photon microscopy seems incompatible with many naturalistic behaviors, even with virtual reality methods. However, other behavioral studies should benefit from head-fixation, since it can restrict and simplify behavioral responses. Still, head-fixation seems likely to evoke greater stress responses than naturalistic behavior with a head-mounted device. We expect future roles for both two-photon and integrated microscopy in brain imaging.
We observed synchronization of up to > 30 Purkinje neurons, but selectively during motor behavior and spatially organized by microzone boundaries. Although ~50–100 μm wide in the medial-lateral dimension, microzones extend millimeters in the rostral-caudal dimension23
, suggesting that during microzone activation hundreds to thousands of Purkinje neurons could be acting simultaneously with the ~600-μm long subsets observed via the integrated microscope. Such concerted activity of large neural ensembles at the millimeter-scale has few if any precedents. Imaging studies have typically lacked either the resolution of individual neurons' spikes, or the field of view required to observe mesoscale synchronization. During animal inactivity, individual Purkinje neurons exhibit ongoing Ca2+
spiking, whereas the ensemble activation stops or persists at a much-reduced rate. These observations prompt many questions regarding the extent and mechanisms of the large-scale synchronization, the information encoded, and its role in motor behavior.
Neurophysiologic control of motor behavior likely involves large neuronal ensembles acting in an organized fashion. Each microzone is mapped to part of the body plan23
, and coherent microzone activation may carry signals of especial importance for control of this part. The prominence of the microzone's ensemble activation likely enhances its detection and downstream impact in the deep cerebellar nuclei, the sole recipients of Purkinje neuron output from the vermis.
An attractive possibility is that microzone activation provides a means for reliably encoding motor errors in the presence of ongoing Ca2+
spiking. Purkinje neurons' Ca2+
spikes are driven by climbing fiber axons from the inferior olive, an area where excitatory neurons can synchronize via gap junction networks23
and are thought to convey motor error information to cerebellar cortex. As our and prior recordings4,6,38
show, individual Ca2+
spikes persist in the absence of body movement and so seem unlikely to signal motor errors in a straightforward way. Ensemble activation of the microzone is nearly exclusive to movement periods and could provide a motor error signal that is readily identifiable against individual cells' baseline spike rates. Beyond the cerebellum, large-scale synchrony in mesoscale neuronal ensembles could be a fundamental means in the brain to reliably engage downstream circuitry and evoke animal behavior.
In addition to its usages in behaving animals, the integrated microscope is a multipurpose instrument for epi-fluorescence imaging. For in vitro applications, the microscope has the potential to enable large-scale parallelism, allow imaging inside other instrumentation such as incubators, and reduce costs. Moreover, a high-resolution microscope that can be held by the fingertips and readily ported should stimulate new imaging applications in biology, including portable usages where microscopy is presently challenging or infeasible. In our personal experience, we have found the integrated microscope with its laptop computer can be easily transported on commercial air flights and immediately used at the remote destination. This opens up possibilities for mobile assays, such as for ecological studies or biomedical diagnostics of potential significance for developing nations.
As our cell counting assays show, the integrated microscope can attain counting accuracies comparable to flow cytometry or conventional instrumentation based on digital imaging. Our array of four microscopes points the way to larger arrays that could enable massively parallel strategies for high-content screening. Today, most image-based screens involve one or a few microscopes for serial inspection that ultimately limit throughput. Currently available massively parallel approaches generally do not involve imaging and use simpler screening criteria. When combined with computational tools for analyzing massive data sets, arrays of integrated microscopes might combine the best of high-content screening and massive parallelism. For instance, screens involving high-speed Ca2+
-imaging in neurons or myocytes are generally prohibitive today with conventional plate readers but should be feasible with an array of integrated microscopes. For screens requiring diffraction-limited resolution, it should be straightforward to substitute the existing micro-objective with one designed for diffraction-limited imaging39
Integrated technologies usually advance rapidly, which motivated our choice of a design that can capitalize on upcoming sensor advances to improve resolution, sensitivity, and dynamic range. Multi-color imaging should also be feasible. Since CMOS technology underpins most modern electronics, ‘intelligent’ integrated microscopes seem likely to emerge with sensors having built-in electronic capabilities to facilitate analysis, screening, data compression, or diagnostic evaluations.