Obtaining high signal to noise ratio is challenging in wide-field two photon microscopy and one must ensure the mouse brain can be imaged safely under high laser power. Here, we demonstrated a simultaneous thermal imaging and two photon imaging technique. The maximum temperature of the cortex was below 39°C using 400mW laser power with a 5 x 5mm field of view. Together with the brain activities under hind paw stimulation and at rest, we argued that high laser power for wide-field two-photon imaging can potentially be used while keeping the temperature under safety limit.
By paying careful attention to optical and mechanical design, a two-photon microscope (TPM) can image a large field of view (FOV) with high spatial and temporal resolution. Successfully applying such a TPM for functional neuroimaging requires careful consideration of the signal properties, physiological limitations, and image processing techniques. We present our thermal imaging results as well as functional imaging results in awake mice using a fast, large FOV TPM, and discuss how the system expands the spatial bandwidth available for studying functional connectivity.
Quick and accurate parcellation of neural networks has been a goal spanning multiple decades of research in the functional MRI world in order to organize and understand the overwhelmingly complex human brain with high statistical rigor. The same mathematical development in the mouse brain, which is frequently studied to understand human conditions, has been lagging. To this end, we perform high-throughput fluorescence imaging (GCaMP6f) in healthy mice in a cell-specific and non-invasive manner during rest and sensorimotor tasks in order to map healthy networks and understand patterns due to disease processes.
Though optical imaging of human brain function is gaining momentum, widespread adoption is restricted in part by a tradeoff among cap wearability, field of view, and resolution. To increase coverage while maintaining functional magnetic resonance imaging (fMRI)-comparable image quality, optical systems require more fibers. However, these modifications drastically reduce the wearability of the imaging cap. The primary obstacle to optimizing wearability is cap weight, which is largely determined by fiber diameter. Smaller fibers collect less light and lead to challenges in obtaining adequate signal-to-noise ratio. Here, we report on a design that leverages the exquisite sensitivity of scientific CMOS cameras to use fibers with ∼30 × smaller cross-sectional area than current high-density diffuse optical tomography (HD-DOT) systems. This superpixel sCMOS DOT (SP-DOT) system uses 200-μm-diameter fibers that facilitate a lightweight, wearable cap. We developed a superpixel algorithm with pixel binning and electronic noise subtraction to provide high dynamic range (>105), high frame rate (>6 Hz), and a low effective detectivity threshold (∼200 fW / Hz1/2-mm2), each comparable with previous HD-DOT systems. To assess system performance, we present retinotopic mapping of the visual cortex (n = 5 subjects). SP-DOT offers a practical solution to providing a wearable, large field-of-view, and high-resolution optical neuroimaging system.
Conventional two-photon microscopy (TPM) is capable of imaging neural dynamics with subcellular resolution, but it is limited to a field-of-view (FOV) diameter <1 mm. Although there has been recent progress in extending the FOV in TPM, a principled design approach for developing large FOV TPM (LF-TPM) with off-the-shelf components has yet to be established. Therefore, we present a design strategy that depends on analyzing the optical invariant of commercially available objectives, relay lenses, mirror scanners, and emission collection systems in isolation. Components are then selected to maximize the space-bandwidth product of the integrated microscope. In comparison with other LF-TPM systems, our strategy simplifies the sequence of design decisions and is applicable to extending the FOV in any microscope with an optical relay. The microscope we constructed with this design approach can image <1.7-μm lateral and <28-μm axial resolution over a 7-mm diameter FOV, which is a 100-fold increase in FOV compared with conventional TPM. As a demonstration of the potential that LF-TPM has on understanding the microarchitecture of the mouse brain across interhemispheric regions, we performed in vivo imaging of both the cerebral vasculature and microglia cell bodies over the mouse cortex.
Spontaneous neuronal activity has been measured at cellular resolution in mice, zebrafish, and C. elegans using optical sectioning microscopy techniques, such as light sheet microscopy (LSM) and two photon microscopy (TPM). Recent improvements in these modalities and genetically encoded calcium indicators (GECI’s) have enabled whole brain imaging of calcium dynamics in zebrafish and C. elegans. However, these whole brain microscopy studies have not been extended to mice due to the limited field of view (FOV) of TPM and the cumbersome geometry of LSM. Conventional TPM is restricted to diffraction limited imaging over this small FOV (around 500 x 500 microns) due to the use of high magnification objectives (e.g. 1.0 NA; 20X) and the aberrations introduced by relay optics used in scanning the beam across the sample.
To overcome these limitations, we have redesigned the entire optical path of the two photon microscope (scanning optics and objective lens) to support a field of view of Ø7 mm with relatively high spatial resolution (<10 microns). Using optical engineering software Zemax, we designed our system with commercially available optics that minimize astigmatism, field curvature, chromatic focal shift, and vignetting. Performance of the system was also tested experimentally with fluorescent beads in agarose, fixed samples, and in vivo structural imaging. Our large-FOV TPM provides a modality capable of studying distributed brain networks in mice at cellular resolution.
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