Fast widefield imaging of neuronal structure and function with optical sectioning in vivo

Abstract

Optical microscopy, owing to its noninvasiveness and subcellular resolution, enables in vivo visualization of neuronal structure and function in the physiological context. Optical-sectioning structured illumination microscopy (OS-SIM) is a widefield fluorescence imaging technique that uses structured illumination patterns to encode in-focus structures and optically sections 3D samples. However, its application to in vivo imaging has been limited. In this study, we optimized OS-SIM for in vivo neural imaging. We modified OS-SIM reconstruction algorithms to improve signal-to-noise ratio and correct motion-induced artifacts in live samples. Incorporating an adaptive optics (AO) module to OS-SIM, we found that correcting sample-induced optical aberrations was essential for achieving accurate structural and functional characterizations in vivo. With AO OS-SIM, we demonstrated fast, high-resolution in vivo imaging with optical sectioning for structural imaging of mouse cortical neurons and zebrafish larval motor neurons, and functional imaging of quantal synaptic transmission at Drosophila larval neuromuscular junctions.

Multicolor structured illumination microscopy and quantitative control of polychromatic coherent light with a digital micromirror device

“…The maximum achievable pattern display rate is limited by the physical translation and rotation of the grating or the refresh rate of the SLM. Here, we provide a new theoretical framework to use a DMD as the diffractive optic for multi-wavelength coherent SIM, extending previous work (25, 26). This opens the possibility for quantitative, multi-wavelength pattern formation at rates up to 20 kHz for a factor of ~5 lower cost than SLM based units.…”

“…Digital mirror devices (DMD’s) are a promising alternative to SLM’s for coherent SIM which offer several advantages, including a potential factor of 5–10 imaging rate increase, less experimental complexity, and lower cost (24–26). However, DMD use is currently limited by a computationally expensive forward model (25) to evaluate the blazed grating effect, which imposes severe restrictions on multicolor operation.…”

“…However, DMD use is currently limited by a computationally expensive forward model (25) to evaluate the blazed grating effect, which imposes severe restrictions on multicolor operation. To date, all published work has used incoherent projection (27, 28) or one coherent wavelength (25, 26, 29).…”

“…Digital micromirror devices (DMD’s) are a promising alternative to ferroelectric and LCD SLM’s for a variety of wavefront shaping tasks. DMD’s offer several advantages, including a potential factor of 5–10 imaging rate increase, less experimental timing complexity, a factor of 2–10 lower cost, and enhanced diffraction efficiency (36–38). DMD’s can reach frame rates of up to 30 kHz, exceeding the rate achievable in ferroelectric and liquid crystal SLM’s by a factor of ∼5 and ∼10 respectively.…”

Squid: Simplifying Quantitative Imaging Platform Development and Deployment

“…Higher power LEDs (>10W optical power at the LED) widely used in 3D printing and projectors are available at low cost, and can be used if appropriate heat sink is implemented. Further more, using the configuration in Figure 2B, we demonstrated patterned illumination with a DLP projector ( Figure 10), which, along with its LCoS alternatives, may be used to perform targeted photo-stimulation [24][25][26][27]158] and structured illumination for optical sectioning and/or super resolution [19,21,25,52,137,159,160]. Alternatively, lasers may be used in place of the LED for higher efficiency or for coherent SIM [20,[161][162][163][164].…”