This review presents a selection of advanced microscope designs employing acousto-optical deflectors (AODs). In the designs covered, AODs are used as tunable diffraction gratings to control complex illumination patterns at the Fourier plane of an objective lens. This approach allows us to generate desired illumination patterns at the focal plane of a light microscope. In simple terms, I will describe two established designs, the 3D Random-Access Multi-Photon Microscope and the Standing-Wave Super-Resolution Microscope, as well as two new schemes including the Random-Access STED Microscope and the Frequency-Encoded Multi-Beam Microscope. All instruments mentioned here were designed to overcome the throughput limitations of previously used light microscopes in experimental Neuroscience.
Proc. SPIE. 10069, Multiphoton Microscopy in the Biomedical Sciences XVII
KEYWORDS: Signal to noise ratio, Surface plasmons, Multiphoton microscopy, Signal attenuation, Microscopy, Luminescence, Calcium, Functional imaging, In vivo imaging, Deep tissue imaging, Neuroimaging, Neurons, Brain imaging, Brain
We demonstrate that three-photon microscopy (3PM) with 1300-nm excitation enables functional imaging of GCaMP6s labeled neurons beyond the depth limit of two-photon microscopy (2PM) with 920-nm excitation. We quantitatively compared 2PM and 3PM imaging of calcium indicator GCaMP6s by measuring correlation between activity traces, absolute signal level, excitation attenuation with depth, and signal-to-background ratio (SBR). Compared to 2PM imaging of GCaMP6s-labeled neurons, 3PM imaging has increasingly larger advantages in signal strength and SBR as the imaging depth increases in densely labeled mouse brain, given the same pulse energy, pulse width, and repetition rate at the sample surface. For example, 3PM has comparable signal strength as 2PM and up to two orders of magnitude higher SBR as 2PM in mouse cortex around 700-800um. We also demonstrate 3PM activity recording of 150 neurons in the hippocampal stratum pyramidale (SP) at 1mm depth, which is inaccessible to non-invasive 2PM imaging. Our work establishes 3PM as a powerful tool for calcium imaging at the depth beyond 2PM limits.
Optogenetic methods developed over the past decade enable unprecedented optical activation and silencing of specific neuronal cell types. However, light scattering in neural tissue precludes illuminating areas deep within the brain via free-space optics; this has impeded employing optogenetics universally. Here, we report an approach surmounting this significant limitation. We realize implantable, ultranarrow, silicon-based photonic probes enabling the delivery of complex illumination patterns deep within brain tissue. Our approach combines methods from integrated nanophotonics and microelectromechanical systems, to yield photonic probes that are robust, scalable, and readily producible en masse. Their minute cross sections minimize tissue displacement upon probe implantation. We functionally validate one probe design in vivo with mice expressing channelrhodopsin-2. Highly local optogenetic neural activation is demonstrated by recording the induced response—both by extracellular electrical recordings in the hippocampus and by two-photon functional imaging in the cortex of mice coexpressing GCaMP6.