remote path  (Unicode Inc)

Journal: Nature Communications

Article Title: Fast and light-efficient remote focusing for volumetric voltage imaging

doi: 10.1038/s41467-024-53685-5

Figure Lengend Snippet: a Conceptual layout of our proposed design. Current polarizing beamsplitter based designs (left) lose half of the light intensity because unpolarized fluorescence is split into a beam dump and the remote focusing objective. Our design (right) uses half the available FOV for the incoming light and a retroreflector in the image plane of the remote objective to fold the image to the other side of the FOV. A knife edge mirror in the primary image plane is then able to direct the refocused light onto the camera. b Light path at the level of the object, retroreflector and image of two point sources located at different z-depths. Moving the retroreflector along the optical axis brings either of them in focus. c Picture of the microscopic retroreflector (R) together with the coverslip (C) and parts of the remote objective (RO). d Layout of the entire microscope for volumetric voltage imaging. A light sheet is generated with a cylindrical lens (CL) and projected onto the sample via a folding mirror (FM), galvo mirror (G), scan and tube lens (SL, TL) and illumination objective (IO). The galvo mirror controls the z-position of the light sheet. Fluorescence is collected via the primary imaging objective (PO) and imaged onto the knife edge mirror (KM) in the primary image plane and directed into the remote focusing path. The remote focusing path consists of a tube lens, remote objective (RO) and voice coil (VC) on which the retroreflector is mounted. After refocusing the image is relayed with a 1:1 relay (RL) onto the camera (C).

Article Snippet: Detection and remote focusing path: Fluorescence was detected with a 16×0.8 NA water immersion objective (Nikon LWD 16x, NA 0.8) and filtered with a longpass filter to reject light from the excitation laser (Chroma ET542LP).

Techniques: Fluorescence, Microscopy, Imaging, Generated

Journal: Nature Communications

Article Title: Fast and light-efficient remote focusing for volumetric voltage imaging

doi: 10.1038/s41467-024-53685-5

Figure Lengend Snippet: a Schematic of the pipeline for volumetric voltage imaging data acquisition and signal extraction. 8 frames are acquired during the downward and another 8 during the upward stroke of the refocusing cycle. Frames at each z-position are then motion corrected in x-y and the time-average frame for each z-position is aligned with frames above and below to generate one single volume (see methods for details). This volume is then used for 3D segmentation. The obtained 3D ROIs are then used as the basis for the extraction of the fluorescent time traces (see methods for details). b Average fluorescence of 8 planes spanning the entire volume of 50 μm of spinal cord tissue of a 4 dpf zebrafish larva. animals are expressing UAS:Voltron2-ST under the control of HuC:Gal4 and are stained with JF-526 dye. Footprints of select neurons shown in ( c ). Most footprints are visible over several z-sections but numbered only once. Shown is a representative example of an experiment with 4 fish. c Fluorescent traces corresponding to the spatial footprints shown in b (z-scored), ordered by z-position. Several neurons show clear single spikes while others show distinct oscillations likely corresponding to fictive swimming activity. Color of footprints in b and traces in c denote z-position in the volume. d Average bleaching of baseline fluorescence in recordings from 3 fish (n cells per fish = 28-114). e Closeup of several spikes of one example neuron (trace 6 in c) at the beginning (0 s –2.5 s) and end (18.5 s – 20 s) of the recording. Visible is the fast timing of individual spikes and only a small reduction in SNR at the end of the recording.

Article Snippet: Detection and remote focusing path: Fluorescence was detected with a 16×0.8 NA water immersion objective (Nikon LWD 16x, NA 0.8) and filtered with a longpass filter to reject light from the excitation laser (Chroma ET542LP).

Techniques: Imaging, Extraction, Fluorescence, Expressing, Control, Staining, Activity Assay