Wide-field imaging was performed on adult OMP-GCaMP3 mice (Isogai et al., 2011 (link)) as described previously (Rokni et al. 2014 (link)) using a 10X objective (Olympus, NA 0.3). Blue light from an LED (M470L3, Thorlabs) was used for excitation, and the emitted light was filtered (500–550nmMDFGFP, Thorlabs) and collected with a CMOS camera (DMK 23U274, The Imaging Source GmbH). Odors were delivered using a homemade automated olfactometer (Rokni et al., 2014 (link)). All odorants were diluted to 30% v/v in diethyl phthalate and then further diluted 16 fold in air. The 16 odors and their mixtures were presented in a random order for 2 seconds (inter-stimulus interval of 45 seconds).
Dmk 23u274
Manufactured by Imaging Source
The DMK 23U274 is a USB3 uEye industrial camera produced by Imaging Source. It features a CMOS sensor with a resolution of 2.3 megapixels and captures images at a frame rate of up to 45 frames per second. The camera supports various image formats and has a USB3 interface for data transfer.
Automatically generated - may contain errors
3 protocols using dmk 23u274
1
Wide-field Calcium Imaging of Olfactory Responses
2
Multimodal Imaging Microscopy Setup
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All imaging were carried out using a Nikon Ti‐E setup for both phase contrast and epifluorescence microscopy. The microscope was equipped with 100× CPI Plan Apo Lambda (Nikon). Phase contrast images were acquired using a dmk23u274 (The Imaging Source). Bright‐field and fluorescence images were acquired using a Zyla 4.2 PLUS sCMOS (Andor).
For wide‐field epifluorescence‐based phenotyping, a 300 ms excitation [shuttered using an AOTFnC (AA Opto Electronics)] from a 514‐nm CW‐laser at 415 W/cm2 (Fandango, Cobolt) was used. The laser light was reflected on a zt514.5rdc (Chroma) dichroic before hitting the sample. The Ypet emitted light was transmitted through the above dichroic and filtered through a BrightLine Fluorescence 542/27 (Semrock) before hitting the sCMOS camera. The genotyping and DAPI imaging were carried out using LED white light source (Sola, Lumencore) together with the appropriate filter cubes. Filter cube for Cy3 detection: excitation filter: FF01‐543/22 (Semrock), dichroic mirror: FF562‐Di03 (Semrock), emission filter: FF01‐586/20 (Semrock). Filter cube for Cy5 detection: excitation filter: FF01‐635/18 (Semrock), dichroic mirror: FF652‐Di01 (Semrock), emission filter: FF01‐680/42 (Semrock).
For wide‐field epifluorescence‐based phenotyping, a 300 ms excitation [shuttered using an AOTFnC (AA Opto Electronics)] from a 514‐nm CW‐laser at 415 W/cm2 (Fandango, Cobolt) was used. The laser light was reflected on a zt514.5rdc (Chroma) dichroic before hitting the sample. The Ypet emitted light was transmitted through the above dichroic and filtered through a BrightLine Fluorescence 542/27 (Semrock) before hitting the sCMOS camera. The genotyping and DAPI imaging were carried out using LED white light source (Sola, Lumencore) together with the appropriate filter cubes. Filter cube for Cy3 detection: excitation filter: FF01‐543/22 (Semrock), dichroic mirror: FF562‐Di03 (Semrock), emission filter: FF01‐586/20 (Semrock). Filter cube for Cy5 detection: excitation filter: FF01‐635/18 (Semrock), dichroic mirror: FF652‐Di01 (Semrock), emission filter: FF01‐680/42 (Semrock).
3
Super-resolution Fluorescence Microscopy Setup
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Imaging experiments were performed using Nikon inverted microscope (Ti-Eclipse) system. Four laser lines (MLC 400B, Agilent Technologies, equipped with 405, 488, 561, 640 nm) were combined through single mode optical fiber and focused on the back focal plane of the objective lens (APO 100×, NA 1.49, Nikon) which was used for illumination and collection of photons. Samples were loaded on xyz-translational stage (xy: motorized, z: piezo). Focus was maintained by Nikon perfect focus system (PFS). For 3D measurement a cylindrical lens (fcyl = 10 m) was put in the slide-in port between the objective lens and the tube lens to generate astigmatism.28 (link) Quad-band dichroic mirror (Chroma, ZT405-488-561-640RPC) was used for separating excitation and fluorescence emission, band pass filters were used for each spectrum (447/60, 525/50, 600/50, and 680/40). A back illuminated EMCCD camera (DU897, Andor Technology) was used for recording fluorescence signal. For drift correction, 760 nm LED was used for illuminating the sample from the top and transmitted IR signal was collected by separate IR sensitive CMOS camera (DMK 23U274, The Imaging Source) after reflected by 750 nm long pass dichroic mirror placed above the quad-band dichroic mirror. For image acquisition and device control Nikon NIS Element and IC capture were used.
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