A custom-built two-photon microscope was used for in vivo imaging. Fluorophores were excited and imaged with a water immersion objective (20×, 0.95 NA, Olympus) at 920 nm using a Ti:Sapphire laser (Mai Tai HP, Spectra-Physics). Images were acquired at 16-bit resolution and 4–8 frames/s. The pixel size was 0.6 μm OSN somata imaging and 1.2–2.4 μm for imaging glomeruli. Fields of view ranged from 180 × 180 μm in the epithelium to 720 × 720 μm in the glomerular layer. The point-spread function of the microscope was measured to be 0.51 × 0.48 × 2.12 μm. Image acquisition and scanning were controlled by custom-written software in LabView (National Instruments). Emitted light was routed through two dichroic mirrors (680dcxr, Chroma and FF555- Di02, Semrock) and collected by a photomultiplier tube (R3896, Hamamatsu) using filters in the 500–550 nm range (FF01–525/50, Semrock).
Ff555 di02
Manufactured by IDEX Corporation
The FF555-Di02 is a laboratory instrument designed for precision flow measurement. It is capable of accurately measuring the flow rate of liquids and gases within a specific range. The core function of this device is to provide reliable and consistent flow data for various experimental and analytical applications.
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5 protocols using ff555 di02
1
In Vivo Two-Photon Microscopy for Olfactory Imaging
2
Two-Photon Imaging of Granule Cell Odor Responses
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A custom-built two-photon microscope104 was used for in vivo imaging. Fluorophores were excited and imaged with a water immersion objective (20×, 0.95 NA, Olympus) at 950 nm using a Ti:Sapphire laser (Mai Tai HP, Spectra-Physics). Frame rates were typically 4 Hz. Image acquisition and scanning were controlled by custom-written software in Labview. Emitted light was routed through two dichroic mirrors (680dcxr, Chroma and FF555-Di02, Semrock) and collected by 2 photomultiplier tubes (R3896, Hamamatsu) using filters in the 500–550 nm range (green channel, FF01-525/50, Semrock) and 572–642 nm range (red channel, FF01-607/70, Semrock).
To identify sparsely labeled granule cells that allow imaging of both their soma and dendrites the laser was tuned to 1020 nm and the red channel was used to record a detailed z-stack with 1 μm step size. The entire dendritic tree of a given GC was then examined to ensure that it could be clearly separated from processes of neighboring cells and faithfully traced to its corresponding soma. After choosing one imaging plane containing the soma and one imaging plane containing several branches of the same dendrite the laser was tuned back to 950 nm for imaging odor-evoked activity in either plane.
To identify sparsely labeled granule cells that allow imaging of both their soma and dendrites the laser was tuned to 1020 nm and the red channel was used to record a detailed z-stack with 1 μm step size. The entire dendritic tree of a given GC was then examined to ensure that it could be clearly separated from processes of neighboring cells and faithfully traced to its corresponding soma. After choosing one imaging plane containing the soma and one imaging plane containing several branches of the same dendrite the laser was tuned back to 950 nm for imaging odor-evoked activity in either plane.
3
In Vivo Two-Photon Imaging of Olfactory System
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A custom-built two-photon microscope was used for in vivo imaging. Fluorophores were excited and imaged with a water immersion objective (20X, 0.95 NA, Olympus) at 920 nm using a Ti:Sapphire laser with dispersion compensation (Mai Tai HP, Spectra-Physics). Images were acquired at 16-bit resolution and 4–8 frames/s. The pixel size was 0.6 μm for OSN somata and axon imaging. Fields of view ranged from 180 × 180 μm in the epithelium to 720 × 720 μm in the OB. The point-spread function of the microscope was measured to be 0.51 × 0.48 × 2.12 μm. Image acquisition and scanning were controlled by custom-written software in LabView (National Instruments). Emitted light was routed through two dichroic mirrors (680dcxr, Chroma, and FF555- Di02, Semrock) and collected by a photomultiplier tube (R3896, Hamamatsu) using filters in the 500–550 nm range (FF01–525/50, Semrock).
4
Two-Photon Microscopy for In Vivo Imaging
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A custom-built two-photon microscope was used for in vivo imaging. Fluorophores were excited and imaged with a water immersion objective (20× , 0.95 NA, Olympus) at 920 nm using a Ti:Sapphire laser (Mai Tai HP, Spectra-Physics). Images were acquired at 16-bit resolution and 4 frames/s. The pixel size was 1.218 μm, and fields of view were typically 365 × 365 μm. The point-spread function of the microscope was measured to be 0.51 × 0.48 × 2.12 μm. Image acquisition and scanning were controlled by custom-written software in Labview. Emitted light was routed through two dichroic mirrors (680dcxr, Chroma and FF555- Di02, Semrock) and collected by a photomultiplier tube (R3896, Hamamatsu) using filters in the 500–550 nm range (FF01–525/50, Semrock).
5
In Vivo Two-Photon Microscopy Imaging
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A custom-built two-photon microscope (Wienisch et al., 2011 (link)) was used for in vivo imaging. Fluorophores were excited and imaged with a water immersion objective (20x, 0.95 NA, Olympus) at 950 nm using a Ti:Sapphire laser (Mai Tai HP, Spectra-Physics). The point spread function of the microscope was measured to be 0.66 × 0.66×2.26 µm. Image acquisition and scanning were controlled by custom-written software in Labview. Emitted light was routed through two dichroic mirrors (680dcxr, Chroma and FF555- Di02, Semrock) and collected by two photomultiplier tubes (R3896, Hamamatsu) using filters in the 500–550 nm range (green channel, FF01-525/50, Semrock) and 572–642 nm range (red channel, FF01-607/70, Semrock). Fields of view were 75 × 75 µm square spanning 800 × 800 pixels. Z-stacks of approximately 30 µm depth with a 1 µm z step for both channels (16 bit) were taken every 3 min (0.5 Hz frame rate with 3x averaging during acquisition) for periods of 30–90 min. Two or three fields of view were imaged in each mouse.
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