After whole cell recordings, AII or ONα morphology was imaged using two photon microscopy (800 nm Chameleon laser) under a 20X water immersion objective (Olympus XLUMPlanFL N, NA 1.00) for cell identification. Fixed tissues were imaged on a Zeiss LSM 800 laser scanning confocal microscope equipped with a 40X oil immersion objective (Plan-Apochromat, NA 1.3). nNOS, TdTom and ChAT labeling were imaged at 488, 568 and 647 nm excitation, respectively. All confocal images were collected with spacing of 0.5 μm in the z axis.
Xlumplanfl n
Manufactured by Olympus
Sourced in Japan
The XLUMPlanFL N is a high-performance microscope objective lens designed for use in a variety of laboratory applications. It features a long working distance, high numerical aperture, and low distortion. The XLUMPlanFL N is optimized for bright-field and fluorescence microscopy.
Automatically generated - may contain errors
Lab products found in correlation
Movable objective microscope, by Olympus (3 mentions)
Fluorescein conjugated dextran, by Merck Group (3 mentions)
Mai tai hp, by Spectra-Physics (3 mentions)
Bx51wi, by Olympus (2 mentions)
Lsm 800, by Zeiss (1 mentions)
Plan apochromat, by Zeiss (1 mentions)
Matlab, by MathWorks (1 mentions)
Fastcam sa1, by Photron (1 mentions)
Vt1000, by Leica (1 mentions)
Fv1000mpe, by Olympus (1 mentions)
Ad gfp, by Vector Biolabs (1 mentions)
10 protocols using xlumplanfl n
1
Morphological Imaging of AII and ONα Cells
2
Imaging Cerebral Vasculature in Mice
3
Tracking Hair Bundle Dynamics
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Experiments were performed with an upright optical microscope (Olympus BX51WI) mounted on a vibration-isolation table (Technical Manufacturing Corporation) and imaged with a water immersion objective (Olympus XLUMPlanFL N, 20×, 1.00 NA). Images were optically magnified (∼400× total magnification) and recorded with a high-speed CMOS camera (Photron FASTCAM SA1.1) at 1000 frames per second. A hair bundle’s movement was tracked using software written in MATLAB (The MathWorks), and its position was determined by the center of gravity of the bundle’s intensity profile along a row of pixels. This calculation was performed with at least 10 rows of adjacent pixels to enhance the signal-to-noise ratio. The time-dependent position trace of a hair bundle’s motion was acquired by plotting the mean position for each frame of the recording.
4
Imaging Cerebral Vasculature in Mice
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Mice were briefly anesthetized with isoflurane (5% in oxygen) and retro-orbitally injected with 50 μL 5% (weight/volume in saline) fluorescein-conjugated dextran (70 kDa, Sigma-Aldrich, cat.no.: 46945), and then fixed on a spherical treadmill. Imaging was done on a Sutter Movable Objective Microscope with a 20×, 1.0 NA water dipping objective (Olympus, XLUMPlanFLN). A MaiTai HP (Spectra-Physics, Santa Clara, CA) laser tuned to 800 nm was used for fluorophore excitation. All imaging with the water-immersion lens was done with room temperature distilled water between the PoRTS window and the objective. All the 2PLSM measurements were started at least 20 min after isoflurane exposure to reduce the disruption of physiological signals due to anesthetics. High-resolution image stacks of the vasculature were collected across a 500 by 500 μm field and up to a depth of 250 um from the pial surface. All the images were acquired with increasing laser power up to 100 mW at a depth of ~200 μm. Lateral sampling was 0.64 um per pixel and axial sampling was at 1 um steps between frames. Shortly (within 20 min) after the imaging, the mouse was perfused with FITC filling for STPT based ex vivo vasculature imaging.
5
Two-Photon Microscopy of Fluorescent Samples
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Two-photon images (see in the results section) were done using a laser scanning two-photon microscope (Femto2D-galvo, Femtonics, Budapest, Hungary). The microscope contained a 20× water immersion objective lens with 1.00 NA and 2 mm WD (XLUMPlanFL N, Olympus, Evident, Tokyo, Japan). The samples were excited at wavelenths between 830 and 920 nm with a femtosecond-pulsed two-photon laser (Chameleon, Coherent, Santa Clara, CA, USA) and the fluorescence was collected using two GaAsP photomultipliers (PMTs) for green and blue detection (H11706P-40, Hamamatsu, Herrsching am Ammersee, Germany). The figures were made with a MATLAB-based program (MES, Femtonics, Budapest, Hungary).
6
Intravital Imaging of Draining Lymph Nodes
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We used a custom-made multiphoton intravital microscope Ultima Pro Multiphoton Microscopy System, Bruker Technology, WI, United States) equipped with two Mai Tai Titanium-Sapphire lasers (Newport Spectra-Physics, Irvine, United States) for simultaneous coaxial illumination. One of the lasers was set at 850 nm wavelength (to visualize YFP+ cells) or 760 nm wavelength (to visualize DsRed+ cells), and the other laser was set at 900 nm wavelength for second harmonic generation to visualize the dLN capsule in a completely separated channel, a convenient tool to analyze the anatomical distribution of the cells within the dLN. Image acquisition was performed using Prairie View Software (Bruker Technology). A 20× water immersion objective (NA: 0.95, Olympus, XLUMPlanFL N, Tokyo, Japan) was used to capture images of the mandibular dLNs with volumes of 70 × 596 × 596 μm at 3 μm Z-steps with 30 s intervals over 30 min.
7
Visualizing GFP-labeled Organoids
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GFP-labeled organoid was prepared by labeling day 35 organoids with a 1/500 dilution of human adenovirus type 5 expressing eGFP under control of a CMV promoter (Vector Biolabs; Ad-GFP). Three days after viral labeling, organoids were fixed and SHIELD-processed in supernatant of 2% P3PE solution, (as described above) but without clearing. Organoids were then embedded in low melt agarose and sliced at 200 µm thickness by vibratome (VT1000S, Leica Biosystems, Germany). Organoid slices were subject to passive clearing using the 0.2 M SDS, 50 mM phosphate (pH7.3) clearing buffer at 37C for 24 h and extensively washed by PBST. Fluorescence in situ hybridization-hybridization chain reaction (FISH-HCR) of GFP was performed as described before26 (link) using 50nt GFP probes and B1 Alexa 647 hairpin. FISH-stained sample was imaged by an Olympus confocal microscope (FV1000MPE) with 20X 1.0 NA water objective (XLUMPlanFL N).
8
In vivo Calcium Imaging of Neural Responses
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In vivo calcium imaging recordings were done with an epifluorescent microscope (Olympus BX51WI) equipped with a 20x (NA 1.00) water immersion objective (Olympus XLUMPlanFLN). Images were acquired by a 512x512 pixel 16-bit electron-multiplying CCD camera (Photometrics Evolve 512). The preparation was alternately excited with 340 and 380 nm monochromatic light (TILL Photonics Polychrome V), and data were acquired ratiometrically. A dichroic mirror (420nm) together with an emission filter (490-530nm) was used to separate the excitation and emission light. We used 2x2 binning on chip (pixel size ~1x1μm), and each recording consisted of 150 double frames at a sampling frequency of 10 Hz with 25–35 ms and 10–15 ms exposure times for light of the two respective wavelengths. Each recording was of 15 s duration, started 3 s prior to onset of the odor stimulus, and the time interval between each recording was 60 s.
9
Two-Photon Microscopy for Fluorescence Imaging
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Two‐photon microscopic images were taken using a laser scanning two‐photon microscope (Femto2D‐galvo, Femtonics). The microscope contained a 20× water immersion objective lens with 1.00 NA and 2 mm WD (XLUMPlanFL N, Olympus). The samples were excited between 830 and 920 nm wavelength with a femtosecond‐pulsed two‐photon laser (Chameleon, Coherent) and the fluorescence was collected using two GaAsP photomultipliers (PMTs) for green and blue detection (H11706P‐40, Hamamatsu). The figures (Figures 7 and 8 ) were made with a MATLAB‐based program (MES, Femtonics).
10
In Vivo Vasculature Imaging of Mice
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Mice were briefly anesthetized with isoflurane (5% in oxygen) and retro-orbitally injected with 50 µL 5% (weight/volume in saline) fluorescein-conjugated dextran (70 kDa, Sigma-Aldrich, cat.no.: 46945), and then fixed on a spherical treadmill. Imaging was done on a Sutter Movable Objective Microscope with a 20X, 1.0 NA water dipping objective (Olympus, XLUMPlanFLN). A MaiTai HP (Spectra-Physics, Santa Clara, CA) laser tuned to 800 nm was used for fluorophore excitation. All imaging with the water-immersion lens was done with room temperature distilled water between the PoRTS window and the objective. All the 2PLSM measurements were started at least 20 minutes after isoflurane exposure to reduce the disruption of physiological signals due to anesthetics. High-resolution image stacks of the vasculature were collected across a 500 by 500 µm field and up to a depth of 250 um from the pial surface. All the images were acquired with increasing laser power up to 100 mW at a depth of ~200 µm. Lateral sampling was 0.64 um per pixel and axial sampling was at 1 um steps between frames. Shortly (within 20 minutes) after the imaging, the mouse was perfused with FITC filling for STPT based ex vivo vasculature imaging.
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