Raman spectroscopic analysis was performed with a CRM 300 WITec micro-Raman setup (WITec, Ulm) equipped with a 600 lines/mm grating. A frequency-doubled cw Nd-YAG laser beam with an excitation wavelength of 532 nm and a power of 15 mW before passing the objective was focused on the sample via a 60× Nikon water immersion objective with NA 1.0. The 180° backscattered light was detected by a back illuminated CCD camera (DV401 BV, Andor Technology Ltd, Belfast) with 1024 × 127 pixels cooled to −60°C.
Water immersion objective
Manufactured by Nikon
Sourced in Japan
The 16x water immersion objective is a high-magnification lens designed for microscopy applications. It provides a magnification of 16x and is optimized for use with water-based samples. The objective is constructed to minimize optical distortions and deliver high-quality images.
Automatically generated - may contain errors
Lab products found in correlation
Aavretro h2b mcherry, by IDEX Corporation (2 mentions)
A1r two photon microscope system, by Nikon (2 mentions)
Maitai deepsee, by Spectra-Physics (2 mentions)
Two photon laser scanning microscope, by Thorlabs (2 mentions)
Physiological monitoring system, by Harvard Apparatus (2 mentions)
Dv401 bv, by Oxford Instruments (1 mentions)
Confocal raman microscope alpha300r, by WITec (1 mentions)
Du 970 n bv353, by Oxford Instruments (1 mentions)
Analog to digital converter, by National Instruments (1 mentions)
Two photon 8 khz resonant scanner, by Bruker (1 mentions)
Sp8x confocal microscope, by Leica (1 mentions)
Investigator 2 photon microscope, by Bruker (1 mentions)
Gaasp pmt, by Hamamatsu Photonics (1 mentions)
Orca flash 4, by Hamamatsu Photonics (1 mentions)
Eclipse te2000 e, by Nikon (1 mentions)
Ti e microscope, by Nikon (1 mentions)
A1 confocal microscope, by Nikon (1 mentions)
Eclipse ti, by Nikon (1 mentions)
Oil immersion objective, by Nikon (1 mentions)
Plan apochromat objective, by GE Healthcare (1 mentions)
32 protocols using water immersion objective
1
Micro-Raman Spectroscopy Protocol
2
Two-Photon Imaging of BA Neuron Activity
3
Two-photon Calcium Imaging in Mice
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A commercial Nikon A1R two-photon microscope system was used to perform two-photon calcium imaging. Two-photon excitation beam was emitted by a mode-locked Ti: Sa laser (model “Mai-Tai Deep See”, Spectra Physics). We utilized a water-immersion objective (Nikon) with 25 ×/1.10NA to perform imaging. The excitation wavelength was set to 910 nm for Gcamp6f calcium imaging experiments. For somatic imaging, the dimension of field-of-view (FOV) was set 200 μm × 200 μm. We acquired images of 512 × 512 pixels at 30-Hz frame rate. The average power reaching the cortical surface ranged from 30 to 40 mW, depending on the expression efficiency of virus and depth of imaging. Within an imaging time window of ∼3 min (∼1 min per time, three times in total) for each imaging, no sign of photo-damage was observed. Before the experiment, the mice were fixed under microscope objective frequently with the chamber to adapt to the imaging state for real calcium activity.
4
Two-photon Imaging of Mouse Brain
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A Ti:Sapphire 2-photon laser scanning microscope was used with a Nikon water-immersion objective (25x, 1,10 NA) and Nikon NIS Elements AR 4.20.03 (Build 995; Düsseldorf, Germany). Imaging was performed under isoflurane anesthesia (1.5%, flow ∼800 mL/min). The mouse was put onto a heating blanket and rectal temperature was kept constant at 37°C. All images were taken using 920 nm wavelength for EGFP and RFP. An overview stack (x, y, z: 522 × 522 × 75 μm; 1 μm z-step size; pixel size, 1.02 μm/pixel) was taken for orientation, before areas of interest were randomly chosen. Z stacks for each region of interest (x, y, z: 155 × 155 × 70 μm) with a pixel size of 0.3 μm/pixel and a z-spacing of 0.5 μm were acquired. For time-lapse recordings, images were taken every 30 s.
5
Two-Photon Calcium Imaging Setup and Procedure
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A commercial Nikon A1R two-photon microscope system was used to perform two-photon calcium imaging. Two-photon excitation beam was emitted by a mode-locked Ti: Sa laser (model “Mai-Tai Deep See”, Spectra Physics). We utilized a water-immersion objective (Nikon) with 25 ×/1.10NA to perform imaging. The excitation wavelength was set to 910 nm for Gcamp6f calcium imaging experiments. For somatic imaging, the dimension of field-of-view (FOV) was set 200 µm × 200 µm. We acquired images of 512 × 512 pixels at 30-Hz frame rate. The average power reaching the cortical surface ranged from 30 to 40 mW, depending on the expression efficiency of virus and depth of imaging. Within an imaging time window of ~3 min (~1 min per time, three times in total) for each imaging, no sign of photo-damage was observed. Before the experiment, the mice were fixed under microscope objective frequently with the chamber to adapt to the imaging state for real calcium activity.
6
Two-Photon Imaging of BA Neuron Activity
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Two-photon imaging was performed using a resonant-scanning two-photon microscope (Neurolabware) at 15.5 frames/second and 796 × 512 pixels/frame. Imaging was performed with a 16× 0.8 NA water-immersion objective (Nikon) for mice implanted with 1.5 pitch GRIN lens (GRINtech: NEM-050–25-10–860-S-1.5p; see above) or a 4× 0.28 NA air objective (Olympus) for mice implanted with 1.0 pitch 2.6× GRIN lens (GRINtech: NEM-050–25-10–860-DM; see above). Imaging fields of view were at a depth of 100–300 μm below the face of the GRIN lens. A Mai Tai DeepSee laser or InSight X3 laser (Spectra-Phsyics) was used. We imaged using an excitation wavelength of 960 nm for all calcium imaging of BA cell bodies and 940 nm for VTA axon imaging (using pre-chirp compensation for dispersion as much as possible). Laser power ranged between 40–60 mW at the front aperture of the objective (the power at the sample was substantially less because of partial transmission via the GRIN lens). GCaMP6 signals were collected using a green emission filter (510/84 nm; Semrock). For all BA neuron imaging, a prior injection of AAVretro-H2b-mCherry in NAc allowed red labeling of nuclei of NAc-projecting BA neurons (red emission filter: 607/70 nm; Semrock). We collected both green and red light using PMTs (H10770B-40; Hamamatsu).
7
Raman Imaging of Biological Samples
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WITec Confocal Raman Microscope Alpha 300 R (WITec Inc., Ulm, Germany) is used to collect Raman spectra. a frequency-doubled Nd:YAG laser (Newport, Evry, France) with 532 nm wavelength and 50 mW power provided sample excitation. A 60x NIKON water immersion objective (numerical aperture of NA = 1.0.) focused laser beam on PBS immerged cells. an electron-multiplying charge-coupled device (EMCCD) camera (DU 970 N-BV353, Andor, Hartford, USA) captured the scattered signals. Using the formula rlateral = 1.22·λlaser/2·NA gives the spatial resolution of the system 325 nm. For the axial resolution, raxial = 1.4·λlaser·n/NA2 (where n is the index of refraction 1.33 for the water-based objective) gives 991 nm. WITec Image Plus software performed data acquisition and processing. Calcium fluoride (CaF2) substrate was employed due to its characteristic Raman peak at 320 cm−1 to avoid extra Raman signal interfering with cells signature. Each Raman scan contains more than twenty thousand single spectra. The recorded Raman spectra collected from each voxel (300 nm × 300 nm × 900 nm) contain the sample biochemical fingerprint under the laser spot of 1 µm.
8
In Vivo Calcium Imaging of Hippocampal Neurons
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In vivo calcium imaging movies were acquired with a custom two‐photon laser scanning microscope (based on Thorlabs Bergamo) using a femtosecond‐pulsed laser (Coherent Fidelity 2, 1070 nm) and a 16x water immersion objective (0.8 NA, Nikon). Imaging sessions were started 3–4 weeks after surgery to allow for recovery and for optimal viral expression. During imaging sessions, mice were head‐fixed to the microscope with a titanium headbar and the microscope stage was adjusted so that the hippocampal window was perpendicular to the axis of the objective for optimal imaging conditions. Mice were awake and walking on a manually rotated treadmill belt throughout imaging. The treadmill contained four zones with different textures as previously described (Jordan et al., 2021 (link)). Ten‐minute videos were acquired at 15.253 fps with a 343.6 × 343.6 μm field of view. Treadmill data were acquired using National Instruments analog‐to‐digital converter and synchronized with the imaging data using Thorsync software. To track the same field of view, the initial location was noted on the first imaging day using a coordinate system and by taking an image of the field. During following sessions, the coordinates and initial field image were used to match as close as possible to the same field.
9
Two-Photon Calcium Imaging of Hippocampal Neurons
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In vivo calcium imaging movies were acquired with a custom two-photon laser scanning microscope (based on Thorlabs Bergamo) using a femtosecond-pulsed laser (Coherent Fidelity 2, 1070 nm) and a 16x water immersion objective (0.8 NA, Nikon). Imaging sessions were started 3–4 weeks after surgery to allow for recovery and for optimal viral expression. During imaging sessions, mice were head-fixed to the microscope with a titanium headbar and the microscope stage was adjusted so that the hippocampal window was perpendicular to the axis of the objective for optimal imaging conditions. Mice were awake and walking on a manually rotated treadmill belt throughout imaging. The treadmill contained four zones with different textures as previously described (52 (link)). Ten-minute videos were acquired at 15.253 fps with a 343.6 × 343.6 μm field of view. Treadmill data was acquired using National Instruments analog-to-digital converter and synchronized with the imaging data using Thorsync software. To track the same field of view, the initial location was noted on the first imaging day using a coordinate system and by taking an image of the field. During following sessions, the coordinates and initial field image were used to match as close as possible to the same field.
10
Two-Photon Imaging of Neuronal Structures
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Single- and dual-color imaging of PNs were performed at room temperature using a custom-built two-photon microscope (Prairie Technologies) with a Chameleon Ti:Sapphire laser (Coherent) and a 16 X water-immersion objective (0.8 NA; Nikon). Excitation wavelength was set at 920 nm for GFP imaging, and at 935 nm for co-imaging of mGreenLantern and JF570-Halo. z-stacks were obtained at 4 µm increments (10 µm increments for Figure 5—video 1 ). Images were acquired at a resolution of 1024 × 1024 pixel2 (512 × 512 for Figure 5—video 1 ), with a pixel dwell time of 6.8 µs and an optical zoom of 2.1, and at a frequency every 20 min for 8–23 hr.
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