0.8 na objective

Manufactured by Nikon

Sourced in Japan

The 16× 0.8 NA objective is a high-magnification objective lens designed for use in Nikon's laboratory equipment. It provides a 16x magnification and a numerical aperture (NA) of 0.8, which is a measure of the lens's ability to gather light and produce a high-resolution image. This objective is suitable for a wide range of microscopy applications that require detailed observation and analysis.

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Lab products found in correlation

Tissuecyte 1000, by Spectra-Physics (2 mentions) Gaasp photomultiplier tube, by Hamamatsu Photonics (1 mentions) Two photon microscope, by Sutter Instruments (1 mentions) Texas red conjugated dextran, by Merck Group (1 mentions) Ti sapphire laser, by Spectra-Physics (1 mentions) Timeharp 260, by PicoQuant (1 mentions) Bergamo two photon microscope, by Thorlabs (1 mentions) Bergamo 2, by Thorlabs (1 mentions) Deepsee, by Spectra-Physics (1 mentions) B scope, by Thorlabs (1 mentions) Maitai hp laser, by Spectra-Physics (1 mentions) Pmt2101, by Thorlabs (1 mentions) Ni pcie 6323, by National Instruments (1 mentions)

12 protocols using 0.8 na objective

Two-Photon Imaging in Anesthetized Mice

Cited 4 times

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Two-photon imaging in anesthetized mice was performed as described previously (Wachowiak et al., 2013 (link); Economo et al., 2016 (link)). Mice were initially anesthetized with pentobarbital (50–90 mg/kg) then maintained under isoflurane (0.5–1% in O₂) for data collection. Body temperature and heart rate were maintained at 37°C and ~400 beats per minute. Mice were double tracheotomized and isoflurane was delivered passively via the tracheotomy tube without contaminating the nasal cavity (Eiting and Wachowiak, 2018 (link)). Two-photon imaging occurred after removal of the bone overlying the dorsal olfactory bulb.
Imaging was carried out with a two-photon microscope (Sutter Instruments or Neurolabware) coupled to a pulsed Ti:Sapphire laser (Mai Tai HP, Spectra-Physics; or Chameleon Ultra, Coherent) at 920–940 nm and controlled by either Scanimage (Vidrio) or Scanbox (Neurolabware) software. Imaging was performed through a 16×, 0.8 N.A. objective (Nikon) and emitted light detected with GaAsP photomultiplier tubes (Hamamatsu). Fluorescence images were acquired using unidirectional resonance scanning at 15.2 or 15.5 Hz. For dual-color imaging, a second laser (Fidelity-2; Coherent) was utilized to optimally excite jRGECO1a (at 1,070 nm) and emitted red fluorescence collected with a second PMT, as described previously (Short and Wachowiak, 2019 (link); Moran et al., 2021 (link)).

Moran A.K., Eiting T.P, & Wachowiak M. (2021). Circuit Contributions to Sensory-Driven Glutamatergic Drive of Olfactory Bulb Mitral and Tufted Cells During Odorant Inhalation. Frontiers in Neural Circuits, 15, 779056.

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Non-invasive Detection of Cerebellum GFP

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To directly detect the GFP fluorescent signal ex vivo (without previous immunostaining) and perform high-speed imaging of the entire cerebellum, a dose of 7×10¹¹g.c. of exo-AAV9-CBA-GFP (self-complentary) was injected into BALB/c mice. After 3 weeks, the mice were perfused with 50ml of PBS (10mM phosphate buffer) and 50 ml of 4% PFA at a rate of 9ml/min, before injecting a solution of 2% gelatin (SIGMA G-1890) added with 0.02% of Dextran, Texas Red 75kDa in order to visualize the brain vasculature. The entire mice were then incubated for 15 minutes in ice for dissecting the brain and storing it in 4%PFA.
The brain was embedded in a 4.5% oxidized agarose block and was subsequently placed in a solution of 0.5% sodium borohydride in 0.5 M sodium borate buffer to covalently link the brain to the agarose (27 (link)). Imaging was achieved with the TissueCyte 1000 coupled to a Spectra Physics Mai-Tai HP operating at 920nm. A Nikon 16× 0.8 NA objective was used and in the XY plane sampling was 1.2 microns. A total of 852 optical planes with 5 micron spacing were taken to image the entire cerebellum. The emitted fluorescent light was separated into three spectral channels in order to isolate the Texas Red and GFP signals.

Hudry E., Martin C., Gandhi S., György B., Scheffer D.I., Mu D., Merkel S.F., Mingozzi F., Fitzpatrick Z., Dimant H., Masek M., Ragan T., Tan S., Brisson A.R., Ramirez S.H., Hyman B.T, & Maguire C.A. (2016). Exosome-associated AAV vector as a robust and convenient neuroscience tool. Gene therapy, 23(4), 380-392.

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In Vivo Imaging of Arteriolar Dynamics

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Prior to imaging, mice were briefly anesthetized with isoflurane and retro-orbitally injected with 50 µL of 2.5% w/v of Texas-red conjugated dextran (40 kDa; Sigma-Aldrich), then head-fixed upon a spherical treadmill. The treadmill was coated with a slip-resistant tape and connected to a rotary encoder (US Digital, E7PD-720-118) to monitor changes in velocity of the treadmill. The changes in velocity (acceleration) were used to identify periods of rest and motion. Images were collected under a Sutter moveable objective microscope with either a 16 × 0.8 NA objective or a 20 × 1.0 NA objective (Nikon). A MaiTai HP laser tuned to 920 nm was used to excite the YFP and the Texas-Red. The power exiting the objective was between 30 and 70 mW. Arteries were visually identified by their more rapid blood flow, rapid temporal dynamics of their response to locomotion, and vasomotion [51 (link), 72 (link), 73 (link)]. A two-channel photomultiplier setup was used to collect fluorescence from YFP and Texas-red. Images were collected at a nominal frame rate of 3–8 Hz. All the data was collected at a depth of 30–120 µm below the pial surface, and none of the arterioles imaged bifurcated before the depth at which the measurements were made.

Kedarasetti R.T., Turner K.L., Echagarruga C., Gluckman B.J., Drew P.J, & Costanzo F. (2020). Functional hyperemia drives fluid exchange in the paravascular space. Fluids and Barriers of the CNS, 17, 52.

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Two-Photon Imaging of Mouse Brain

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Imaging experiments were performed through a 16×/0.8 NA objective (Nikon) using a Sutter MOM microscope equipped with the Resonant Scan box module. A Ti:Sapphire laser tuned to 930 nm (Mai Tai, Spectra Physics) was raster scanned using a resonant scanning galvanometer (8 kHz, Cambridge Technologies) and images were collected at 512 × 512 pixel resolution over FOVs of 670 μm × 670 μm at 30 Hz. Sample plane power used for recordings ranged from 30 to 70 mW and recordings were performed midway between the pial surface and the Purkinje cell body layer, at depths of ~75 μm. The microscope was controlled using ScanImage (v.2015, Vidrio Technologies) and tilted to ~10° so that the objective was orthogonal to the surface of the brain and coverglass. Blood vessel landmarks were used to approximately find the same FOV across imaging sessions and fine scale adjustments were made to maximize day-to-day overlap by taking short imaging movies (10 s) and aligning them to the previous day’s recordings.

Kostadinov D., Beau M., Pozo M.B, & Häusser M. (2019). Predictive and reactive reward signals conveyed by climbing fiber inputs to cerebellar Purkinje cells. Nature neuroscience, 22(6), 950-962.

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Two-Photon FLIM Interaction Measurement

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The interaction between GFP::ACT-1 and RHGF-1::tagRFP was measured in day 1 adults using two-photon FLIM. Briefly, GFP was excited with a Ti:sapphire laser (Chameleon, Coherent) at a wavelength of 920 nm and a power of 1.0–2.0 mW. Fluorescence lifetime images were obtained using a Bergamo two-photon microscope (Thorlabs) equipped with a Time-Correlated Single Photon Counting board (Time Harp 260, Picoquant). Emission was collected with a 16 × 0.8 NA objective (Nikon), divided with a 565-nm dichroic mirror (Chroma) and detected with two PhotoMultiplier Tubes with low transfer time spread (H7422-40p, Hamamatsu). Images were collected by 128 × 128 pixels and acquired at 2 ms/line, averaged over 24 frames. The fluorescence lifetime of GFP was measured by curve fitting using custom software written with C# as described previously (Laviv et al., 2020 (link)).

Avivi Kela S., Sethi K., Tan P.Y., Suresh D., Ong H.T., Castaneda P.G., Amin M.R., Laviv T., Cram E.J., Faix J, & Zaidel-Bar R. (2022). Tension-dependent RHGF-1 recruitment to stress fibers drives robust spermathecal tissue contraction. The Journal of Cell Biology, 222(2), e202203105.

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In vivo Two-Photon Calcium Imaging

Cited 3 times

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In vivo two-photon calcium imaging^{95 (link)} was performed with a customized commercially available Bergamo II (Thorlabs) two-photon laser scanning microscope^{96 (link)} using a pulsed femtosecond Ti:Sapphire laser (Mai Tai HP Deep See, Spectra-Physics) and controlled by ScanImage 4 (ref. ^{97 (link)}). The calcium indicator GCaMP6m^{98 (link)} and the structural marker mRuby2 (ref. ^{99 (link)}) were both excited with a wavelength of 940 nm. Emitted photons were filtered for reflected laser light (720/25 short-pass filter), spectrally separated using a dichroic beamsplitter (FF560) and two band-pass filters (500–550 nm for GCaMP6m; 572–642 nm for mRuby2) and detected using two GaAsP photomultiplier tubes. Laser power was kept between 18 and 35 mW, depending on the depth of imaging and the quality of the chronic window. Images were acquired from two alternating planes, 40 μm apart, using a ×16 0.8-NA objective (Nikon) mounted on a piezoelectric stepper (Physik Instrumente). The xy image dimensions were 325 × 250 μm (512 × 512 pixels), and each image plane was acquired at a rate of ~15 Hz (total frame rate of ~30 Hz).

Goltstein P.M., Reinert S., Bonhoeffer T, & Hübener M. (2021). Mouse visual cortex areas represent perceptual and semantic features of learned visual categories. Nature Neuroscience, 24(10), 1441-1451.

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Two-photon Imaging of Calcium Dynamics

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Two-photon imaging was conducted for a head-fixed awake mouse through a 16 × 0.8 NA objective (Nikon) mounted on a commercial two-photon microscope (B-scope, Thorlabs) and using a 925-nm laser (Ti:sapphire laser, Newport). Images were acquired at ~29 Hz and a resolution of 512 × 512 pixels, covering either 944 × 1016 μm (Fig 5b) or 189 × 203 μm (Fig 5c). Acquired images were motion corrected offline. For quantification of Ca²⁺ signals from cell bodies, fluorescence time course of each cellular ROI and its surrounding neuropil ROI was extracted using Suite2P package. Then fluorescence signal of a cell body was estimated with F_cellbody = F_cellROI − 0.7 * F_neuropilROI as described previously.[28 (link)] ΔF/F was computed as (F_cellbody − F₀)/F₀, where F₀ is the 8th percentile of the intensity distribution during 5 min recording session. For quantification of Ca²⁺ signals at the depth of 50 μm and 250 μm, we drew ROIs along the edges of each electrode.

Lu Y., Liu X., Hattori R., Ren C., Zhang X., Komiyama T, & Kuzum D. (2018). Ultra-low Impedance Graphene Microelectrodes with High Optical Transparency for Simultaneous Deep 2-photon Imaging in Transgenic Mice. Advanced functional materials, 28(31), 1800002.

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Large Animal 2P Microscope Design

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The 2P microscope for imaging swine was designed with four key specifications: 1) ability to accommodate large animals with considerable flexibility, 2) intensity and fluorescence lifetime imaging without switching detection hardware and scan path, 3) externally triggered high speed volumetric scanning, and 4) low-cost construction from off-the-shelf parts. The overall design of the microscope was based heavily on the previously published open source TIMAHC (Two-photon Imaging that is Modular, Adaptable, High-performance and Cost-effective) design^{14 (link)} with exceptions detailed below. All 2P imaging was performed using excitation light from a MaiTai HP laser (Spectra Physics, Milpitas, CA) 16× , 0.8 N.A. objective (Nikon Instruments, Tokyo, Japan). Emission light was filtered through 440/80 (cyan fluorescent protein and the chloride-sensitive organic dye), 525/50 (YFP and GFP), or 605/70 (TurboRFP and tdTomato, Chroma Technology, Bellows Falls, VT).

Costine-Bartell B.A., Martinez-Ramirez L., Normoyle K., Stinson T., Staley K.J, & Lillis K.P. (2023). 2-Photon imaging of fluorescent proteins in living swine. Scientific Reports, 13, 14158.

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Two-Photon Microscopy Imaging Protocol

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Images were collected with a resonant scanning two-photon microscope from Neurolabware, together with acquisition software by Scanbox. The scan rate was set to a 15 Hz frame rate. We used a Chameleon Ultra laser set to 920 nm. The beam size was adjusted to slightly overfill the back aperture of the ×16, 0.8 NA objective (Nikon).

Chen Y., Ko H., Zemelman B.V., Seidemann E, & Nauhaus I. (2020). Uniform spatial pooling explains topographic organization and deviation from receptive-field scale invariance in primate V1. Nature Communications, 11, 6390.

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Two-Photon Calcium Imaging in Mouse Cortex

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Ca²⁺ imaging in the mouse cortex was performed with a custom-built two-photon microscope based on an open-source design (MIMMS 2.0, janelia.org/open-science) and controlled with Scanimage 5.7_R1 software (Vidrio Technologies) and National Instrument electronics. The Ti:Sapphire excitation laser (Tiberius, Thorlabs) was tuned to 930 nm and focused with a 16×0.8 NA objective (Nikon) below the cortical surface. The laser power (typically 25 mW measured at the objective) was modulated with Pockels Cells (350-80-LA-02, Conoptics) and calibrated with a Ge photodetector (DET50B2, Thorlabs). A 550 µm by 550 µm area of cortex was scanned at ≈30 frames/s using a resonant-galvo scanning system (CRS8K/6215H, CRS/671-HP Cambridge Technologies). Emitted fluorescence was detected with GaAsP photomultiplier tubes (PMT2101, Thorlabs) and the acquired 512 × 512 pixel images written in 16 bit format to disk. Behavioral event (trial start and stimulus onset) TTL pulses issued by the Bpod State Machine were received as auxiliary inputs to the Scanimage electronics and their timestamps saved in the headers of the acquired images. The timestamps were used to temporally align neuronal data to behavioral events.

Alonso I., Scheer I., Palacio-Manzano M., Frézel-Jacob N., Philippides A, & Prsa M. (2023). Peripersonal encoding of forelimb proprioception in the mouse somatosensory cortex. Nature Communications, 14, 1866.

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