Liver tissue was fixed in 10% formalin and then embedded in Tissue-Tek optimal cutting temperature compound. Frozen liver tissues sectioned to 30 μm thick using Cryostat (Leica CM 3000) were subjected to hsSRS imaging analysis as previously described (53 (link), 62 (link), 63 (link)). Briefly, the spectral focusing–based hsSRS system was used, with a dual-output femtosecond laser (InSight DeepSee; Spectra-Physics) providing pump (800 nm) and Stokes (1040 nm) pulses at an 80 MHz repetition rate. An electro-optical modulator (EO-AM-R-C2; Thorlabs) was used to modulate the Stokes laser at a resonant frequency of 10.5 MHz. The time delay line controlled by a motorized stage was employed, and the microscope (BX51; Olympus) equipped with a water objective (UPLSAPO 60XW; Olympus) was used for laser scanning and imaging. The pump beam was detected by a photodiode (S3994-01; Hamamatsu) with two installed short-pass filters (ET980SP; Chroma), and the SRS signals were acquired by a lock-in amplifier (HF2 LI; Zurich Instruments). The laser power of pump and Stokes beams were set to 50 and 70 mW (measured before galvanometer), respectively.
Insight deepsee
Manufactured by Spectra-Physics
Sourced in United States
The InSight DeepSee is a multiphoton laser scanning microscope. It is designed for deep tissue imaging with high resolution and speed. The core function of the InSight DeepSee is to provide a powerful and versatile platform for advanced biological and biomedical research applications.
Automatically generated - may contain errors
Lab products found in correlation
Xlpln25xwmp2, by Olympus (3 mentions)
Trim scope 2, by Miltenyi Biotec (3 mentions)
Lcv mpe, by Olympus (2 mentions)
Rdm690, by Olympus (2 mentions)
Ba460 500, by Olympus (2 mentions)
Ba520 560, by Olympus (2 mentions)
Dm505, by Olympus (2 mentions)
Dm570, by Olympus (2 mentions)
Picoharp 300 tcspc module, by PicoQuant (2 mentions)
Ff775 di01 dichroic mirror, by IDEX Corporation (2 mentions)
Ff01 790 sp 25 shortpass filter, by IDEX Corporation (2 mentions)
R943 02 photomultiplier tube, by Hamamatsu Photonics (2 mentions)
Uplansapo, by Olympus (2 mentions)
Fv10 asw software, by Olympus (2 mentions)
Two photon moveable objective microscope, by Sutter Instruments (2 mentions)
Gaasp pmt, by Hamamatsu Photonics (2 mentions)
Cm 3000, by Leica (1 mentions)
Uplsapo60xw, by Olympus (1 mentions)
Hf2li, by Zurich Instruments (1 mentions)
1.05 na objective, by Olympus (1 mentions)
28 protocols using insight deepsee
1
High-Speed Hyperspectral SRS Imaging of Liver Tissue
2
In Vivo Two-Photon Imaging of Neuronal Activity
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For in vivo two-photon imaging, GCaMP6s and JF585–HaloTag were excited at 940 nm and 1100 nm, respectively, using a femtosecond laser source (InSight DeepSee, Spectra-Physics), and imaged using an Olympus 25× 1.05 NA objective and a homebuilt two-photon microscope53 . Images were acquired from 200 to 550 μm below the pia with post-objective power ranging between 20 and 60 mW. No photobleaching or photodamage of tissue was observed. Typical imaging settings were composed of 256 × 256 pixels, with 1.2 μm per pixel, and a ~3 Hz frame rate. The time-lapse calcium images of spontaneous neuronal activity in awake, head fixed mice were recorded and analyzed with custom programs written in MATLAB (Mathworks). Lateral motion present in head-fixed awake mice was corrected using a cross-correlation-based registration algorithm54 , where cross-correlation was calculated to determine frame shift in x and y directions. Cortical neurons were outlined by hand as regions of interest (ROIs). The fluorescence time course of each ROI was used to calculate its calcium transient as ΔF/F (%) = (F−F0)/F0 × 100, with the baseline fluorescence F0 being the mode of the fluorescence intensity histogram of this ROI. For the Pearson correlation coefficient calculation, the JF585 (red channel) and GCaMP6s (green channel) fluorescence signals in each ROI were averages from 1000 imaging frames (3 Hz).
3
Intravital Multiphoton Microscopy of CTLs
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Intravital microscopy was performed with a multiphoton microscope from Olympus (FVMPE-RS, Tokyo, Japan) with two pulsed lasers (Spectra Physics: Insight DeepSee 690–1300 nm, MaiTai Ti:Sa 690–1040 nm) and a water immersion objective (25×/NA 1.05). The Insight DeepSee was set to 1020 nm for the CellTracker™ Orange-labeled CTLs (545–620 nm). The MaiTai Ti:Sa laser was set to a wavelength of 835 nm for H2B Cerulean (460–500 nm). Anesthetized mice (with 1.5–2% isoflurane via a mask, controlled with a pulse oximeter) or 35 mm Ibidi® dishes with spheroids and CTLs were placed to the heated stage/insert. Images were captured with 640 × 640 pixels (509 µm), a total layer thickness of 60 µm, and a step size of 5 µm. The recording time was 30 min with a time interval of 30 s.
4
Long-term Cochlear Imaging Technique
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For long-term organ-scale imaging, we partially cut off the capsule adjacent to the apex tip of cochlear duct using tweezers carefully and the semicircular canals were removed. The isolated cochlea was put onto the dish as described above. For microscopy, we used an incubator-integrated multiphoton fluorescence microscope system (LCV-MPE, Olympus) with a × 25 water-immersion lens (NA = 1.05, WD = 2 mm, XLPLN25XWMP2, Olympus). The excitation wavelengths were set to 840 nm for CFP (InSight DeepSee, Spectra-Physics). Imaging conditions for the FRET biosensor were as follows: scan size: 800 × 800 pixels; scan speed: 10 μs/pixel; IR cut filter: RDM690 (Olympus); dichroic mirrors: DM505 and DM570 (Olympus); and emission filters: BA460-500 for CFP and BA520-560 for FRET detection (Olympus).
5
Two-Photon Imaging of Awake Head-Fixed Mice
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All imaging was performed on head-fixed awake mice after at least two weeks of recovery in single or paired housing. Before imaging and one week after surgery, mice were habituated to experimental handling and head fixation. During both habituation and imaging, each mouse had its body restrained under a half-cylindrical cover, which reduced struggling and prevented substantial body movements such as running28 (link). Habituation was repeated 3–4 times for each animal, and each time for 15–60 min. Each imaging session lasted 45 min to 2 h, with data collected from multiple imaging planes within the same mouse. GCaMP6s and H2B-GCaMP6s fluorescence was excited at 940 nm (InSight Deepsee, Spectra-Physics) under a Olympus 4× 0.28-NA, a Olympus 10× 0.6-NA, or a Nikon 16× 0.8-NA objective using a homebuilt two-photon fluorescence microscope, with all images collected at 3 Hz49 (link).
6
Two-Photon Fluorescence Lifetime Imaging
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We measured fluorescence lifetime on a homebuilt beam-scanning two-photon microscope with an 80 MHz, 100 fs tunable pulsed laser (SpectraPhysics Insight DeepSee). Pulse compression in the sample plane was performed through maximizing the fluorescence intensity of a bead-sample, using the motorized prism compressor built into the laser. The citrine measurements were performed at 1040 nm excitation wavelength with a time-averaged excitation power of 30 mW, or 0.4 nJ per pulse, focused down to a ~500 nm spot with a 1.2 NA water immersion objective (Olympus UplanSapo) for a time-averaged intensity of 15 MW cm−2; measurements were performed for linear speeds of the scanned spot varying between 0 and 30 cm s−1 without any measurable effect on fluorescence lifetime. Excitation light and fluorescence were separated using a FF775-Di01 dichroic mirror and FF01-790/SP-25 shortpass filter (Both Semrock); fluorescence was detected using a Hamamatsu R943-02 photomultiplier tube in photon counting mode, cooled to −20 °C. THe PMT signal was amplified through an SRS PR325 amplifier and discretized with a Hamamatsu Photon counting unit C9744, before being fed into a Picoharp 300 TCSPC module (Picoquant). The setup was controlled by Labview software written in-house.
7
Visualizing TAGLN2 and Actin Dynamics in Raji Cells
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Raji cells were transiently transfected with 2 μg of an expression plasmid encoding GFP-tagged TAGLN2 (OriGene) and LifeAct-TagRFP (ibidi, Martinsried, Germany) using the Amaxa Cell Line Nucleofector Kit V (Lonza). For the preparation of cell images, cells 24 h after transfection were placed on a 35-mm glass dish with medium containing liquid collagen (Cellmatrix type 1-A, Nitta Gelatin Inc.), RPMI 1640, HEPES, NaHCO3, and NaOH (5 x 10^6 cells/ mL). In these experiments, the cells were stimulated with 10 μg/ mL F(ab’) anti-human IgM + IgG (eBioscience). Imaging data were acquired using an Incubator Fluorescence Microscope equipped with a multi-photon laser scanning system (LCV110 and FV1200MPE, Olympus) with a 25 x 1.05 NA water-immersion objective (XLPLNWMP, Olympus) and FV-10ASW software (Olympus). Fluorescence excitation was performed with a pulsed laser (InSight™ DeepSee™, Spectra Physics). The cells were kept at 37°C in 5% CO2 during the experiments. XYZT scanning data were acquired at 1.0-μm intervals in the Z-dimension and by Free Run in the T-dimension. Figures were processed using an open source Java image processing program, ImageJ (https://imagej.net/Welcome ).
8
Laser-Mediated Axotomy Microscopy
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Axotomies were carried out at the Cambridge Advanced Imaging Centre on a TriM Scope II two-photon Scanning Fluorescence Microscope using Imspector Pro software (LaVision Biotec) and a near-infrared laser source (Insight DeepSee, Spectra-Physics). The laser light was focused by a 25x, 1.05 Numerical Aperture water immersion objective lens (XLPLN25XWMP2, Olympus). Axotomies were carried out by focusing the laser at a 6 × 6 μm section of the posterior lateral line using 100% laser power.
9
Two-Photon Spectral Characterization of T-GECO1
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The two-photon excitation spectra and two-photon absorption cross-sections of T-GECO1 were measured using a previously described protocol.29 (link) Briefly, a tunable femtosecond laser (InSight DeepSee, Spectra-Physics, Santa Clara, California) was coupled to a PC1 Spectrofluorometer (ISS, Champaign, Illinois). The quadratic power dependence of the fluorescence intensity was verified across the spectrum for both proteins and standards. The two-photon cross-section ( ) of the anionic form of the chromophore was determined for both the -free and -bound states, as previously described.30 As a reference standard, a solution of fluorescein in water at pH 12 was used. Fluorescence intensities of the sample and reference were measured for two-photon excitation at 900 nm and for one-photon excitation at 458 nm ( laser line). Fluorescence measurements utilized a combination of filters (770SP and 520LP). The two-photon absorption spectra were normalized based on the measured values.
10
Two-Photon Imaging of Neural Signals
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Fluorescence signals were recorded using a two-photon microscope (ThorLabs Bergamo II, 12 kHz scanner) with a Nikon 16× water-immersion objective (NA 0.8) giving a field of view of 540 × 540 µm. The scope was operated using ThorImageLS software (v4.0.2019.8191, Thorlabs). In most experiments the microscope was rotated by 45° from vertical. Owing to physical constraints, pIC and S1 two-photon imaging was carried out in different animals and sessions. The excitation laser (InSight DeepSee, Spectra-Physics) was tuned to 940 nm, and the power never exceeded 100 mW. Emitted photons were bandpass filtered 525/50 (green) onto a GaAsP photomultiplier tube. Multi-plane (512 × 512 pixels) acquisition was controlled by a fast piezoelectric objective scanner, with planes spaced 45 μm apart in depth. Seven planes were acquired sequentially, and the scanning of the entire stack was repeated at about 5 Hz. Beam turnarounds at the edges of the image were blanked. Acquisition trials lasted 26 s and had an inter-trial interval of 30–60 s. Stimuli generation and hardware synchronization were carried out on a computer with a National Instrument card running custom-written Python code.
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