Intravital imaging through a titanium/glass mammary imaging window were based on previously published protocols (Messal et al., 2021 (link)). Intravital imaging through our custom-made PDMS imaging windows was performed as previously described (Jacquemin et al., 2021 (link)). Briefly, mammary imaging windows were surgically implanted over the fourth mammary glands of 5-6-week-old mice at the indicated times after TAM administration. Mice were anaesthetized using isoflurane (1.5% isoflurane/medical air mixture) and placed in a facemask with a custom designed holder to stabilize windows during imaging acquisition. Imaging was performed on an upright Nikon A1R multiphoton microscope equipped with a Spectra-Physics Insight Deepsee laser, conventional and resonant scanners, GaAsP non-descanned detectors using 16x NA 0.8 or 25x NA 1.1 PlanApo LambdaS water objectives. An excitation wavelength of 960 nm was used for GFP and TdTomato, in addition to second harmonic generation (SHG) imaging of collagen. Mammary epithelial structures imaged in timelaspe were acquired every 30 min using a Z-step size of 2 μm. For long-term, longitudinal imaging z-stacks (with z-step size of 2 μm) of epithelial structures were taken either 1-2x daily for up to 8 days, or 2-3x weekly for up to 3 weeks (as indicated in figure legends).
Insight deepsee laser
Manufactured by Spectra-Physics
Sourced in France, United States
The Insight Deepsee laser is a high-performance laser system designed for scientific and industrial applications. It is a diode-pumped, solid-state laser that operates at a wavelength of 532 nanometers. The laser provides a stable and reliable output power, making it suitable for a variety of tasks that require precise and consistent illumination.
Automatically generated - may contain errors
Lab products found in correlation
A1r multiphoton microscope, by Spectra-Physics (2 mentions)
Ix70 inverted microscope, by Olympus (2 mentions)
Spcm aqr 14 fc, by PerkinElmer (2 mentions)
Xlpln25xwmp2, by Olympus (2 mentions)
Lcv110 mpe incubator microscope, by Olympus (2 mentions)
Ba460 500, by Olympus (2 mentions)
Ba520 560, by Olympus (2 mentions)
Uplsapo30xs, by Olympus (2 mentions)
Ba685rif 3, by Olympus (2 mentions)
Fv1000 ix83 confocal microscope, by Olympus (2 mentions)
Fv1200mpe ix83 inverted microscope, by Olympus (2 mentions)
Dm505, by Olympus (2 mentions)
A1rmp hd, by Nikon (1 mentions)
Dichroic and emission filters, by IDEX Corporation (1 mentions)
Lcpln20xir, by Olympus (1 mentions)
Two photon microscope, by Bruker (1 mentions)
Lsm 780, by Zeiss (1 mentions)
Eight chambered glass bottom microscopy slide, by Ibidi (1 mentions)
Spcimage software, by Becker & Hickl (1 mentions)
Dm505 dichroic mirror, by Olympus (1 mentions)
15 protocols using insight deepsee laser
1
Intravital Imaging of Mammary Glands
2
Intravital Imaging of Mammary Glands
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Intravital imaging through a titanium/glass mammary imaging window were based on previously published protocols (Messal et al., 2021 (link)). Intravital imaging through our custom-made PDMS imaging windows was performed as previously described (Jacquemin et al., 2021 (link)). Briefly, mammary imaging windows were surgically implanted over the fourth mammary glands of 5-6-week-old mice at the indicated times after TAM administration. Mice were anaesthetized using isoflurane (1.5% isoflurane/medical air mixture) and placed in a facemask with a custom designed holder to stabilize windows during imaging acquisition. Imaging was performed on an upright Nikon A1R multiphoton microscope equipped with a Spectra-Physics Insight Deepsee laser, conventional and resonant scanners, GaAsP non-descanned detectors using 16x NA 0.8 or 25x NA 1.1 PlanApo LambdaS water objectives. An excitation wavelength of 960 nm was used for GFP and TdTomato, in addition to second harmonic generation (SHG) imaging of collagen. Mammary epithelial structures imaged in timelaspe were acquired every 30 min using a Z-step size of 2 μm. For long-term, longitudinal imaging z-stacks (with z-step size of 2 μm) of epithelial structures were taken either 1-2x daily for up to 8 days, or 2-3x weekly for up to 3 weeks (as indicated in figure legends).
3
Optical Stimulation of Arc Transcription in Dentate Granule Cells
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An Ultima 2P laser scanning microscope (Bruker Corp.) equipped with an Insight Deep See laser (Spectra-Physics) tuned to 910-930 nm was used to image Arc transcription in dentate granule cells (GCs) with 512 x 512 pixel resolution using 4 mW laser power measured at the 60X objective (Nikon, 1.0 NA). GCs expressing the PCP-GFP coat protein were imaged at 1X magnification to detect at least one transcribing neuron, which was then chosen as the region of interest (ROI) for 2X magnification. A Z-stack of 25 µm thickness with 0.5 µm steps was taken to assess baseline Arc transcription signals before optical stimulation.
Acute slices showing optimal ChIEF-tdTomato reporter expression (at least 75% of DG was fluorescent) were selected for optical stimulation and imaging. The Invitro Ultima 2P microscope (Bruker Corp.) contains a Coherent 473 nm laser path that delivered optical stimulation of 25 pulses at 25 Hz repeated 20 times every 5 s (8 mW, 2-4 ms pulse duration).
The stimulation area was specifically defined using customized Mark Point software (Bruker Corp.) and was empirically determined based on at least one transcribing neuron in the field of view as described above. After stimulation, Z-stack images of 25 µm thickness with 0.5 µm steps were acquired every 15 min for 4-5 hour.
Acute slices showing optimal ChIEF-tdTomato reporter expression (at least 75% of DG was fluorescent) were selected for optical stimulation and imaging. The Invitro Ultima 2P microscope (Bruker Corp.) contains a Coherent 473 nm laser path that delivered optical stimulation of 25 pulses at 25 Hz repeated 20 times every 5 s (8 mW, 2-4 ms pulse duration).
The stimulation area was specifically defined using customized Mark Point software (Bruker Corp.) and was empirically determined based on at least one transcribing neuron in the field of view as described above. After stimulation, Z-stack images of 25 µm thickness with 0.5 µm steps were acquired every 15 min for 4-5 hour.
4
Quantifying UHRF1-Ubiquitin Interaction via FLIM-FRET
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HeLa cells stably expressing GFP-UHRF1 WT or GFP-UHRF1 C724A-H741A protein, were seeded (105 cells per dish) in a µ-dish (Ibidi) with 35-mm wells. Cells were transfected with 1 µg RFP-Ubiquitin plasmid using jetPEI™ reagent. Cells were fixed with 4% paraformaldehyde. Following fixation, cells were imaged with a homemade two-photon excitation scanning microscope based on an Olympus IX70 inverted microscope with a 60X 1.2 NA water immersion objective operating in the descanned fluorescence collection mode as previously described (60 (link),61 (link)). Two-photon excitation at 930 nm was provided by an Insight DeepSee laser (Spectra Physics, Inc.). Fluorescence photons were collected using a short-pass filter with a cut-off wavelength of 680 nm (cat. no. F75-680; Analysentechnik) and a band-pass filter of 520±17 nm (cat. no. F37-520; Analysentechnik). The fluorescence was directed to a fibre-coupled APD (cat. no. SPCM-AQR-14-FC; Perkin Elmer Inc., USA), which was connected to a time-correlated single photon counting module (cat. no. SPC830: Becker & Hickl). FLIM data were analyzed using SPCImage v 7.3 (Becker & Hickel) and the Förster resonance energy transfer (FRET) efficiency was calculated according to E=1-(τDA/τD), where τDA is the lifetime of the donor (GFP) in the presence of acceptor (RFP) and τD is the lifetime of GFP in the absence of acceptor.
5
Longitudinal Neuronal Tracking During Heroin Addiction
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We visualized and longitudinally tracked dmPFC neurons throughout heroin selfadministration, extinction, and reinstatement using a multiphoton microscope (Bruker Nano Inc.) equipped with: a hybrid scanning core with galvanometers and fast resonant scanners (30 Hz; we recorded with 4 frame averaging to improve spatial resolution), multi-alkali photo-multiplier tubes and GaAsP-photo-multiplier tube photo detectors with adjustable voltage, gain, and offset features, a single green/red NDD filter cube, a long working distance 20x air objective designed for optical transmission at infrared wavelengths (Olympus, LCPLN20XIR, 0.45NA, 8.3mm WD), a software-controlled modular XY stage loaded on a manual z-deck, and a tunable Insight DeepSee laser (Spectra Physics, laser set to 930nm, ~100fs pulse width). Following imaging sessions, raw data were converted into an hdf5 format for motion correction using SIMA (Kaifosh et al., 2014) . Fluorescence traces were extracted from the datasets using Python (Namboodiri et al., 2019; Otis et al., 2019) .
6
Multiphoton Microscopy for Endogenous Fluorescence and Harmonic Imaging
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Measurements were performed on a multiphoton microscope (A1RMP-HD, Nikon Europe B.V., Amsterdam, Netherlands) coupled with an Insight Deepsee laser (Spectra Physics, France), used in the 820–1300 nm range < 120 fs pulse width (APEX platform, INRAE/Oniris UMR 703 PAnTher Nantes, France, Center of Excellence Nikon Nantes). An auxiliary beam at 1040 nm was available in combination with the tunable output for dual wavelength excitation. For all images, an apochromat 25X MP1300 immersion objective was employed (NA 1.10, WD 2.0 mm) and distilled water was used as the immersion medium. A half-wave plate (HWP; MKS-Newport, USA) was rotated to control the laser polarization angle in the range 0°–135°. The microscope was equipped with eight Non-Descanned Detectors (NDD), three GaAsP (gallium arsenide non-descanned) and one photomultiplier tube for backward detection, with the same detectors also configured for forward detection.
The endogenous fluorescence (EF) was probed with Two Photon Excitation (TPE). SHG was detected in both backward and forward channels, whereas THG was detected only in the backward channel. Different dichroic and emission filters from Semrock were used according to the excitation wavelengths and analyzed signals (fluorescence and harmonic signals). More information on the acquisition parameters can be found in Table1 .
The endogenous fluorescence (EF) was probed with Two Photon Excitation (TPE). SHG was detected in both backward and forward channels, whereas THG was detected only in the backward channel. Different dichroic and emission filters from Semrock were used according to the excitation wavelengths and analyzed signals (fluorescence and harmonic signals). More information on the acquisition parameters can be found in Table
7
Two-Photon Imaging of PVT-NAc Neurons
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We visualized GCaMP6m-expressing PVT→NAc projection neurons using a two-photon microscope (Bruker Nano Inc) equipped with a tunable InSight DeepSee laser (Spectra Physics, laser set to 920 nm, ~100 fs pulse width), resonant scanning mirrors (~30 Hz framerate), a ×20 air objective (Olympus, LCPLN20XIR, 0.45NA, 8.3 mm working distance), and GaAsP photodetectors. In some cases, two fields of view (FOVs) were visible through the GRIN lens (separated by >75 µm in the Z-axis to avoid signal contamination from chromatic aberration), in which case we recorded from each FOV during separate imaging sessions. Data were acquired without averaging using PrarieView software, converted into hdf5 format, and motion corrected using SIMA73 . Following motion correction, a motion-corrected video and averaged time-series frame were used to draw regions of interest (ROIs) around dynamic and visually distinct cells using the polygon selection tool in FIJI74 (link). Fluorescent traces for each ROI were then extracted using SIMA, and all subsequent analyses were performed using custom Python codes in Jupyter Notebook6 (link),25 (link). Two-photon imaging was performed during select acquisition sessions (early: days 1–2; middle: days 7–8; late: days 13–14) and extinction sessions (early: days 1–2; late: last 2 days) to simplify data analysis.
8
Fluorescence Lifetime Imaging of E. coli
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Fluorescence lifetime measurements and FLIM were performed on an inverted fluorescence microscope LSM780 (Zeiss), using oil immersion 63× objective (NA = 1.4, Zeiss). Solutions of E. coli cells were diluted to the A600 of ∼0.1 and placed into the eight chambered glass bottom microscopy slide (ibidi). Two photon excitation was achieved using Insight DeepSee laser (λexc = 920 nm; 80 MHz, 150 fs pulse; Newport Spectra Physics). Fluorescence intensity decays were generated using time-correlated single photon counting with SPC-150 modules (Becker & Hickl, Germany). Average lifetimes were calculated using monoexponential fitting with a binning parameter of three in SPCImage software (Becker & Hickl) and averaged to obtain τav. CagFbFP solutions with concentration of ∼0.1 mg/ml were measured using the same experimental setup.
9
Imaging MDCK Cysts in Collagen Gel
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MDCK cysts were generated as previously described (Martin-Belmonte et al., 2007; Pollack et al., 1998; Yagi et al., 2012) . Briefly, 7.5×10 3 MDCK cells were placed on a glass coverslip (13 mm in diameter) coated with 80 μl of polymerized Matrigel (BD Biosciences), and then supplied with culture medium containing 2% Matrigel. For time-lapse imaging, 4 to 6-day-old cysts were incubated with 1.25 mM EDTA/PBS at 37 degrees c for 5 to 10 min to depolymerize the Matrigel, followed by washing three times with PBS. The cysts were centrifuged and suspended in 100 μl of collagen solution containing 66% collagen gel (Cellmatrix Type I-A; Nitta Gelatin, Osaka, Japan), 24% reconstitution buffer (Nitta Gelatin), and 10% 10x culture medium. Cysts suspended in the collagen solution were placed into a 15-mm diameter glassbottom well in a 35-mm diameter plastic dish, and the collagen was allowed to polymerize at 37 degrees c for 20 min. Cysts were imaged with an LCV110-MPE incubator microscope (Olympus) equipped with a 25x/1.05 water-immersion objective lens (XLPLN 25XWMP2; Olympus), and an InSight DeepSee Laser (Spectra Physics, Mountain View, CA). The excitation wavelengths for mCherry and iRFP were 1040 nm and 1300 nm, respectively. The filters and dichroic mirrors used for imaging were as follows: an IR-cut filter (BA685RIF-3) and a DM505 dichroic mirror (Olympus).
10
Two-photon Excitation Microscopy for Live-cell Imaging
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For two-photon excitation microscopy (2PM), we used an FV1200MPE-IX83 inverted microscope (Olympus) equipped with a 30×/1.05 NA silicon oil-immersion objective lens (UPLSAPO 30XS; Olympus), an LCV110-MPE incubator microscope (Olympus) equipped with a 25×/1.05 water-immersion objective lens (XLPLN 25XWMP2; Olympus), and an InSight DeepSee Laser (Spectra Physics). The laser power was set to 3–18%. The scan speed was set between 4–12.5 μs per pixel. Z-stack images were acquired at 1–10 μm intervals. In time-lapse analyses, images were recorded every 1–3 min. The excitation wavelength for CFP was 840 nm. We used an IR-cut filter (BA685RIF-3), two dichroic mirrors (DM505 and DM570), and two emission filters (BA460-500 for CFP and BA520-560 for YFP) (Olympus). Confocal images were acquired with an FV1000/IX83 confocal microscope (Olympus) equipped with a 30×/1.05 NA silicon oil-immersion objective lens (UPLSAPO 30XS; Olympus).
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