Chronic in vivo two‐photon imaging was started after a recovery period of 4 weeks to minimize the effect of microglial activity in response to installation of the cranial window. In order to stain Aβ plaques, Methoxy‐X04 was injected intraperitoneally (0.5 mg/kg) 24 h before each imaging session. During imaging, mice were anesthetized with isoflurane (1% in 95% O2, 5% CO2), placed on a heating pad to keep body temperature at 37°C, and fixed to a custom‐made holder using the glued metal bar. In vivo two‐photon imaging was performed on a LSM 7 MP (Carl Zeiss) equipped with GaAsP (Gallium Arsenide) detectors and a 20× water‐immersion objective (W Plan‐Apochromat 20×/1.0 DIC, 1.0 NA, Carl Zeiss). In each mouse, a 425 × 425 × 200 μm3 large region of interest was reimaged weekly at a resolution of 0.24 × 0.24 × 0.4 μm3. Methoxy‐X04 was excited at 750 nm by a Ti:Sa laser (MaiTai DeepSee, Spectra‐Physics), and emission was collected below 485 nm. In subsequent imaging sessions, the previously imaged brain region was identified using the unique blood vessel pattern. To keep the emitted fluorescence stable at different depths, the laser intensity was adjusted using the z‐correction tool in the microscope control software.
Lsm 7 mp
Manufactured by Zeiss
Sourced in Germany, France
The LSM 7 MP is a laser scanning microscope designed for high-resolution, multiparameter imaging. It features a modular and flexible architecture, enabling the integration of various detector modules and objectives to suit a wide range of applications.
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Lab products found in correlation
Zen software, by Zeiss (7 mentions)
Lsm 880, by Zeiss (6 mentions)
Apochromat 20, by Zeiss (3 mentions)
Deepsee, by Spectra-Physics (2 mentions)
X40 water immersion objective 0.8 na, by Olympus (2 mentions)
Oregon green 488 bapta 1, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 594, by Thermo Fisher Scientific (2 mentions)
Axio observer 7 inverted microscope, by Yokogawa (2 mentions)
Texas red conjugated dextran 70 kda, by Merck Group (2 mentions)
Cf 405m conjugated wheat germ agglutinin, by Biotium (2 mentions)
Lsm 510, by Zeiss (2 mentions)
Axiovert 200m, by Zeiss (2 mentions)
Zen lsm, by Zeiss (2 mentions)
Lsm zen software, by Zeiss (2 mentions)
Zen 2010, by Zeiss (2 mentions)
Efficient navigation zen , by Zeiss (2 mentions)
Zen 2011, by Zeiss (1 mentions)
Volocity software, by PerkinElmer (1 mentions)
492 sp25 nm filter, by IDEX Corporation (1 mentions)
Nm filter, by IDEX Corporation (1 mentions)
56 protocols using lsm 7 mp
1
Longitudinal In Vivo Imaging of Amyloid Plaques
2
Macroscopic and Cellular Calcium Imaging
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Macroscopic imaging of GCaMP signal was performed with the same system used for flavoprotein imaging. Cellular imaging was performed with a two-photon laser-scanning microscope (LSM7 MP, ZEISS) equipped with a Ti:sapphire laser (Cameleon, COHERENT) and a water-immersion objective lens (20×, W Plan-Apochromat, NA 1.0, ZEISS). The laser wavelength was tuned to 1020 nm. Fields of view covering 425 × 425 μm at 100–300 μm from the surface were imaged at a spatial resolution of 256 × 256 pixels and at a frame rate of 6.7 Hz. Imaging under the anesthetized state (0.5 or 2% isoflurane) was performed after that under the awake state on the same days.
3
Imaging Microglia in Hippocampal Slices
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A two-photon laser scanning microscope (Zeiss LSM 7MP) with a Zeiss 20×W/1.0 NA objective coupled to a Coherent Chameleon Ultra II laser was used to image live and fixed hippocampal brain slices. Microglia were imaged in the stratum radiatum of the CA1 region at 150 ± 25 μm below the surface of the slice. At that depth, microglia show no signs of activation for up to 4 h11 (link),107 (link),108 (link). Images for time-lapse analysis were collected at 512 × 512 pixels or 1024 × 1024 pixels using 8- or 16-line averaging. Live time series were imaged as stacks of 30 μm depth with a step size of 2 μm in the z-axis. Fixed images were acquired as stacks of 30-80 μm depth with a step size of 1–2 μm in the z-axis. EGFP was excited at 920 nm and the emission was detected with a photo-multiplier tube after passing through a 490–550 nm emission filter (Chroma, ET520/60 m). Lesions were induced by exposure of high laser power illumination to a restricted area. Inhibitors were applied at concentrations ~10–1,000 times their respective IC50 to account for brain slice (at 150 μm depth) permeability, diffusion and/or breakdown.
4
Long-Term Two-Photon Imaging of Neurons
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Imaging was performed under light anaesthesia (ketamine/xylazine, 0.1/0.0075 mg/g body weigh, i.p). Mice were placed under the microscope in a custom-made holder with fixed position relative to the objective lens. Two-photon imaging data presented here were from 6 consecutive imaging sessions from 9 mice. First imaging took place four weeks after surgery (time point 0), then imaging continued up to 2.5 months in a two-week interval. Imaging was performed using an upright two-photon microscope (Zeiss), equipped with a 20× water immersion objective (1.0 NA; Zeiss). TdTomato was excited with a femtosecond laser (MaiTai DeepSee, Spectra-Physics) at 915 nm and emission was collected at 527–582 nm, respectively (LSM 7MP, Zeiss). Images of cell bodies were acquired at 0.41 μm pixels resolution in xy dimension and 2 μm in z dimension (1024 × 1024 pixels). Same imaging volumes were precisely aligned over the imaging period. The laser intensity was adjusted to keep the emitted tdTomato fluorescence stable.
5
Mouse Islet Transplantation and In Vivo Imaging
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Mouse islet isolation and transplantation to the anterior eye chamber of female 6–8 week old B6-albino or B6.ROSA-tomato mice were performed as previously described [26 (link)]. Human pancreatic islets of five nondiabetic brain-dead organ donors (ESM Table 2 ) were obtained from The Nordic Network for Islet Transplantation, through the Human Tissue Laboratory at Lund University Diabetes Center (Malmö, Sweden), cultured as described previously [29 (link)] and transplanted to the anterior eye chamber of female 6–8 week old NOD.Rag2−/− or NOD.ROSA-tomato.Rag2−/− mice. The Regional Ethics Committee in Lund, Sweden, approved the study according to the Act Concerning the Ethical Review of Research Involving Humans. In vivo imaging was performed as previously described [26 (link), 27 (link)] using an upright laser scanning microscope (LSM 7 MP; Zeiss, Jena, Germany) equipped with a tunable Ti:sapphire laser (Spectra-Physics Mai Tai; Newport, CA, USA) and a long working distance 20×/1.0× water-dipping lens (Zeiss), specified in more detail in the ESM Methods. A 3D analysis of in vivo images of three to five randomly chosen islets per mouse eye was performed using Imaris 9.1 (Bitplane, Zurich, Switzerland) (ESM Fig. 1 ).
6
In vivo Two-photon Imaging of 5XFAD Mice
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Two-photon microscopy
(LSM 7 MP; Carl Zeiss Inc., Goettingen, Germany) equipped with titanium–sapphire
femtosecond laser (Chameleon Ultra; Coherent, Santa Clara, CA), and
20× water immersion objective lens (W Plan-Apochromat 20×/1.0
DIC M27 70 mm, Carl Zeiss Inc. Germany) was used for in vivo imaging. Probe1 or IBC 2 was intraperitoneally
injected (10 mg kg–1) into mice (2–11-month-old
5XFAD Tg mice and 9-month-old litter mates (WT)) 2 h before imaging.
In addition, Dextran-Texas-Red (70 kDa) was intravenously injected
for blood vessel staining (25 mg kg–1) just before
imaging. The laser power was limited to 70 mW to avoid the damage
associated with phototoxicity for in vivo mouse brain
imaging as well as to minimize autofluorescence from tissues. Zen
2011 software (Carl Zeiss Inc.) and Volocity software (PerkinElmer)
were used for image analysis and 3D-reconstructed images.
(LSM 7 MP; Carl Zeiss Inc., Goettingen, Germany) equipped with titanium–sapphire
femtosecond laser (Chameleon Ultra; Coherent, Santa Clara, CA), and
20× water immersion objective lens (W Plan-Apochromat 20×/1.0
DIC M27 70 mm, Carl Zeiss Inc. Germany) was used for in vivo imaging. Probe
injected (10 mg kg–1) into mice (2–11-month-old
5XFAD Tg mice and 9-month-old litter mates (WT)) 2 h before imaging.
In addition, Dextran-Texas-Red (70 kDa) was intravenously injected
for blood vessel staining (25 mg kg–1) just before
imaging. The laser power was limited to 70 mW to avoid the damage
associated with phototoxicity for in vivo mouse brain
imaging as well as to minimize autofluorescence from tissues. Zen
2011 software (Carl Zeiss Inc.) and Volocity software (PerkinElmer)
were used for image analysis and 3D-reconstructed images.
7
Two-Photon and Confocal Microscopy of AGuIX-RhoB
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AGuIX®-RhoB (10 mM) were added to the apical phase of the cells during the indicated time. Two-photon microscopy was performed as described in Sancey et al. [39 (link)], using a LSM 7 MP (Zeiss, Germany) equipped with a 20 × water-immersion objective (NA 1.0; Zeiss) and ZEN 2010 software for detection of the NPs. Laser excitation was done at 800 nm with a Ti:Sapphire laser (Chameleon vision II; Coherent, UK). Fluorescence emissions were detected simultaneously by three non-descanned photomultiplier tubes with a 492/SP25 nm filter (Semrock, US) for blue autofluorescence and Hoechst emission, a 542/50 nm filter (Semrock, US) for green autofluorescence emission, and a 617/73 nm filter (Semrock, US) for AGuIX®-RhoB fluorescence emission. Autofluorescence and second harmonic generation of biological structures could also be collected in the 3 channels due to the presence of collagen, lectin and elastin as example. Confocal microscopy was performed using an LSM 510 (Zeiss Germany) equipped with a 40 × oil-immersion objective (NA 1.2; Zeiss). Laser excitations/emissions were 760 nm biphotonic/400–450 nm for Hoechst, 488 nm/500–550 nm for FITC/GFP, 543 nm/550–600 nm for Rhodamine-B, 633 nm/650–705 nm for Cy-5, respectively.
8
Quantifying Nodes of Ranvier in Sciatic Nerves
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All CARS images were obtained with a two-photon microscope LSM 7 MP coupled to an OPO (Zeiss, France)25 (link) and complemented by a delay line68 . A ×20 water immersion lens (W Plan Apochromat DIC VIS-IR) was used for image acquisition. Four consecutive fields (250 mm square, 200 µm deep) were captured on sciatic nerves and an image of 1 cm long/20 µm deep was then reconstructed using Zen software (Zeiss, France). The number of nodes of Ranvier was determined for each reconstructed image. The results were expressed as a density of nodes of Ranvier over the number of fibers per field.
9
Two-Photon Imaging of Skeletal Muscle Calcium Dynamics
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Two-photon images were acquired from tibialis anterior muscle with a laser scanning system (LSM 7 MP, Carl Zeiss) equipped with 2 types of water-immersion objective lens (×10 and ×20, with numerical apertures of 0.5 and 1.0, respectively; Carl Zeiss) and a Ti:sapphire laser (Mai Tai HP, Spectra-Physics) operating at a wavelength of 950 nm (36 (link), 37 (link)). Continuous 4000-frame Ca2+ imaging was repeated for each imaging field. The imaged fields were 848.54 by 848.54 μm (original scan) or 425.1 by 425.1 μm (×2.0 digital zoom). The pixel size was 1.657 or 0.83 μm (×2.0 digital zoom), and the frame duration was 968 ms. FRET imaging of tibialis anterior from the surface to a maximum depth of 250 μm (pixel size, 0.83 μm; frame duration, 3.87 seconds; depth interval, 2 μm) was recorded at an excitation wavelength of 830 nm (38 (link)). Fluorescence was separated by a 509-nm dichroic mirror with 460- to 500-nm (cyan channel: for CFP fluorescence detection) and 520- to 560-nm (yellow channel: for YFP fluorescence detection) emission filters.
10
Imaging Lead Levels in Cardiomyocytes and Drosophila Brain
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For in-cell or in vivo FRET ratio imaging of Pb within iPSC-derived cardiomyocytes or in the Drosophila brain, an inverted microscope (Axiovert 200 M, Zeiss, Germany) or a stereomicroscope (MVX-10, Olympus, Japan) equipped with a 440 nm light source and a W-View module (Gemini, Hamamatsu, Japan; with filters 542/27 nm for YFP and 483/32 nm for CFP; Figure S1 and Figure 1 C,D) and a CMOS camera (ORCA-Flash4.0, Hamamatsu, Japan) controlled by HCImage software was used. The fluorescent signals of cp173Venus and ECFP(ΔC11) from samples that expressed Met-lead were rapidly acquired from the in-cell or in vivo FRET Y/C ratiometric imaging systems.
For the high-resolution FRET Y/C ratiometric imaging of larval CNS, a two-photon microscope was used. An 850-nm two-photon laser was applied as an excitation source within a multiphoton microscope (Zeiss LSM 7 MP, with 20×, NA 1.0 water objectives, Germany). The emission signals of YFP (530–630 nm) and CFP (460–500 nm) were acquired separately (Figure S1 and Figure 1 C,D).
For the high-resolution FRET Y/C ratiometric imaging of larval CNS, a two-photon microscope was used. An 850-nm two-photon laser was applied as an excitation source within a multiphoton microscope (Zeiss LSM 7 MP, with 20×, NA 1.0 water objectives, Germany). The emission signals of YFP (530–630 nm) and CFP (460–500 nm) were acquired separately (
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