Paraffin-embedded sections were imaged using a Zeiss LSM 780 confocal microscope with a 3 watt Chameleon Vision II (Coherent Inc, Santa Clara, CA) Ti:Sapphire oscillator used to generate 140 fs laser pulses at 820 nm with a 80 MHz repetition rate. A 40× 1.4 numerical aperture (NA) PlanApo oil immersion objective (Zeiss 420762-9900) and 1.2 NA water immersed condenser (Zeiss, Oberkochen, Germany) were used. The 410nm, second harmonic signals generated in the tissue were detected using a 400 ± 20 nm filter (Chroma Technology Corp., Bellows Falls, VT), and a 525 ± 50 nm filter (Chroma Technology Corp.) was used for two-photon excited fluorescence (TPEF) detection. TPEF was used to visualize elastin autofluroescence and a nondescanned detection (NDD) system was used for detection of SHG in the forward direction using a photomultiplier tube (LSM NDD; Zeiss) and in the reverse direction using a GaAsP detector (LSM BiG; Zeiss) to visualize collagen.33 (link), 34 (link)
Chameleon vision 2
Manufactured by Coherent Inc
Sourced in Germany
The Chameleon Vision II is a high-performance laser system designed for a wide range of scientific and industrial applications. It features an ultrafast tunable laser with a wide spectral range, providing researchers and engineers with a flexible tool for their experiments and projects.
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Lab products found in correlation
Lsm 880 airy nlo, by Zeiss (2 mentions)
Imaris 8, by Oxford Instruments (2 mentions)
Lsm big, by Zeiss (1 mentions)
Imagej, by Leica (1 mentions)
Imaris, by Leica (1 mentions)
On stage incubator, by Okolab (1 mentions)
Plan apochromat 20 0.8 m27, by Zeiss (1 mentions)
Lsm 880 nlo, by Zeiss (1 mentions)
Plan apochromat 10 0.8 m27, by Zeiss (1 mentions)
5 protocols using chameleon vision 2
1
Multimodal Imaging of Tissue Structure
2
Collagen Fiber Orientation Analysis via SHG Imaging
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Second harmonic generation (SHG) images were obtained using a multiphoton confocal microscopy system (LSM 880 Airy NLO, Carl Zeiss, Oberkochen, Germany) with excitation laser (Chameleon Vision II, wavelengths: 680–1080 nm; repetition rate: 80 MHz pulse width: 140 fs, Coherent Inc., Santa Clara, CA, USA) and objective lens (Plan-Apochromat 10x/0.8 M27, Carl Zeiss, Oberkochen, Germany). The excitation wavelength for observing collagen fibers was 880 nm. From the image obtained, a region of 200 μm × 200 μm square at the implant neck (A and F) was extracted as the region of interest. High-precision image analysis software (Imaris8.4, Bitplane AG, Zürich, Switzerland) was used to trace and measure the angles of the collagen fiber bundles.
3
Visualizing 3D Tumor Cell Invasion
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For time-lapse experiments, CAFs were stained with a lyophilic carbocyanine dye (Vybrant DiI-Cell Labeling Solutions; ThermoFisher) according to the manufacturer’s recommendation. Cells were embedded in collagen as described in the Invasion assay section. The dish was incubated at 5% CO2 and 37°C in the on-stage incubator (Okolab). For fixed and live 3D samples, images were acquired with an inverted AOBS two-photon laser-scanning confocal microscope (SP8; Leica) coupled with a femtosecond laser (Chameleon Vision II; Coherent Inc.) using a 25×/1.0 NA water immersion objective. The microscope was equipped with three nondescanned HyD detectors: NDD1 (500–550 nm), NDD2 (≥590 nm), and NDD3 (405 nm). Fluorescence channels were recorded simultaneously using the excitation wavelength 980 nm. Collagen was visualized by either second harmonic generation using the excitation wavelength 910 nm or by confocal reflectance microscopy that does not interfere with DAPI staining, using light at a wavelength of 488 nm and a standard photomultiplier tube detector at a low gain (500 V). Images were recorded every 10 min up to 72 h. 3D stacks were obtained at a step size of 2-µm intervals. The images were processed with the Leica Application Suite, ImageJ, and Imaris (Bitplane).
4
Quantifying Collagen Fibre Bundles in Mandibular Muscle
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Second harmonic generation (SHG) images were acquired using a multiphoton confocal microscope system (LSM880 NLO, Carl Zeiss, Jena, Germany) with an excitation laser (Chameleon Vision II; wavelengths: 680–1080 nm, repetition rate: 80 MHz, pulse width: 140 fs; Coherent Inc., Santa Clara, CA, USA) and an objective lens (Plan-Apochromat 20×/0.8 M27, Carl Zeiss). Following image acquisition, the collagen fibre bundles were quantitatively evaluated using Imaris 8.4 software (Bitplane AG, Zurich, Switzerland). Collagen fibres with a thickness greater than 375 nm, which can be imaged with multiphoton excitation phase-contrast microscopy, were extracted as collagen fibre bundles. Evaluation was carried out by tracing the collagen fibre bundles within the bone in approximately 200 µm2 of the mandibular masseter muscle prominence and calculating the mean diameter [27 (link)].
5
Quantifying Collagen Fiber Anisotropy
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Second harmonic generation (SHG) images were acquired by using a multiphoton confocal microscopy system (LSM 880 Airy NLO; Carl Zeiss) with an excitation laser (Chameleon Vision II, wavelengths: 680–1080 nm; repetition rate: 80 MHz; pulse width: 140 fs; Coherent Inc., Santa Clara, CA, USA) and an objective lens (Plan-Apochromat 10×/0.8 M27; Carl Zeiss). The excitation wavelength for collagen fiber observation was 880 nm. The images thus obtained were used to measure the angle between the Z-axis and the negative direction of the collagen fiber bundles in an area measuring 200 μm × 200 μm centered on the implant central region (B and D). Since the collagen fiber bundles are drawn as curves, angle computation was conducted as a straight line connecting the ends of the curves in the area of observation. Collagen fiber bundle tracing and angle measurement were carried out using high-precision image analysis software (Imaris 8.4; Bitplane AG, Zürich, Switzerland). The variation in collagen fiber bundle angle was taken as an index of anisotropy.
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