For fluorescence microscopy, selected tissue slides were deparaffinized, and the nuclei were counterstained with 4’,6‐diamidino‐2‐phenylindole (DAPI, Prolong Diamond, Thermo Fisher Scientific). Stained slides were dried overnight, in the dark, and imaged using a custom setup inverted digital fluorescence microscope (DM6B, Leica Biosystems, Wetzlar, Germany) equipped with a highly sensitive Leica DFC9000GT camera (4.2‐M pixel sCMOS camera), a metal halide LED light source (X‐Cite 200DC, Excelitas Technologies, Waltham, MA, USA) for DAPI imaging, and a xenon arc lamp LB‐LS/30 (Sutter Instrument, Novato, CA, USA) for NIR imaging of IRDye800CW. Image acquisition and processing was done using LAS X software (Leica Biosystems).
X cite 200dc
Manufactured by Excelitas
Sourced in United States
The X-Cite 200DC is a compact and versatile fluorescence illumination system designed for a wide range of microscopy applications. It features a high-intensity mercury-based light source that provides stable and consistent illumination across a broad spectral range. The system offers precise control over light intensity and exposure time, allowing users to optimize imaging conditions for their specific research needs.
Automatically generated - may contain errors
Lab products found in correlation
Las x software, by Leica Biosystems (3 mentions)
Lb ls 30, by Sutter Instruments (3 mentions)
Dfc9000gt, by Excelitas (2 mentions)
Prolong diamond, by Thermo Fisher Scientific (2 mentions)
M165 fc, by Excelitas (2 mentions)
Dmi600b fluorescence microscope, by Excelitas (2 mentions)
Odyssey infrared imaging system, by LI COR (2 mentions)
Pm100d, by Thorlabs (1 mentions)
Ckx41, by Olympus (1 mentions)
Prolong gold, by Thermo Fisher Scientific (1 mentions)
Ixon3 897, by Oxford Instruments (1 mentions)
Eclipse ti, by Nikon (1 mentions)
Dmi6000b, by Leica Biosystems (1 mentions)
Hq800795lp, by Chroma Technology (1 mentions)
Tricaine, by Merck Group (1 mentions)
Celldiscoverer 7, by Zeiss (1 mentions)
Dfc7000t, by Leica Microsystems (1 mentions)
Lsm 900, by Zeiss (1 mentions)
M205fa, by Leica Microsystems (1 mentions)
Dfc450 c camera, by Leica camera (1 mentions)
13 protocols using x cite 200dc
1
Fluorescence Microscopy of Tissue Slides
2
Particle Dynamics Microscopy Characterization
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We followed the particle dynamics using an inverted microscope (Zeiss 200M) equipped with a high numerical aperture oil objective lens (×60 1.25 NA, ×100 1.3 NA, Oil, EC Plan NeoFluar) and with a mercury lamp (X-Cite 200 DC, Excelitas Technologies, USA), and a Nikon inverted fluorescence microscope equipped with a CCD camera (Olympus CKX41). Light that was sent through the microscope objective lens and the intensity of light was uniform across the area of interest. Moreover, the light source is equipped with a manual knob to control the intensity of light. Different bandpass filters were used to restrict light emission to 370–385 nm (UV) and 535–565 nm (green). With our current experimental setup, we found that the time required to switch between two wavelengths is about 0.6 s. This is fast enough for the current study, as the time scale of switching needed to be smaller than the persistence time (≈35 s, 1/Dr, rotational diffusion of the self-propelled particle) for the self-propulsion. The intensity of light emission was measured with a photo detector (Thorlabs, PM 100D). We acquired images with frame rates ranging between 5 and 50 fps. The brightness and contrast of the images were adjusted using ImageJ. The particle’s centroid was extracted from time-lapsed images using ImageJ plugin, Trackmate24 (link).
3
Fluorescence Microscopy of Tissue Slides
4
Multi-channel Fluorescence Microscopy Protocol
5
Fluorescence Microscopy Protocol for FFPE Tissue
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For fluorescence microscopy, 4 μm FFPE tissue sections were deparaffinized, rehydrated and stained with Hoechst to visualize nuclei (33258; Invitrogen, Thermo Fisher Scientific). Fluorescence microscopy was performed using an inverted wide-field microscope (63-100x magnification, immersion oil; DMI6000B, Leica Biosystems GmbH, Nussloch, Germany), with a LED light source that is able to excite up to 900 nm (X-Cite 200DC; Excelitas Technologies, Waltham, MA, USA), a monochrome camera also sensitive in the NIR range (1.4M Pixel CCD, DFC365FX; Leica Biosystems GmbH) and an adapted filter set (two band-pass filters 850-90m-2p and a long-pass emission filter HQ800795LP; Chroma Technology). All tissue slides were assessed using the same settings to enable visual comparison. Following acquisition, the images were processed with LAS-AF2 software (Leica Microsystems).
6
Fluorescence Microscopy of DAPI-Stained Tissue
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For fluorescence microscopy, selected tissue slides were deparaffinized, and the nuclei were counterstained with 4’,6-diamidino-2-phenylindole (DAPI, Prolong Diamond, Thermo Fisher Scientific). Stained slides were dried in the dark at 4 °C overnight. The slides were imaged using a custom set-up inverted digital fluorescence microscope (DM6B, Leica Biosystems, Wetzlar, Germany) equipped with a highly sensitive Leica DFC9000GT camera (4.2 M Pixel sCMOS camera), a metal halide LED light source (X-Cite 200DC, Excelitas Technologies, Waltham, Massachusetts, USA) for DAPI imaging, and a xenon arc lamp LB-LS/30 (Sutter Instrument, Novato, California, USA) for NIR imaging of IRDye800CW. Image acquisition and processing was done through LAS X software (Leica Biosystems).
7
Quantitative Fluorescence Imaging of Cryo-Anaesthetized Embryos
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Embryos were placed in a black 96-well plate (Falcon; Thermo Fisher, Dreieich, Germany) and cryo-anaesthetized by submersion into ice water. Images for quantitative fluorescence analysis were acquired using the following equipment: a fluorescence stereo microscope (M205 FA, Leica Microsystems, Wetzlar, Germany) in combination with a fluorescence illuminator (X-Cite, 200DC, Excelitas Technologies, Waltham, MA, USA) and a microscope camera (DFC7000 T, Leica Microsystems). Quantification of fluorescence was performed using Fiji ImageJ version 2.1.0. Fluorescent Integrated Density (FID) was determined by counting the number of pixels with fluorescence intensity greater than the threshold (10) and multiplying the count with its mean intensity. For confocal imaging, embryos were anaesthetized through immersion in tricaine (945 µM, Sigma-Aldrich) and immobilized in 1.2% low-melting agarose in a flat-bottom 96-well plate. Confocal images and time-lapse series were acquired using the Celldiscoverer 7 with LSM 900 (Zeiss) together with the 5×/0.35 Plan-APOCHROMAT objective.
8
Live Imaging of Zebrafish Embryos
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Live imaging was carried out on 2, 3, and 5 dpf embryos. Before 24 hpf, 1-phenyl-2-thiourea (75 mM, Sigma, #P7629) was added to inhibit melanogenesis (Karlsson et al., 2001 (link)). For imaging, embryos were anesthetized with 42 mg/l tricaine (Sigma, #A5040) and embedded in 0.8% low melting agarose (Thermo Fischer, #16520100) dissolved in embryo medium. Embryo medium containing tricaine was layered on top of the agarose once solidified for overnight imaging. Additionally, embryos were kept at 28°C during overnight imaging. Embryos were imaged with an inverted Leica SP8 microscope using a ×20/×0.75 dry objective or a ×40/1.1 water immersion objective detection and employing Leica LAS X 3.5.7.23225 software. Scoring of PLs or TD fragments was performed using a Leica M165 FC and an X-Cite 200DC (Lumen Dynamics) fluorescent light source. Confocal stacks were processed using Fiji-ImageJ version 1.52 g. Brightfield images were taken using an Olympus SZX16 microscope and a LEICA DFC450 C camera. Images and figures were assembled using Adobe Illustrator. All data were processed using raw images with brightness, colour, and contrast adjusted for printing.
9
Imaging Techniques for Biological Samples
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Images of live embryos, embryonic fish and adult brain whole mounts were imaged with a Leica SP8 microscope using 10x or 20x dry objectives and 25x and 40x water immersion objectives. Scoring of ccbe1 mutant embryos (Figure 4E ) was performed using a Leica M165 FC and an X-Cite 200DC (Lumen Dynamics) fluorescent light source. Cryosections were imaged with a Zeiss Airyscan 880 using a 63x oil immersion objective. In situ hybridizations (ISH) were imaged with a Nikon Eclipse Ni using 10x and 20x objectives. Brains from double fluorescent ISH were imaged on a Leica SPE with 20x objective. Confocal stacks were processed using Fiji-ImageJ version 1.51g or Imaris version 7.7.2. Images and figures were assembled using Microsoft Power Point and Adobe Photoshop and Adobe Illustrator. All data was processed using raw images with brightness, colour and contrast adjusted for printing.
10
Optogenetic Stimulation of Sensory Neurons
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Channelrhodopsin (ChR2) was expressed in parvalbumin (Pv)-lineage sensory neurons [PvIRES-Cre; R26LSL-ChR2(H134R)-EYFP (Ai32)] and tdTomato in Atoh1-lineage neurons (Atoh1P2A-FLPo; R26FSF-tdTom). Transverse slices were prepared as described above from P10 to P14 mice to avoid expression of ChR2 in PV+ spinal cord neurons. For optogenetic stimulation, blue light (465–495 nm) from an X-Cite 200 DC (Lumen Dynamics) lamp was used. The voltage pulse to activate the shutter had a time threshold of 1.6–2.4 ms. The shutter itself has an average 6 ms latency to open and an average 6 ms latency to close. Therefore, light stimulation-to-oEPSC (optogenetic EPSC) latencies should be considered as apparent latencies. The latency to oEPSC was determined for each cell from the onset of the electrical pulse to open the shutter (7 sweeps/cell for Atoh1-lineage medial cells, 10 sweeps for Atoh1-lineage lateral cells). The standard deviation of the latencies is the jitter value for each cell.
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