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Dfc9000 gtc

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The Leica DFC9000 GTC is a high-performance camera for microscopy applications. It features a large sensor, high-resolution image capture, and advanced imaging capabilities. The core function of the DFC9000 GTC is to provide detailed and accurate digital imaging for a variety of microscopy workflows.

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Lab products found in correlation

Dmi8 microscope, by Leica (3 mentions) Spectra x light engine, by Leica (2 mentions) Dmi8 epifluorescence microscope, by Lumencor (2 mentions) Thunder imager 3d cell culture epifluorescence microscope, by Leica (2 mentions) Vt1000, by Leica (1 mentions) Permount, by Thermo Fisher Scientific (1 mentions) Citrisolv, by Thermo Fisher Scientific (1 mentions) Aperio at2, by Leica (1 mentions) Spectra x, by Leica (1 mentions) Matlab script, by MathWorks (1 mentions) Las x software, by Leica (1 mentions) Dmi8 inverted fluorescence microscope, by Leica (1 mentions) Lsm 880, by Zeiss (1 mentions) Lsm 800, by Zeiss (1 mentions)

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DFC9000 GTC camera (19 citations) DFC9000 (11 citations) DFC9000 GTC sCMOS camera (7 citations) DFC9000 GTC CMOS camera (4 citations) Leica DFC9000 GTC VSC-09991 camera (3 citations)

11 protocols using dfc9000 gtc

1

Picrosirius Red Staining of Muscle Tissue

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Picrosirius red staining was done similarly to other studies [14 (link), 15 (link), 38 , 39 (link)]. Before sectioning, fixed muscles were embedded in 4% agarose. 200 μm thick longitudinal sections were sliced using Leica VT1000S. Sections were washed, dried for 60 min, and stained for 60 min in 0.1% (weight/volume) Direct Red 80 (Fisher) dissolved in saturated aqueous picric acid (Fisher). Sections were washed twice for 60 s in 0.5% acetic acid, then dehydrated with three 60 s washes of 100% ethanol. The sections were then cleared using CitriSolv (Fisher Scientific) for 3 min, and blotted with Permount (Fisher Scientific).
Full longitudinal sections were imaged via a 20X objective with brightfield illumination on a Leica DMi8 microscope and DFC9000GTC camera. Linearly polarized light imaging required a rotating polarizer in the beam path before and after the sample. A sequence of ten tiling scans were imaged at angles from 0 to 90° in increments of 10°. ECM architecture was quantified using a custom MATLAB script providing MicroECM alignment and MacroECM deviation parameters as described previously [14 (link)] in conjunction with the polarized light images.
Brashear S.E., Wohlgemuth R.P., Hu L.Y., Jbeily E.H., Christiansen B.A, & Smith L.R. (2022). Collagen cross-links scale with passive stiffness in dystrophic mouse muscles, but are not altered with administration of a lysyl oxidase inhibitor. PLOS ONE, 17(10), e0271776.
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2

Autofluorescence Imaging of Unlabeled Tissue Slides

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The autofluorescence images of the unlabeled autopsy tissue slides were acquired using a Leica DMI8 microscope with a 40×/0.95 NA objective lens (Leica HC PL APO 40×/0.95 DRY), controlled using Leica LAS X microscopy automation software. Two fluorescence filter cubes, DAPI (Semrock OSFI3-DAPI5060C, EX377/50 nm EM 447/60 nm) and TxRed (Semrock OSFI3-TXRED-4040C, EX 562/40 nm EM 624/40 nm), were used to capture the autofluorescence images at different excitation-emission wavelengths. Each image was captured with a scientific complementary metal-oxide-semiconductor (sCMOS) image sensor (Leica DFC 9000 GTC) with an exposure time of ∼100 ms for the DAPI channel and ∼300 ms for the TxRed channel. Following the standard histochemical H&E staining procedure, the stained tissue slides were then digitized by a brightfield slide scanner (Leica Biosystems Aperio AT2).
Li Y., Pillar N., Li J., Liu T., Wu D., Sun S., Ma G., de Haan K., Huang L., Zhang Y., Hamidi S., Urisman A., Keidar Haran T., Wallace W.D., Zuckerman J.E, & Ozcan A. (2024). Virtual histological staining of unlabeled autopsy tissue. Nature Communications, 15, 1684.
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3

Spatially Resolved Transcriptomics: HybISS Protocol

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HybISS was performed as reported by Gyllborg et al.5 (link). The protocol and materials used were as described in protocols.io (10.17504/protocols.io.xy4fpyw). Probe sequences are included in Supplementary Tables 3 and 4. For subtype/cell state markers, kits from 10x Genomics were provided along with an accompanying protocol (High Sensitivity kit). In summary, the tissue was fixed, and then the direct RNA probe mixture was added (incubated overnight at 37 °C). The section was subsequently washed, and ligation mix was added (incubated at 37 °C for 2 h). After washing, rolling circle amplification was performed at 30 °C overnight. Lastly, rounds of labeling and stripping were done for detection.
Imaging was performed with a Leica DMi8 epifluorescence microscope equipped with an LED light source (Lumencor SPECTRA X), sCMOS camera (Leica DFC9000GTC) and ×20 objective (HC PL APO, 0.80). Each field of view (FOV) was imaged with 24 z-stack planes with 0.5 μm spacing and 10% overlap between FOVs.
Li X., Andrusivova Z., Czarnewski P., Langseth C.M., Andersson A., Liu Y., Gyllborg D., Braun E., Larsson L., Hu L., Alekseenko Z., Lee H., Avenel C., Kallner H.K., Åkesson E., Adameyko I., Nilsson M., Linnarsson S., Lundeberg J, & Sundström E. (2023). Profiling spatiotemporal gene expression of the developing human spinal cord and implications for ependymoma origin. Nature Neuroscience, 26(5), 891-901.
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4

Quantifying Collagen Fibers in Muscle Tissue

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Picrosirius red staining and imaging was done similarly to previous studies [20] (link), [21] (link), [23] , [69] (link), [70] . Diaphragm, EDL, and soleus muscles were sectioned at −20 °C into 10 μm transverse slices. Sections were placed on glass slides and circled with a hydrophobic PAP pen before fixing in 4% paraformaldehyde for 10 min. Sections were then rinsed with distilled water three times for 5 min each and air dried for 15 min. Samples were stained in picrosirius red solution for 60 min and then washed twice in acidified water for 1 min each. Three 1-minute washes of 100% ethanol were applied to dehydrate the samples. Citrisolv was then applied for 3 min and Permount was left on the sections before a coverslip was placed on top.
Transverse sections were imaged with a 20X objective using brightfield illumination on a Leica DMi8 microscope and DFC9000GTC camera. Linearly polarized light imaging was utilized with a rotating polarizer in the beam path before and after the sample. Sirius Red Area and Collagen fiber density were quantified using a custom MatLab script (Mathworks, Natick, MA) and as previously described [21] (link).
Wohlgemuth R.P., Feitzinger R.M., Henricson K.E., Dinh D.T., Brashear S.E, & Smith L.R. (2023). The extracellular matrix of dystrophic mouse diaphragm accounts for the majority of its passive stiffness and is resistant to collagenase digestion. Matrix Biology Plus, 18, 100131.
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5

Microscopic Imaging of Coacervate Droplets

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Microscopy was performed on a Leica Thunder inverted widefield microscope equipped with a sCMOS camera Leica DFC9000 GTC using a ×63/NA 1.47 objective. Fluorescence channels were λEx = 484–496 nm/λEm = 507–543 nm for FAM, and λEx 629–645 nm/λEm 669–741 nm for Cy5. The sample stage was warmed to 30°C. Samples were loaded into clear-bottomed 384-well plates (Greiner µclear, medium binding). Coacervate droplets were formed by combining a solution of RNA with a solution containing poly(L-lysine), buffer and magnesium chloride and mixing using a pipette in a PCR tube. The mixture was incubated on ice for 5 min, then 5 µL was loaded into the well plate. Individual wells were sealed with a drop of silicon oil to prevent evaporation. After loading, plates were immediately incubated at 30°C. Imaging was performed as soon as the coacervate suspension had settled, typically first at 30 min after mixing. A 3 × 3 image grid, centred at the middle of the well, was captured for each sample at various time points. In most cases multiwell plates were passivated using Pluronic F-68 to prevent droplet wetting and adhesion.
Le Vay K.K., Salibi E., Ghosh B., Tang T.D, & Mutschler H. (2023). Ribozyme activity modulates the physical properties of RNA–peptide coacervates. eLife, 12, e83543.
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6

Multiplexed Spatial Transcriptomics Imaging

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Images were acquired with Leica DMi8 epifluorescence microscope equipped with an external LED light source (Lumencor SPECTRA X light engine), automatic multi-slide stage (LMT200-HS), sCMOS camera (Leica DFC9000 GTC), and objective (HCX PL APO 40 X/1.10 W CORR). A series of images (10% overlap between neighboring images) were obtained, and maximum intensity projected to two-dimensional images. These images were aligned between cycles and stitched together using Ashlar algorithm. Stitching was followed by retiling to get smaller (6000x6000 pixel) images. Those images were used for decoding using PerRoundMaximumChannel Decode Spots Algorithm. The resulting spots were filtered based on minimum quality. The preprocessing (Langseth et al., 2021 ) and decoding (Langseth and Marco, 2021 ) pipeline can be found on the Moldia GitHub page (https://github.com/Moldia).
Yaghmaeian Salmani B., Lahti L., Gillberg L., Jacobsen J.K., Mantas I., Svenningsson P, & Perlmann T. (2024). Transcriptomic atlas of midbrain dopamine neurons uncovers differential vulnerability in a Parkinsonism lesion model. eLife, 12, RP89482.
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7

Multispectral Imaging of Biological Specimens

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All images were obtained with a Leica DMi8 epifluorescence microscope equipped with an external LED light source (Lumencor® SPECTRA X light engine), automatic multi-slide stage (LMT200-HS), sCMOS camera (Leica DFC9000 GTC), and objectives (HC PL APO 10X/0.45; HC PL APO 20X/0.80; HCX PL APO 40X/1.10 W CORR). Multispectral images were captured with microscope equipped with filter cubes for 6 dye separation and an external filter wheel (DFT51011). Image scanning was performed by outlining ROIs that could be saved for multi-cycle imaging tiled imaging with 10% overlap. Z-stack imaging of 10 µm at 0.5 µm steps to cover the depth of the tissue.
Lee H., Marco Salas S., Gyllborg D, & Nilsson M. (2022). Direct RNA targeted in situ sequencing for transcriptomic profiling in tissue. Scientific Reports, 12, 7976.
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8

Epifluorescence Microscopy Protocols

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Epifluorescence microscopy was performed using a Leica THUNDER Imager 3D cell culture epifluorescence microscope equipped with a Leica LED8 multi-LED light source and sCMOS camera (Leica DFC9000 GTC). All epifluorescence images were acquired without THUNDER computational clearing and are displayed with instrument noise subtracted but without background subtraction. See Table S6 for details on the objective, excitation wavelengths and filters used for each experiment.
Schwarzkopf M., Liu M.C., Schulte S.J., Ives R., Husain N., Choi H.M, & Pierce N.A. (2021). Hybridization chain reaction enables a unified approach to multiplexed, quantitative, high-resolution immunohistochemistry and in situ hybridization. Development (Cambridge, England), 148(22), dev199847.
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9

Live Cell Fluorescence Imaging Workflow

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Video recording was performed on a Leica DMi8 inverted fluorescence microscope equipped with an environmental control chamber for live cell imaging. The light-emitting diode excitation power was set to 1% and additionally attenuated by 85% using a neutral density filter to minimize light exposure. Images were acquired in a rapid time-lapse fashion (1 Hz) over 3 min with the excitation shutter closed between the frames, using Leica LAS X software and a monochrome cooled camera (Leica DFC9000GTC). In the thapsigargin experiments, a field of view was selected blindly and imaged before and 5 min after application of the inhibitor. In other experiments, the starting field of view was similarly selected and the culture imaged following a predetermined geometrical pattern around the first field, using the computerized stage control. Each field of view was recorded once, and a field diaphragm was used to limit excitation light exposure to the area being currently imaged.
Maly I.V., Hofmann W.A., & Pletnikov M. (2021). Experimental and computational analysis of calcium dynamics in 22q11.2 deletion model astrocytes.
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10

Gel-Liquid Phase Separated Vesicle Formation

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Gel-liquid phase separated vesicles were prepared from mixtures of DOPC:DPPC in a 1:1 molar ratio 73 with 0.5 mol% DiD. 20 nmol of total lipids were evenly distributed on Pt electrodes and dried under a vacuum for 15 minutes. Electroformation (300 Hz, 2.5 V, 65 °C) was conducted in 1 mM HEPES pH 7
and 300 mM sucrose for 3 hours followed by 30' of lower frequency current to promote GUV formation (2 Hz, 2.5 V, 65 °C).
GUVs were visualized in a home-made microscopy chamber with temperature control in isosmotic solution of glucose and buffer. A Leica DMi8 confocal microscope coupled with a camera (Leica DFC9000 GTC) was used for image acquisition. For both confocal as well as camera acquisition a 63x water-immersed objective was used.
For confocal image acquisition, 488 nm (SybrGold) and 635 nm (DiD) lasers were used at low power (<0.5%) to avoid photobleaching and the signal was collected using hybrid detectors (500 -550 nm for SybrGold and 650 -750 nm for DiD). Z-scans were acquired as 4 -8 averaged images per layer and Zprojected as a sum of the slides using FIJI software 74 .
Temperature ramps were captured using a camera. 1.5 sec of exposure without binning was used to acquire each channel and measurements were repeated every 5 seconds. Temperature changes were registered and calculated to be 3.4 °C/min. Image analysis was conducted in FIJI software.
Czerniak T., & Sáenz J. (2021). Lipid membranes modulate the activity of RNA through sequence-dependent interactions.
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