Live worms, colorimetric WISH stains, and whole-worm H3P stains were imaged on a Leica M165 fluorescent dissecting microscope. dFISH stains were imaged on a Leica DMIRE2 inverted fluorescence microscope with a Hamamatsu Back-Thinned EM-CCD camera and spinning disc confocal scan head. H3P cell counts were quantified using freely available ImageJ software (http://rsb.info.nih.gov/ij/ ) with the cell counter function, and muscle lesions were labelled by hand, using control RNAi animals as a reference so as to only label muscle gaps larger than those found in controls, and quantified using the ImageJ area measurement function. Piwi-1+ and H3P+ cell counts were performed in cross-section using Imaris (Bitplane, South Windsor, CT, USA). Cross-sections were taken from the same regions in control and knockdown animals (examples shown in Fig 3A ), and “normal” and “ectopic” regions were determined manually using DAPI staining to mark the boundary (as shown in Fig 3B ). Collagen+ cells, TUNEL+ cells, and neoblast subclass markers were also quantified using Imaris. Significance was determined by a two-tailed unequal variance student’s t-test. All images were post-processed in a similar manner using Adobe Photoshop. Heatmaps were made in R studio using data downloaded from https://compgen.bio.ub.edu/PlanNET/planexp [61 (link)].
Back thinned em ccd camera
Manufactured by Hamamatsu Photonics
The Back-Thinned EM-CCD camera is a specialized imaging device designed for low-light applications. It features a back-thinned, high-quantum-efficiency sensor that enhances the camera's sensitivity and enables the detection of faint signals. The camera utilizes electron multiplying technology to amplify the signal, providing improved signal-to-noise ratio and enabling the detection of single photon events.
Automatically generated - may contain errors
Lab products found in correlation
Dmire2 inverted fluorescence microscope, by Hamamatsu Photonics (5 mentions)
Axiovert 200m microscope, by Zeiss (3 mentions)
Sp8 lightning confocal dmi6000 microscope, by Leica (2 mentions)
Motorized xy stage, by Leica (2 mentions)
Superz galvo, by Leica (2 mentions)
M165 fluorescent dissecting microscope, by Leica (1 mentions)
Ep0162, by Thermo Fisher Scientific (1 mentions)
Annexin 5 alexa fluor 647 conjugate, by Thermo Fisher Scientific (1 mentions)
Gentamicin, by Thermo Fisher Scientific (1 mentions)
Volocity software, by PerkinElmer (1 mentions)
Gentamicin, by Wisent (1 mentions)
Dsu spinning disc confocal, by Olympus (1 mentions)
Volocity 6, by PerkinElmer (1 mentions)
11 protocols using back thinned em ccd camera
1
Quantifying Planarian Regeneration Markers
2
Comprehensive Imaging Techniques for Developmental Biology
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Whole-mount ISH (WISH), and double fluorescent ISH (dFISH), and immunostainings were performed as previously described [29 (link),73 (link),74 (link)]. Colorimetric WISH stains were imaged on a Leica M165 fluorescent dissecting microscope. dFISH and fluorescent phospho-histone H3 (rabbit monoclonal to H3ser10p, 1:500, Millipore) immunostains were imaged on a Leica DMIRE2 inverted fluorescence microscope with a Hamamatsu Back-Thinned EM-CCD camera and spinning disc confocal scan head. BrdU (Sigma B5002-5G, 25 mg/ml) was dissolved in 50% ethanol and fed to animals and was stained as previously described [24 ]. TUNEL was performed as previous described [39 (link)] with the Terminal Deoxynucleotidyl Transferase enzyme (Thermo, EP0162). All cell counts and co-localizations were quantified using freely available ImageJ software (http://rsb.info.nih.gov/ij/ ) with the cell counter function. Positive cells were visually distinguished manually. Significance was determined by a two tailed unequal variance pairwise student’s t-test. All images were post-processed in a similar manner using Adobe Photoshop.
3
BrdU Incorporation and In Situ Hybridization
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BrdU (Sigma B5002-5G, 25 mg/ml) was dissolved in 50 % ethanol and injected into the gut of animals. Animals were fixed 4 h later and BrdU was stained as previously described [20 (link)]. In situ hybridizations were performed as previously described [18 (link), 55 (link)]. Colorimetric WISH samples were imaged on a Leica M165 fluorescent dissecting microscope. dFISH and tFISH samples were imaged on a Leica DMIRE2 inverted fluorescence microscope with a Hamamatsu Back-Thinned EM-CCD camera and spinning disc confocal scan head with Volocity software. Raw images were opened in ImageJ and saved as tiffs and resolution, brightness, and contrast were adjusted in Adobe Photoshop.
4
Visualizing Listeria Invasion in HeLa Cells
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HeLa cells were plated at 2×105 cells per well in 6-well tissue culture plates with glass coverslips 48 h prior to infection. At 24 h prior to infection, cells were transfected with LifeAct-RFP. Cells were then infected with wild type Lm expressing GFP (DP-L1039) at an MOI of 100 in DMEM. After 60 min of invasion at 37°C, cells were washed three times with phosphate buffered saline (PBS) with Calcium and Magnesium (Wisent #311-420-CL) followed by the addition of growth media containing 50 μg/ml Gentamicin (Wisent #400-130-IG). At 6 h post infection, coverslips were washed with PBS with Calcium and Magnesium (Wisent #311-420-CL) and transferred into spaceships and RPMI-1640 (Wisent #350-025-CL) with 10% heat-inactivated FBS (Wisent), 50 μg/ml Gentamicin, 2.5 mM CaCl2 and 2% (v/v) Annexin V Alexa Fluor 647 Conjugate (Invitrogen). HeLa cells were maintained at 37°C during imaging. A Leica DMIRE2 inverted fluorescence microscope equipped with a Hamamatsu Back-Thinned EM-CCD camera and spinning disk confocal scan head with a 63X objective and LSM 510 software was used. Volocity software (Improvision) was used to acquire images.
5
Spinning Disk and Lightning Confocal Imaging
6
Visualizing Lysosome Motility Dynamics
7
Spinning Disk Confocal Microscopy Protocol
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Unless otherwise indicated, cells were imaged using a Quorum spinning disk microscope with a ×63 oil immersion objective (Leica DMIRE2 inverted fluorescence microscope equipped with a Hamamatsu Back-Thinned EM-CCD camera or Hamamatsu CMOS FL-400 camera, spinning disk confocal scan head) and Volocity 6.3 acquisition software (Improvision)). Confocal z-stacks of 0.3 μm were acquired and images were analyzed with Volocity 6.3 software.
8
Multimodal Imaging and Quantification of Worm Cells
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WISH, dFISH, and immunostaining were performed as previously described (Currie et al., 2016 (link)). Colorimetric WISH stains were imaged on a Leica M165 fluorescent dissecting microscope. dFISH and fluorescent mouse-anti-PIWI-1 (gift from Dr. Jochen Rink) stains were imaged on a Leica DMIRE2 inverted fluorescence microscope with a Hamamatsu Back-Thinned EM-CCD camera and spinning disc confocal scan head. Cell counts and co-localizations were quantified using freely available ImageJ software (http://rsb.info.nih.gov/ij/ ). Significance was determined by a two-tailed Student’s t-test. All experiments were, at minimum, triplicated and at least five worms were used per stain and per time point. All labeling images were post-processed using Adobe Photoshop.
9
Imaging HCV Localization in Organoids
10
Spinning Disk and Lightning Confocal Imaging
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Spinning disk confocal imaging was performed with a Quorum spinning disk confocal head on a Zeiss Axiovert 200M microscope, equipped with a 63× NA 1.4 oil objective and 25× NA 0.8 multi-immersion objective, a 1.5× tube lens, and a back-thinned EM-CCD camera (C9100–13, Hamamatsu). Acquisition settings were controlled using Volocity software and were acquired at equal laser and exposure settings within experiments across compared conditions. Lightning microscopy was performed on a Leica SP8 Lightning Confocal DMI6000 microscope utilizing a 100× 1.4 NA (O, STED) objective and two HyD detectors. Acquisitions were driven by a Leica motorized XY stage and Leica SuperZ Galvo with Adaptive Focus Control. Acquisition settings were controlled using the Leica LAS, Lightning Module software, with matched settings within experiments.
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