Orca ag camera

Images were acquired using an Olympus IX71 inverted light microscope using a 20x/0.45-N.A objective (LUCPFLN) or 4x/0.13-N.A objective (UPFLN). The images were captured using a Hamamatsu Orca-AG camera (C4742-80-12AG) in 1024 × 1024 pixel 8-bit mode and saved as.tif files.

The morphology of neuronal cell bodies, axon debris, regenerating axons, and muscles was based on high-magnification Z-stacks using a Zeiss ×60, 1.4 NA oil-immersion objective. We mounted individual animals on 2% agar pads and anaesthetized them with 3 mM sodium azide, the lowest possible concentration to keep adult animals immobilized. Laser axotomy was performed and worms were recovered within 10 minutes of sodium azide treatment. Recovered worms were placed on fresh plates with bacterial foods and imaged 2.5–12 h (Fig. 1c) or 24 h after axotomy using a Hamamatsu ORCA AG camera.
The axon length of regenerating neurons was quantified 24 h after surgery. Axon lengths were calculated as the actual contour length between the injury site and axon termini measured along the cylindrical surface of each worm, by tracing the axon through a 3-dimensional image stack. P values for the length measurements were calculated using a Student’s t-Test. All experiments were carried out in duplicate but only one result was shown.

For all microscopy experiments, overnight cultures grown at 30 C in YPD medium were re-suspended at a final OD₆₀₀ of 0.2 and allowed to reach mid-log phase prior treatment and image acquisition. Live cell imaging was performed by briefly centrifuging the cells (800 g for 3 min), followed by resuspending in a minimal volume of growth media, spotting onto glass slides and coverslipping prior to imaging. All images were acquired using a Leica DMI 6000 florescent microscope (Leica Microsystems GmbH, Wetzler Germany), equipped with a Sutter DG4 light source (Sutter Instruments, California, USA), Ludl emission filter wheel with Chroma band pass emission filters (Ludl Electronic Products Ltd., NY, USA) and Hamamatsu Orca AG camera (Hamamatsu Photonics, Herrsching am Ammersee, Germany). Images were acquired at 0.2 µM steps using a 63× oil-immersion objective with a 1.4 numerical aperture. Deconvolution and analysis were performed using Velocity Software V4 (Perkin Elmer). For most images, representative images of the middle section and compressed image stack are shown. Numerical insets represent the indicated quantifications of at least 100 cells from 2 to 3 independent experiments unless indicated otherwise.

Conidiospores were inoculated in 35 mm glass-bottom microwell dishes (MatTech) containing minimal media with glucose as the carbon source and urea as the nitrogen source. Imaging was carried out at room temperature using an Ultraview ERS spinning disk confocal system (Perkin-Elmer) fitted with an Orca-AG camera (Hamamatsu) on a TE2000-U inverted microscope (Nikon) with a 60× 1.40 NA Plan Apochromatic objective (Nikon). For some experiments, imaging was with an Ultraview Vox spinning disk confocal system (Perkin-Elmer) fitted with dual C9100-13 cameras (Hamamatsu) run by Volocity software (Perkin-Elmer). Data are displayed as maximal intensity profiles. Benomyl (Sigma) was used at a concentration of 2.4 µg/ml which is sufficient to depolymerize all microtubules [131] (link). Image analysis, quantification, pixel line intensity profiles and kymograph generation was carried out using ImageJ freeware (Rasband, WS, ImageJ, US National Institutes of Health, Bethesda, MD, USA, http://rsb.info.nih.gov/ij/, 1997–2008). To quantify histone H1 fluorescence levels the average pixel intensity less the background was determined for an identical sized area for each cell. Each time series was aligned for mitotic entry and values normalized relative to the last frame in G2 before mitotic entry which was set to 100%. Data points represent the mean +/− standard deviation.

H&E staining was performed on 4% paraformaldehyde fixed and paraffin embedded tumor sections (5 μm thick). Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining was executed according to the manufacturer’s recommendations using the ApopTag Peroxidase In Situ Apoptosis Detection kit (Millipore). Bright field images were taken on a Zeiss wide-field Axioplan microscope equipped with a 40×/1.3 oil Plan-NEOFLUAR lens (Carl Zeiss Microimaging) and SPOT Insight QE color camera (Diagnostic Instruments). TUNEL optical density was quantitated by taking the ratio of TUNEL positive staining to the total tissue area. CD31 (1:100, Abcam) immunofluorescence staining was performed with antigen retrieval using 20 mg/kg proteinase K. An anti-rat Alexa Fluor 647 secondary antibody was used (1:300, Life Technologies). Images were taken with an Axiovert 200M microscope equipped with a 20x/0.8 Plan-APOCHROMAT lens (Carl Zeiss Microimaging) and ORCA-AG camera (Hamamatsu Photonics) using Micro-Manager software[22 ]. CD31 blood vessel density was quantitated by taking the ratio of CD31-red positive staining to the total DAPI-blue positive staining.