A Hamamatsu C9100-13 camera (−94°C, 0.63 MHz, 16-bit ADC) was used typically with EM gain of 50 (conversion factor 0.044 e−/ADU, readout noise 0.470 e− RMS, thermal current 0.014 e−/sec, established experimentally) (see Berry and Burnell 2005 ). IR was blocked with a FF01-750/SP filter (Semrock). A Zeiss AxioObserver Z1 microscope (Carl Zeiss Microscopy) was equipped with a Zeiss plan-apochromat 150× NA1.35 glycerin immersion objective, P-737 piezoelectric stage (Physik Instrumente), Zeiss Colibri LED illuminator, and custom fluorescence cubes (see Supplemental Table S3 for light sources and filters used for wide-field fluorescence imaging). Yeast cells were grown in complete darkness in the CSM + adenine medium (250 rpm at 25°C, final OD600 ≤ 0.3), manipulated only under dim red light (660 nm), and imaged in CellAsic Y04C microfluidic chambers (CellAsic). Narrowband illumination (671 nm; Edmund Optics, no. 65-233) was used for differential interference contrast. To minimize phototoxicity, low-level excitation (∼7 W/cm2, 1- to 5-sec exposure) was used for fluorescence imaging, and 405-nm light (∼0.7 W/cm2, 7–10 sec) was used for tdEos photoconversion. Typically, Z-stacks consisted of 13 steps, 333-nm apart.
C9100 13 camera
Manufactured by Hamamatsu Photonics
The C9100-13 is a high-performance scientific camera manufactured by Hamamatsu Photonics. It features a large-format, back-illuminated sCMOS sensor with a resolution of 2048 x 2048 pixels. The camera is capable of high-speed image acquisition at up to 100 frames per second, making it suitable for a variety of scientific and industrial applications that require high-speed, high-resolution imaging.
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Lab products found in correlation
Axio observer z1 microscope, by Zeiss (1 mentions)
Tcs sp5 2 laser, by Leica camera (1 mentions)
Ultraview vox spinning disk confocal imaging system, by PerkinElmer (1 mentions)
Vectashield, by Vector Laboratories (1 mentions)
Rabbit anti aldh1l1, by Abcam (1 mentions)
Leica dm5500 b epifluorescence microscope, by Leica Microsystems (1 mentions)
Rabbit anti olig2, by Merck Group (1 mentions)
Mouse anti gad67, by Merck Group (1 mentions)
Chicken anti map2, by Abcam (1 mentions)
Rabbit anti foxp1, by Abcam (1 mentions)
Mouse anti tuj1, by Abcam (1 mentions)
Dylight 488 or 549, by Jackson ImmunoResearch (1 mentions)
Axioobserver, by PerkinElmer (1 mentions)
Neurotrace fluorescent nissl stain, by Thermo Fisher Scientific (1 mentions)
Mdy 64, by Thermo Fisher Scientific (1 mentions)
Dmi 6000 b inverted microscope, by Hamamatsu Photonics (1 mentions)
4 protocols using c9100 13 camera
1
Quantitative Live-Cell Fluorescence Imaging
2
Immunofluorescence Imaging of Adherent Cells
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Cells were plated onto 50 µg/ml fibronectin-coated glass coverslips, fixed in 4% paraformaldehyde, and then permeabilized in 0.1% Triton X-100. Cells were then incubated with primary antibody followed by species appropriate Alexa Fluor 488 or 568 secondary antibodies and DAPI and Alexa Fluor 647 Phalloidin (Life Technologies). Samples were then mounted to glass slides using Vectashield (#H-1100; Vector Laboratories). Cells were visualized using an UltraView VOX Spinning Disk Confocal Imaging System (PerkinElmer) with a DM1-6000 SD inverted microscope (Leica) and an electron multiplying charge coupled device C9100-13 camera (63×/NA 1.47 oil; Hamamatsu Photonics) or a TCS SP5 II laser scanning confocal imaging system with five spectral detectors (63×/NA 1.40 oil; Leica). 3D reconstruction and nuclear height measurements were performed with Volocity Software version 6.1.1.
3
Immunofluorescence Imaging of Neural Markers
4
Visualizing C. albicans Vacuole Integrity and Cytoskeletal Dynamics
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C. albicans vacuole integrity was assessed using the lipophilic vacuole membrane dye MDY-64 (Molecular probes, Fisher Scientific) following the manufacturer's recommended procedure. Briefly, cells were grown overnight on RPMI liquid medium with pH 4.5 at 30°C. Cells were pelleted and washed twice with fresh RPMI pH 4.5 and resuspended in the same medium at an OD595 of 0.1. VPA was added at different concentrations (10, 50, and 100 μg/ml). Cells were incubated for 2 h at 30°C under agitation. Aliquots were taken from VPA-treated and non-treated cultures and the MDY-64 was added at a final concentration of 10 μM. Cells were incubated at room temperature for 3 min prior to confocal microscopy visualization. Images were acquired with a 1.3-numerical-aperture (NA) 63x objective on a Leica DMI6000B inverted microscope connected to a Hamamatsu C9100-13 camera.
Pan1-green fluorescent protein (GFP), End3-GFP and LIFEACT-GFP (Epp et al., 2013 (link)) were visualized using confocal microscopy as follow: an overnight culture was diluted in SC supplemented with 10 or 50 μg/ml VPA to an OD595nm of 0.05 and grown for four generations at 30°C under agitation. Cells were imaged as described for the vacuole staining experiments.
Pan1-green fluorescent protein (GFP), End3-GFP and LIFEACT-GFP (Epp et al., 2013 (link)) were visualized using confocal microscopy as follow: an overnight culture was diluted in SC supplemented with 10 or 50 μg/ml VPA to an OD595nm of 0.05 and grown for four generations at 30°C under agitation. Cells were imaged as described for the vacuole staining experiments.
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