Fluorescence microscopy was performed using a DMIRB inverted microscope (Leica Microsystems), with a cooled charge-coupled device camera (Princeton Instruments). Images were collected with a 20× objective. To perform ‘prismless’ TIRF microscopy a 60×1.45 numerical aperture oil-immersion objective (Olympus America) was used. The evanescent field decay length was 100–250 nm. MetaMorph software (Universal Imaging) was used for image processing and quantification as previously described [35 (link)].
Cooled charge coupled device camera
Manufactured by Teledyne
Sourced in Israel, United States
The Cooled charge-coupled device camera is a specialized imaging device that utilizes a charge-coupled device (CCD) sensor. The camera is designed to operate at low temperatures, typically achieved through integrated cooling mechanisms, in order to minimize thermal noise and improve image quality. The core function of this camera is to capture high-quality, low-noise images for scientific and research applications.
Automatically generated - may contain errors
Lab products found in correlation
Metamorph software, by Universal Imaging (3 mentions)
1.45 numerical aperture oil immersion objective, by Olympus (2 mentions)
Dmirb inverted microscope, by Leica (2 mentions)
Dh 2000 bal, by OceanOptics (1 mentions)
V 670 uv vis spectrometer, by Jasco (1 mentions)
Zyla scmos 5.5 megapixel camera, by Oxford Instruments (1 mentions)
Axioplan microscope, by Zeiss (1 mentions)
Inverted microscope, by Leica (1 mentions)
Softworx, by Cytiva (1 mentions)
Deltavision elite imaging system, by Cytiva (1 mentions)
Inspeck, by Thermo Fisher Scientific (1 mentions)
Wib cube, by Olympus (1 mentions)
5 protocols using cooled charge coupled device camera
1
Prismless TIRF Microscopy for Imaging
2
Krypton Sorption and Optical Characterization
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Krypton sorption isotherms were recorded on a BEL Japan Inc. Belsorp-max system at 77 K. Samples were dried on a Belprep-vac II at 140 °C and ~2 Pa overnight prior to the gas sorption measurements. Optical absorption was measured using a JASCO V-670 UV-VIS spectrometer. The ionization energy of SpCz and PSpCz was determined by atmospheric pressure photoelectron spectroscopy using a Riken Keiki AC-2.
For determining transmission and reflection spectra a Deuterium Halogen lamp (DH-2000-BAL, OceanOptics) and a spectrometer with a range from 186 nm to 1041 nm (USB 2000 + XR1-ES) were used.
The photoluminescence (PL) of the samples was measured in the setup shown inFig. 2a . For excitation, a diode pumped solid state laser (λ = 355 nm, 11 mW/cm2 (link)) or a UV laser diode (λ = 405 nm, 35 mW/cm2 (link)) was used. The PL signal was coupled into a monochromator and detected by a cooled charge coupled device camera (Princeton Instruments).
For determining transmission and reflection spectra a Deuterium Halogen lamp (DH-2000-BAL, OceanOptics) and a spectrometer with a range from 186 nm to 1041 nm (USB 2000 + XR1-ES) were used.
The photoluminescence (PL) of the samples was measured in the setup shown in
3
High-Resolution TIRF Microscopy Protocol
4
Visualizing Membrane Protein Localization
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Images of Pma1-RFP and Can1-GFP distribution at the plasma membrane were taken with a DeltaVision Elite Imaging system (Applied Precision, Issaquah, WA). A Z-stack was acquired with 200-nm intervals, and the images were deconvolved in SoftWoRx (Applied Precision, Issaquah, WA). Images of DHE were taken with an inverted microscope (Leica, Deerfield, IL) equipped with a cooled charge-coupled device camera (Princeton Instruments, Trenton, NJ) and MetaMorph software (Universal Imaging, West Chester, PA). To visualize DHE, a specially designed filter cube (Chroma Technology, Brattleboro, VT) was used with a 355-nm (20-nm bandpass) excitation filter, a 365-nm long-pass dichromatic filter, and a 405-nm (40-nm bandpass) emission filter. All other images were taken with an Axioplan microscope (Carl Zeiss, Thornwood, NY) equipped with a Zyla sCMOS 5.5-megapixel camera (Andor, Belfast, United Kingdom) and micromanager software (University of California, San Francisco, San Francisco, CA). Filipin was imaged using a 4′,6-diamidino-2-phenylindole filter set; GFP-tagged proteins were imaged using a GFP filter set; RFP-tagged proteins and FM4-64 were imaged using an RFP filter set. Images were merged and pseudocolored in ImageJ (National Institutes of Health, Bethesda, MD).
5
FITC-LG Cellular Uptake Imaging
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Samples (1 × 107 cells) were incubated with FITC-LG (1 mg) at the designated temperature for 30 min. The cells were centrifuged and washed with PBS (pH 7.4) three times. Samples were fixed with paraformaldehyde (4%) for 30 min and washed with PBS (pH 7.4). Samples were then examined using a confocal microscope; optical sections were obtained with an epifluorescence inverted microscope (Olympus IX-70) equipped with a cell scanner (EPR™ system, Scanalytics, Billerica, MA) and a water immersion apochromatic lens (60x PSF: 1.2-NA, Olympus, France). Scanning along the optical axis was performed with a piezoelectric z-axis focus device (z spacing 0.25 mm). Images were collected on a cooled charge-coupled device camera (12 bits, Princeton Instruments, USA). With a filter set (WIB cube, Olympus), we selected the fluorescence excitation (460–490 nm) and the integral part of the emission spectrum (BA515). An image intensity-calibration kit (InSpeck, Molecular Probes, Eugene City, OR) was used for calibration. A blank image of the detector dark current and the background were removed from each image acquired pixel by pixel.
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