Confocal MicroscopyFluorescence Microscopy
Spinning Disk Confocal Microscopy
Confocal microscopy addresses two significant challenges in biological imaging that conventional fluorescence microscopy cannot overcome. Firstly, biological specimens are 3-dimensional structures so to fully understand them we often need to construct 3-dimensional images. Secondly, many processes biologists would want to study occur inside biological structures, but other cell features can block a clear view.
Confocal microscopy uses optical sectioning to take multiple, thin, 2-dimensional slices of a sample to construct a 3-dimensional model from them. This is made possible through the addition of a pinhole into the same focal plane as the sample to block out-of-focus light. Spinning disk confocal microscopy increases the speed of this technique by using multiple pinholes etched into an opaque disk which, when spun, scans the pinholes across the entire image.
Spinning disk confocal microscopy is, essentially, a light rejection technique so one of the main challenges is to collect as many of the emitted photons as possible so light intensity can be reduced to lessen the impact of photobleaching and photodamage on samples, which means that high camera sensitivity is of critical importance.
Extremely sensitive, 95% quantum efficient sCMOS camera with 11 µm pixels and EMCCD level detection.
Go beyond EMCCD for spinning disk confocal microscopy with the back-illuminated Prime 95B, which features an equivalent level of detection but with a faster speed, larger field of view and no EM-gain aging or excess noise.
The Prime 95B allows exposure times to be lowered significantly to increase acquisition speed and reduce photobleaching and photodamage to the lowest levels possible on an sCMOS camera.
The results of a comparison between the Prime 95B and EMCCD for spinning disk confocal microscopy can be seen below.
AlexaFlour488-Phalloidin labelled actin cytoskeletal structure was imaged using spinning disk confocal microscopy.
The signal-to-noise improvement of the Prime 95B can be clearly seen. The Prime 95B image looks cleaner, details are sharper and structural elements are identified that can’t be seen with the EMCCD camera.
High sensitivity, 95% quantum efficient, sCMOS camera with 6.5 µm pixels and 1.0 e– read noise.
The high quantum efficiency and low read noise combined with the balanced 6.5 µm pixel size offers high sensitivity imaging whilst achieving Nyquist sampling with the most popular objective magnifications used for spinning disk confocal microscopy.
The Prime BSI offers an alternative to the Prime 95B when resolution is more important than extreme sensitivity.