Confocal Microscopy
Confocal Microscopy (+)
Confocal microscopes work to block out of focus light by utilising the pinhole and therefore generate clearer images than those from a widefield system. Confocals are either laser scanning confocal microscope (LSCM) configurations utilising a detector and point scanning system, or spinning disk microscope which uses a array of pinholes configured on a disk which spins.
Confocal Microscope Systems
Leica SP5 LSCM
Zeiss 800 LSCM
Zeiss 880 LSCM
Zeiss 980 LSCM with MP
Zeiss 980 LSCM with MP
The Zeiss 980 LSCM microscope is the newest confocal system in the facility, having additional capabilities alongside standard confocal mode, namely; LSM plus, Airyscan and multiphoton capabilities.
General Specification
Inverted microscope
Environmental control - temperature, humidity & CO2
Definite focus & Software focus
Airyscan detector
Objective Lenses
- 25 x - NA 0.8
- 63 x - NA 1.4
Excitation Laser Lines
405nm
488nm
543nm
639nm
Mai Tai HP Ti:Sapphire multiphoton laser (Tuning Range: 690 - 1040nm)
Zeiss Airyscan
Airyscan technology delivers superresolution with high sensitivity (4-8x) at 140 nm laterally and 400 nm axially resolution (1.7x) whilst preserving precious emission light normally rejected at a closed pinhole. This gentle superresolution imaging with increased sensitivity and speed reduces phytotoxicity and bleaching and allows long-term live cell imaging without artefacts caused by influencing the sample’s viability and biological function.
Instruments
What does the Airyscan do
The Airyscan detector draws on the fact that a fluorescence microscope will image a point-like source of light as an extended Airy disk or Airy pattern.
In a standard confocal microscope the out-of-focus emission light is rejected at the pinhole. You get a sharper image by closing the pinhole to reject out-of-focus light, however, it’s also much dimmer as a great deal of light is then lost. The smaller the pinhole, the higher the resolution, but the greater the loss in light. Normally on a confocal a compromise is required.
The Airyscan solves this trade off between resolution and light efficiency by imaging the entire Airy disk onto a concentrically-arranged hexagonal detector array consisting of 32 single detector elements, all of which act like very small pinholes. The signals from all the detector elements are then reassigned to their correct position, producing an image with increased signal-to-noise ratio and super-resolution.
Airyscan imaging capitalises on the scanning and optical sectioning capabilities of a confocal and therefore works with standard samples, standard dyes and importantly even with thicker samples such as tissue sections or whole animal mounts that need a higher penetration depth.