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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

+ functional imaging capabilities; FLIM and FCCS
staff member using Leica SP5 confocal microscope

Zeiss 800 LSCM

+ Airyscan
a student sitting at a confocal microscope

Zeiss 880 LSCM

+ Airyscan
Zeiss 880 laser scanning confocal microscope

Zeiss 980 LSCM with MP

+ Airyscan + Multiphoton
Confocal microscope

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.

Confocal microscope

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

Zeiss 980 LSCM

Zeiss 880 LSCM

Ziess 800 LSCM

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.