Laser Scanning Confocal Microscopy (LSCM)

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Technique in brief

Laser scanning confocal microscopy (LSCM)is a system used in epifluorescence and reflected light imaging. A finely focused beam of laser light is scanned across a sample and the resultant light emitted passed through a pinhole aperture to exclude any out of focus light. The image thus produced includes only the plane of focus for a given objective lens (hence a clearer image). Additionally, by varying the stage height, a series of sequential images through the thickness of a single sample can be collected and used to project a three-dimensional image.

A LSCM will typically include at least 3 different wavelength lasers which can be used simultaneously or sequentially. For imaging fluorescence in a sample this means multiple stains (fluorophores) can be used to highlight specific features of interest.

Current instrumentation

The current LSCM at the AAC is a BioRad Radiance 2000 with the following configuration:

Laser configuration

Argon ion - 488nm(14mW); 514nm(11mW)
Green HeNe - 543nm(1.5mW)
Red laser diode - 638nm(5mW)

Emission Filters

Detector (photomultiplier - PMT) Emission filters (wavelength in nm)

1 Open; 488/10; 500LP; 515/30; 530/60;

2 Open; 515/30; 530/60; 570LP; 590/70; 600/50; 600LP

3 Open; 660LP

It is based around the light optics of a Nikon E600 upright microscope with tungsten transmitted and reflected light sources and a mercury lamp. Current lenses available are; 10, 20 and 40x (cover slip corrected); 50 and 100x (air); 60x oil immersion and 40x (water immersion).

Applications

LSCM is used in many applications for imaging biological specimens. Although it cannot improve on magnifications available to conventional fluorescence microscopy the sharper, clearer images obtained greatly improving image quality. Also, as the system acquires images digitally, timed images may be automatically generated. In samples that are relatively transparent to the lasers the ability to create sequential images through the depth of a specimen (“z-series or stacks) produce 3-dimensional information provides real spatial context to the features of interest. Opaque materials can also be imaged this way, essentially recording reflected light. Thus it is also a useful technique in material sciences for modeling material in 3D.

Sample requirements

For fluorescence imaging specimens need by fixed and stained using fluorphores that will not only attach (label) to the relevant feature of interest but are excited by and emit at wavelengths appropriate to the configuration of lasers and detector filters available.

Samples for reflected laser light applications need be of a size suitable to be viewed on a conventional microscope.