Confocal Laser Scanning Microscopy (CLSM)
Confocal laser scanning microscopy is a special type of light microscopy in which a sample is scanned point by point and then these points are assembled pixel by pixel on a computer to create an image.
By Dr Jakob Bierwagen, AHF analyentechnik AG
The most common application of confocal laser scanning microscopy (CLSM) is in conjunction with fluorescence labelling of biological samples. The advantage is the better contrast compared to "ordinary" wide field microscopy, since the fluorescence from other planes of the samples are strongly suppressed due to the confocal imaging technique. Furthermore, due to this separation of fluorescence from the different layers of the sample, a good resolution in the z-direction is achieved and thus very good 3D images can be obtained. In addition, a slightly better resolution is achieved in the x-y plane of the image. Disadvantages are the more complex setup of the microscopes and the (often) longer acquisition time of the images, since they have to be scanned.
Use of laser light
For confocal microscopy, it is very helpful to use lasers because they emit monochromatic, coherent light. In addition, one needs a uniform laser beam (TEM00 mode) to achive the best possible focussing. Especially the coherence and the uniform wavefront in form of a TEM00-mode are important, because only then the light can be focused diffraction limited with the objective.
Pay attention to high quality mirrors and beam splitters
The wavefront must reach the objective uniformly and may not be distorted by bended dichroic beamsplitters or mirrors. Therefore, it is crucial to use high quality mirrors and beamsplitters that have at least a flatness < 1λ/inch (diameter, the wavelength at which this is measured is 633 nm). Otherwise, the excitation focus of the laser will be severely distorted and you will lose significant signal while bleaching the samples unnecessarily due to high excitation power.
Suitable mirrors and beamsplitters
At AHF analysentechnik you can get suitable mirrors and beamsplitters for confocal microscopy in highest quality. We will be pleased to advise you on the selection of the matching optical components. You will benefit from our many years of interdisciplinary experience in microscopy.