Confocal fluorescence laser-scanning microscopy image depth

An alternative to SEM is the use of fluorescence microscopy. The most common technique is confocal laser scanning microscopy (CLSM). CLSM offers two significant advantages over SEM, namely the ability to produce 3D images in situ and the discrimination of species based on fluorescence. Both the x,y resolution and the z penetration depth are to some degree system- and sample-dependant (further discussion of this point can be found in Chapter 4). With the exception of... 

Figure 11.9 Schematic illustration of the principles of confocal fluorescence microscopy singlephoton exdted fluorescence occurs along the entire beam path, however the insertion of a pinhole in the focal plane of the objective ensures that fluorescence from outside the focal point of the laser (dashed line) is prevented from reaching the detector. The laser is scanned in theX-K plane to build up an image of the sample for a given depth. Translation of the sample in the Z-direction allows images (optical sections) to be recorded as a function of depth...

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