Facts about Confocal Laser Scanning Microscopy
In CLSM, a laser light beam is focused to a diffraction-limited spot in the focal plane. Images are captured by scanning the beam over the focal plane, and recording the variations in fluorescence intensity.
A pinhole aperture in front of the fluorescence detectors, placed to correspond with the focal plane (hence confocal), excludes most of the out-of-focus fluorescence. By recording fluorescence from mainly the focal plane, you acquire an image that is an "optical section" of the specimen.
By scanning consecutive focal planes, a series of optical sections can be acquired. These optical sections can be assembled in a ”z-stack”, which can be used for 3D-analysis and 3D-rendering of the fluorescent structures.
What's so good with confocal microscopy?
The immediate gain in confocal microscopy, when compared with conventional epifluorescence microscopy, is the superior depth resolution. With a high-resolution objective (numerical aperture 1,3-1,4) it is possible to acquire an image in which most of the signal comes from a ca. 0,5 µm tick optical section! With the epifluorescence microscope (using the same objective) the image would contain light from the entire thickness of the specimen. Thus, the confocal microscope improves the signal-to-noise ratio (S/N) tremendously, making it easier to distinguish small structures in the specimen.
Please note, though, that resolution in the image plane (x/y) is only about 25% better than in conventional light microscopy. The improvement depends on that coherent laser light can be focussed into a smaller spot than non-coherent light from e.g. a Hg-lamp – in all other respects is it "the same microscope optics". However, the combination of a dramatic improvement of S/N due to the optical sectioning, and a marginal improvement in lateral resolution, gives a superior detection of e.g. co-localized fluorescent markers.
And which are the limitations?
It is of course not possible to acquire high-resolution images of deep structures at any depth in a specimen. First, the objective's working distance (distance between lens surface and focal point) is a physical constraint. In practice, however, it is more usual that factors like optical density and variations in the refractive index set an absolute limit at depths around 200 µm in biological specimens. If you need to study deeper volumes you should consider using multiphoton microscopy, which may work down to approximately 500 µm.
Confocal laser scanning microscopy is a good tool for studying different types of dynamic processes. However, the scan speed of the laser beam limits how fast process you can study. For studies of biological processes at the cellular level (and some biochemical processes) it may often suffice to "zoom in" and scan only a small area/volume, thus achieving an adequate frame rate. If this approach is not fast enough, you should consider using conventional (wide-field) fluorescence, TIRF, or spinning disk confocal microscopy, where temporal resolution is limited by the read-out rate of the CCD camera.
Finally: as mentioned above, the resolution (x/y) in a confocal microscope is only marginally better than in conventional light microscopy. If you need to resolve, visualize and exactly locate smaller structures using fluorescent markers, you'll have to use one of many so-called super-resolution (or subdiffraction) microscopy methods, like STED, GSD, GSDIM, PALM, STORM, etc. Please note, though, that these methods are very specialized, have great limitations, and demand special (often not commercially available) technically advanced equipment. Another alternative is to use quantum dots that may first be visualized with fluorescence, and then exactly localized with electron microscopy!
In depth support
For dedicated in depth support regarding experimental design and hands-on optimizations for confocal microscopy the Department of Biology recommends you to contact ImageneIT.
Telephone: +46 70-984 93 38
E-mail: bo [at] imagene-it [dot] se
If you are publishing an article with results obtained with the microscopes belonging to the Department of Biology, please make sure to acknowledge us:
"We acknowledge the Microscopy Facility at the Department of Biology, Lund University".
In addition, we would be grateful to receive a pdf version of the paper in which the Microscopy Facility has been acknowledged. Thereby, your paper can also be listed after your permission on this webpage.