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What is Confocal Microscopy?A major advantage of confocal microscopy (CM) is that it enables you to acquire a 3 dimensional image without specimen fixing or preserving steps. This is of obvious importance when your section may be thicker than a few cells, for example when looking at intact biofilms. In addition CM has many other advantages for observing biological specimens.
- Increased resolution of specimens by eliminating 'out of focus' light.
- Detectors can be 'tuned' to look for wavelengths emitted by specific stains.
- Laser light is monochromatic (only one wavelength) allowing specific stains to be targeted for excitation.
- Short dwell times significantly reduce photobleaching.
- CM integrates with molecular techniques such as Fluorescence Protein (FP) technology to study 4D cellular processes and Fluorescence In-Situ Hybridization (FISH) to probe spatial distributions in complex populations.
- Dual or triple-labeled images that can be easily superimposed to allow visualization of multiple parameters.
- Commercially available and self constructed flow cells can be used to image biofilms on opaque coupons such as stainless steel, fibrous materials, and plastics that are too thick to examine under the conventional microscope
Confocal microscopy is a depth-resolving 'optical slicing' technology that allows the in situ study of dimensionally complex (thick), hydrated, living, flowing systems.
- Improved x-y resolution
Resolution of z dimension
- No other microscopic technique yields high resolution 3D structure that is compatible with imaging living cells
- Lasers allow efficient excitation of fluorescent molecules
- Photomultiplier tube (PMT) technology allows amplification of faint signals; diminution of saturated signals
- Confocal pinhole restricts recovery to single (adjustable) optical plane
- This restricts blurred out-of-focus light coming in from other focal planes yielding a crisp image
- System allows x-y coordinates to be kept in perfect register through the sample
Confocal image of a Streptococcus mutans biofilm shows a plan view (square) looking down on the biofilm and side views through the biofilm (left and below). The biofilm was composed of cell clusters separated by water channels. Structural heterogeneity can result in the creation of vastly different microenvironments within very small distances of only tens of microns (1 micron is 1 millionth of a meter).
Dual Leica AOBS confocal system, the upright microscope is in the forground and the inverted in the background.