FISH : Fluorescent in situ hybridization

We've already talked a little about fluorescent in situ hybridization, or FISH, but it perhaps bears a general review. In this method, cells in an environmental sample are fixed with glutaraldehyde, then treated to make them permiable (e.g. with toluene) and mixed with an oligonucleotide probe that contained a fluorsecent tag such as Texas Red or Acridine Orange. The probe will find matches in the DNA and/or RNA of the permiablized cells and stick. Unannealed probe is washed out, and the sample is examined by fluorescent microscopy. If enough probe accumulates in a cell, i.e. if it contains the target to which the probe is designed, it should be fluorescent.

The most common probes for FISH in the microbial world target the rRNA. FISH probes targeting rRNA sequences are sometimes called "phylogenetic probes", because they target specific phylogenetic groups. There are two reasons why rRNAs are a good target for fluorescent in situ hybridization, Firstly, it allows you to search using phylogenetically relevant sequences - often rRNA sequence is all you know about an organism. This also allows you to 'tune' the range of organisms you will label - the more conserved the target region of the rRNA, the wider the phylogenetic range of cells you will label. Secondly, because there are thousands of ribosomes in each cell, a lot of probe can bind to each cell, giving a strong signal. In fact, it is generally seen that only metabolically-active cells contain enough ribosomes to be labeled with an rRNA probe.

Nanoarchaeum equitans stained with a specific Texas Red-labeled FISH probe, and its host Igniococcus stained with an Acridine orange (which is green)-labeled specific FISH probe. from Huber, et al, 2002 Nature 417:63-67

Confocal laser scanning microscopy

Confocal laser scanning microscopy (CLSM) is a method that allows the collection of 3-dimensional data from a fluorescent microscope sample. The laser light is focused on a single point in the field of view (about 1um in diameter), and the resulting fluorescence from that point is visualized and quatitated by the detector. The laser focal point is scanned back-and-forth across the field of view to collect a single plane image, then moved slightly up or down in the sample and another plane image is collected...and again, over and over. The result, then, is a 3-D digital image of the sample.

• For more details, see: http://en.wikipedia.org/wiki/Confocal_laser_scanning_microscopes
• And here is a great simulation of a CLSM image: http://micro.magnet.fsu.edu/primer/virtual/confocal/index.html