3.1.2. Selecting fluorochromes

Recently, various types of fluorochromes have become available which emit fluorescent light at different wavelengths. This allows the simultaneous detection of distinct bacteria using specific probes labelled with different fluorochromes. A high-performance laser scanning microscope (LSM) in combination with multiple types of probes dually-labelled with such fluorochromes increases the number of bacteria that can be detected simultaneously (Valm et al., 2011). However, most of epifluorescence microscopes used routinely in conventional laboratories may be equipped with only a few fluorescence filters. This limits the number of fluorochromes that can be detected unambiguously. Moreover, because insect tissues generally have stronger autofluorescence in shorter (blue to green) wavelengths, signals of fluorochromes emitting fluorescence in this wavelength range tend to be obscured by the autofluorescence of the insect tissue (Fukatsu et al., 1998; Koga et al., 2009). Accordingly, only fluorochromes emitting longer wavelength light are generally suitable as reporter dyes of FISH probes for insect materials (Table 1).

Green fluorochromes are sometimes allocated for counter staining, especially when using LSM, because these microscopes are typically equipped with a fairly inexpensive laser source and filters that detect only blue fluorescence dyes. Different companies offer a broad variety of fluorochromes for oligonucleotide labelling. Alexa Fluor® dyes seem to outperform traditional fluorochromes, such as FITC, TRITC or Cy™ Dyes, in fluorescence intensity and photo stability. However, these patented dyes are relatively expensive, and for most applications, other dyes will be sufficient to generate good results.

Table 1.  Fluorochromes suitable for experiments in Apis mellifera. 1If autofluorescence low in tissues.

 Use

Recommended Fluorochromes   

 Emission colour   

Counter staining DNA

DAPI

Blue

Hoechst33342

Blue

SYTOX Green®

Green

Labeling oligonucleotide probes

Alexa Fluor® 555

Orange

Alexa Fluor® 647

Far-red

CyTM3

Orange

CyTM5

Far-red

TYETM563

Orange

TYETM665

Far-red

Alexa Fluor® 4881

Green