Fluorochromes

Recommended Fluorochromes for the Olympus FV1000 confocal system

The fluorescent dye should have absorption maximum close to the one of the available laser lines. The dye should be resistant to photobleaching and have high quantum efficiency (bright fluorescence). That is why we recommend using the Alexa Fluor 488 dye instead of the traditional FITC (Fluorescein isohtiocyanate). In multi-color labeling it is important to choose dyes with relatively narrow and well separated excitation and emission peaks in order to prevent cross-talk between the detection channels (i.e., the signal from dye 1 being detected in a fluorescent channel for dye 2). While the Cy3 dye is very useful for single-color fluorescence staining, it is not ideal for multi-color fluorescence imaging with green- or red-emitting dyes, because Cy3 excitation and emission spectrum is very broad.

Laser used for excitationRecommended dyesFluorescence Emission ColorAlso useable dyes
405 nm diode DAPI, Hoechst
Alexa Fluor 405
calcofluor white
Blue
458 nm Argon laser CFP Cyan
488 nm Argon laser Alexa Fluor 488, GFP, EGFP,
Acridin Orange, DiO, Cy2
Green Oregon Green, FITC
514 nm Argon laser YFP, EYFP Yellow
543 nm HeNe laser Alexa Fluor 543, mCherry, mOrange, RFP, Cy3 Orange-red Alexa Fluor 594,
633 nm HeNe laser Alexa Fluor 633, Alexa Fluor 647, Cy5, Histochemical indigogenic reaction products (e.g., x-gluc histochemical staining for GUS reporter in plants, BCIP/NBT staining with AP-conjugated secondary antibodies) red, far red

 

MULTI-COLOR LABELING:

For best results, Fluorochromes should be well spectrally separated in order to avoid channel bleed-through and false-positive signal.   Users should confirm specificity of detection by negative controls (one fluorochrome omitted). Sequential scanning is recommended unless it was confirmed that there is no cross-talk between channels.Color combinations that work well on the current system are listed below: 

two colors (in addition to DAPI)

CFP+YFP

GFP + tdTomato

Alexa 488 + Alexa 633 or 647

Cy3 + Cy5

three colors (in addition to DAPI) Alexa 488 + Alexa 598 + Alexa 647

Fixed (=dead, fluorescently stained) specimens should be mounted in a mounting medium containing antifade reagents, in order to minimize photobleaching. Most mounting media contain 50-80% glycerol in order to improve optical properties for imaging with oil immersion objectives. However, some lipophilic membrane dyes, such as DiI, are not compatible with glycerol and thus those specimens should be mounted in a glycerol-free medium or a buffer.

Please be aware that even the glycerol-based mounting medium does not match refractive index of immersion oil. Thus, using oil immersion objectives on these samples results in spherical aberration that will degrade resolution and significantly decrease signal intensity.  A new medium for high-resolution fluorescence mcroscopy, 2,2-thiodiethanol,  can be mixed to precisely match refractive index of oil. It is inexpensive and has anti-fade properties. See Staudt, T., Lang, M. C., Medda, R., Engelhardt, J., & Hell, S. W. (2007) Microsc Res Tech 70, 1-9.  

Be aware that antifade reagents containing p-phenylenediamine should NOT be used with cyanine dyes (such as Cy2, Cy3) because the p-phenylenediamine will cleave the dye molecule and cause loss of fluorescence, increased background and diffuse straining after only few days of storage. Always consult the mounting media data sheet for compatibility. Information on mounting media and antifade reagents, including recipes for making your own compiled by Tonny Collins from Wright Cell Imaging Facility (Toronto, Canada) can be downloaded here: http://www.uhnresearch.ca/facilities/wcif/PDF/Mountants.pdf

For more comprehensive listing of fluorescent dyes and their properties, visit these sites:

Interactive database of Fluorescence Spectra: http://www.mcb.arizona.edu/ipc/fret/default.htm
Olympus fluorchrome data tables: http://www.olympusmicro.com/primer/techniques/fluorescence/fluorotable2.html
Molecular Probes dye spectra viewer: http://probes.invitrogen.com/resources/spectraviewer/