Page 215 - Fundamentals of Light Microscopy and Electronic Imaging

198 FLUORESCENCE MICROSCOPY

separate filter sets to capture different images of each of the two fluorochromes. Bleed-
through can be reduced, but is never completely eliminated, because:

• The excitation spectra of two or more fluorochromes are broad and overlap to a sig-
nificant extent (Figs. 11-5 and 11-6; Color Plate 11-1). Thus, the excitation of fluo-
rescein at 490 nm also causes rhodamine to fluoresce. The solution is to choose
fluorochromes with well-separated excitation spectra so that the excitation peak for
one fluorochrome is many wavelengths shorter than the excitation peak for the sec-
ond fluorochrome.
• The fluorescence spectra of the fluorochromes may overlap, allowing fluorescence
from the shorter-wavelength fluorochrome to contribute to the image of the longer-
wavelength dye. To minimize the problem, a narrow bandpass filter is used as an
emission filter to collect only the peak fluorescence of the lower-wavelength fluo-
rochrome; a long-pass barrier filter is usually used for the longer-wavelength fluo-
rochrome.
• The fluorescence emission of one fluorochrome (fluorescein) may stimulate a sec-
ond longer-wavelength dye (rhodamine) to fluoresce. Selecting well-separated dyes
and assuring that labeling and fluorescence of the dyes are balanced help reduce
this problem.
• If the amount of labeling and the intensity of fluorescence of the two fluorochromes
are not equally balanced, the brighter signal can overwhelm and penetrate the filter
set for the second signal and cause a significant contribution to the image of the sec-
ond dimmer signal. The intensity of fluorescence from dyes such as fluorescein and
rhodamine should be similar and is adjusted according to the amount of dye in the
specimen and the type of illumination used. Microscopists using epi-illumination
with a mercury arc lamp frequently forget that rhodamine is excited 10 more
effectively than fluorescein owing to the bright 546 nm emission line in the mercury
arc spectrum.

Even when these factors are controlled, the amount of signal crossover and bleed-
through generally remains about 10–15%. In experiments involving double staining, you
should always examine single-stained specimens using the filter set for the other fluo-
rochrome to assure that the amount of bleed-through is minimal. In many cases, the main
cause of bleed-through is unequal staining by the two dyes. Remember that it is nearly
always desirable to return to the lab bench to prepare a proper specimen, rather than to use
unequal exposures and image processing on a poorly prepared specimen just to save time.

EXAMINING FLUORESCENT MOLECULES IN LIVING CELLS

Fluorescence microscopy is potentially damaging to living cells, since light sources are
intense and rich in damaging UV and IR wavelengths, and because fluorescence filter
sets are not totally efficient in removing these unwanted wavelengths. The chief concern
is phototoxicity to the cell through absorption of photons by introduced and endogenous
fluorophores and the generation of reactive oxygen species, including singlet oxygen
1

( O ), superoxide (O • ), hydroxyl radical (OH•), various peroxides (ROOR ),
2
2
hydroperoxides (ROOH), and others. They react with oxidizable metabolites and com-
ponents in the cell such as the pyridine nucleotides in nucleic acids, several amino acids,
glutathione, lipids, and ascorbate. Among the immediate effects is damage to membrane