Page 202 - Fundamentals of Light Microscopy and Electronic Imaging

AUTOFLUORESCENCE OF ENDOGENOUS MOLECULES 185

eye, the full fluorescence emission spectrum of fluorescein can be examined. It
begins with the longest of the blue-green wavelengths, reaches a peak in the green
and yellow green, and then tapers off in the orange and red. It is clear that fluo-
rescein re-emits light over a broad spectrum of wavelengths and that the yellow-
green fluorescence color is not due to a single spectral line or narrow band of
yellow-green wavelengths.

Table 11-1 and Figure 11-5 indicate important properties of fluorescent dyes com-
monly used in fluorescence microscopy. New classes of dyes such Alexa dyes (Molecu-
lar Probes, Inc.) and cyanine dyes (Amersham-Pharmacia, Inc.) are especially deserving
of attention, because they have a high quantum efficiency and offer high resistance to
photobleaching. Green fluorescent protein (GFP) isolated from the jellyfish Aequorea
victoria, and its mutated allelic forms, blue, cyan, and yellow fluorescent proteins, are
used to produce fluorescent chimeric proteins that can be expressed in living cells, tis-
sues, and whole organisms. The most recent member of the group, DsRed, or red fluo-
rescent protein, comes from a marine anemone. The topic of fluorescent dyes and their
applications is extensive and complex. Readers wishing to explore the subject in greater
depth can begin by reading the chapters by Tsien and other authors in Pawley (1995) and
by consulting the Handbook of Fluorescent Probes and Research Chemicals, which is
available on the Internet at www.probes.com or on hard copy through Molecular Probes,
Inc. The spectra and properties of many fluorescent labels including cyanine dyes are
available at the Web site of Jackson ImmunoResearch Laboratories, Inc., at www.jack-
sonimmuno.com, and of GFP and DsRed at the Web site of Clontech Laboratories, Inc.,
at www.clontech.com. Another excellent reference is available at the website of Omega
Optical, Inc at www.omegafilters.com.
It is common practice to label cells with multiple fluorescent dyes to examine dif-
ferent molecules, organelles, or cells in the same preparation using different fluores-
cence filter sets. As an example, Figure 11-6 shows the excitation and emission spectra
of three dyes (DAPI, fluorescein, and rhodamine) that are suitable for specimens illu-
minated with different spectral regions of a mercury arc lamp. Many factors influence
the decision regarding dye selection, including spectral region, Stokes shift, quantum
efficiency, solubility, and photostability of the dye; spectral profile of the illuminator;
and availability of suitable filter sets. In the example shown here, DAPI and rhodamine
are each excited by bright spectral lines of the mercury lamp at 366 and 546 nm, respec-
tively; the excitation of fluorescein is not as favorable, but this is compensated by the
fact that fluorescein (or Cy2 or Alexa 488) has a high quantum efficiency.

AUTOFLUORESCENCE OF ENDOGENOUS MOLECULES

All cells contain endogenous metabolites that autofluoresce and contribute background
fluorescence to the image. In some cases, these signals are strong enough that they are
mistaken for the signals of fluorescently tagged molecules. Some common sources of
autofluorescence are the B vitamins, flavins, flavin proteins, and flavin nucleotides
(FAD and FMN), reduced pyridine nucleotides (NADH and NADPH), fatty acids, por-
phyrins, uncoupled cytochromes, lipofuchsin pigments, serotonin, and catecholamines