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 CHAPTER

11
FLUORESCENCE MICROSCOPY

OVERVIEW

With light microscope optics adjusted for fluorescence microscopy, it is possible to

examine the distribution of a single molecular species in a specimen, and under special-
ized conditions, even detect individual fluorescent molecules. In contrast to other forms
of light microscopy based on specimen-dependent properties of light absorption, optical
path differences, phase gradients, and birefringence, fluorescence microscopy allows
visualization of specific molecules that fluoresce in the presence of excitatory light.
Thus, the amount, intracellular location, and movement of macromolecules, small
metabolites, and ions can be studied using this technique. Figure 11.1 shows one such
example: the distribution of several internal components in tissue culture cells. Typi-
cally, nonfluorescent molecules are tagged with a fluorescent dye or fluorochrome
in order to make them visible. Examples of these are DAPI and the Hoechst dyes
used to directly label nuclear DNA, or rhodamine-labeled phalloidin used to indirectly
label cytoplasmic actin filaments. Alternatively, fluorochrome-labeled antibodies can
be used to label fixed, permeabilized cells in a method known as immunofluorescence
microscopy, one of the techniques used in Figure 11.1. These methods are commonly
used to visualize the distribution of certain proteins in a cell or to make visible specific
organelles, vacuoles, filaments, and biochemically distinct membrane regions. A variety
of new tagging methods is also employed, including inserting short DNA sequences of
known epitope into the coding sequences of proteins (epitope tagging), constructing
protein chimeras with green or red fluorescent proteins (GFP, RFP), and several other
methods.

Immunofluorescence image of rat epithelial cells showing microtubules, actin, and DNA.

Fundamentals of Light Microscopy and Electronic Imaging, Second Edition.

Douglas B. Murphy and Michael W. Davidson.
© 2013 Wiley-Blackwell. Published 2013 by John Wiley & Sons, Inc.
199

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 200 FLUORESCENCE MICROSCOPY

Figure 11.1
Demonstration of specific molecular labeling and imaging by fluorescence microscopy. Cul-
tured fibroblast cells from Tahr ovary were fixed, detergent extracted, and labeled with primary
antibodies to Giantin (a Golgi resident protein) and the nuclear pore coat protein (NPCP;
nuclear pores surrounding the nucleus). Secondary antibodies conjugated to high-performance
red and green fluorophores were used to highlight the Golgi and nuclear pores, while a blue
fluorescent fluorophore conjugated to phalloidin was used to mark the filamentous network.
Fluorescence microscopy is commonly used to determine the amount, distribution, and
dynamics of specific macromolecules in cells. Bar = 10 µm.

Fluorescence microscopy has gained in popularity ever since Coons et al. (1941)
developed methods to conjugate proteins to fluorochromes, and improvements in optics,
thin-film technology, and optoelectronics increased the specificity and sensitivity of
detection of emitted fluorescent light. Among the most important advances were the
application of interference filters and dichromatic mirrors and their incorporation in a
versatile epi-illuminator (Ploem, 1967), the introduction of special high numerical
aperture (NA)-corrected objectives, and the emergence of highly sensitive detectors.
Advances continue today in all of these areas. Because of its great specificity and rela-
tive ease of use, fluorescence microscopy is the most frequently employed mode of
light microscopy used today in biomedical research.
Fluorescence microscopes contain special filters and employ a unique method of
illumination to produce images of fluorescent light emitted from excited molecules in
a specimen. The filters are designed to isolate and manipulate two distinct sets of exci-
tation and fluorescence emission wavelengths. A band of shorter excitation wavelengths
from the illuminator and filters is directed to the specimen, while a band of longer fluo-
rescence wavelengths emitted from the specimen forms an image of the specimen in
the image plane. To perform fluorescence microscopy effectively, the microscopist must
be able to select the proper fluorophore, filters, and illuminator for a given application
and evaluate the quality of fluorescence signals. In this chapter, we discuss the physical

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 APPLICATIONS OF FLUORESCENCE MICROSCOPY 201

basis of fluorescence, the properties of fluorescent dyes, the action of filters comprising
a fluorescence filter set, the optical design of epi-illuminators, and the positioning of
this equipment in the optical pathway. We also examine important variables that affect
image quality and discuss methods for examining fluorescence in living cells.

APPLICATIONS OF FLUORESCENCE MICROSCOPY

Fluorescence microscopy is used extensively to study the intracellular distribution,

dynamics, and molecular mechanisms of a large variety of macromolecules and metab-
olites. While it is impractical to discuss specialized labeling techniques and methods
of fluorescence quantitation and analysis, it is important to note some of the principal
applications for which fluorescence microscopy is applied. These include:

• Determination of the intracellular distribution of macromolecules in formed struc-

tures, such as membranes, cytoskeletal filaments, and chromatin. Fluorochrome-
conjugated metabolites, ligands, and proteins can be used to label membrane
channels and ion channels. Target molecules can also be labeled with fluorescent
antibodies (immunofluorescence microscopy) or with biotin or epitope tags, or
conjugated to fluorescent proteins, such as green fluorescent protein (GFP) and
other genetically encoded agents. Multicolor labeling is possible (Fig. 11.1),
whereby several different molecular species are labeled and viewed simultane-
ously using dyes that fluoresce at different wavelengths.
• Study of intracellular dynamics of macromolecules in supramolecular structures
and organelles (fluorescence recovery after photobleaching, or FRAP). FRAP
techniques give the half-time for subunit turnover in a structure, binding constants,
and diffusion coefficients. Related technologies, such as photoactivation, photo-
switching, and photoconversion, can also be used to study cellular dynamics.
• Study of protein nearest neighbors, interaction states, and reaction mechanisms
by fluorescence energy transfer or FRET and by fluorescence correlation
microscopy. In FRET, two different fluorophores are employed, and the excitation
of the shorter-wavelength fluorophore results in the fluorescence of the longer-
wavelength fluorophore if the two moieties come within a short molecular distance
of one another.
• Study of dynamics of single tagged molecules or molecular assemblies in vivo
using an extremely sensitive technique called total internal reflection fluorescence
(TIRF) microscopy. TIRF is used to probe molecular events that occur very close
to the membrane in living cells. The technique has a very short penetration dis-
tance (less than 200 nm) and is thus better suited to monitor fluorescence emitted
by molecules within this distance from the glass coverslip.
• Determination of intracellular ion concentrations, as well as changes in the con-
centrations, for several ionic species, including H+, Na+, K+, Cl−, Ca2+, and many
other metals. Ratiometric dyes or specialized biosensor fluorescent protein com-
binations are used, whose peak fluorescence emission wavelength changes depend-
ing on whether the dye is in the free or bound state. The ratio of fluorescence
amplitudes gives the ion concentration.
• Organelle marking experiments using dyes that label specific organelles and cyto-
skeletal proteins. Specialized fluorophores, such as MitoTracker and LysoTracker,

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 202 FLUORESCENCE MICROSCOPY

can be used to label specific organelles in living cells. Fusions of GFP to targeting
peptides and proteins can also be used to monitor specific regions in cells for
dynamic interactions using time-lapse microscopy. Fixed cells can be specifically
labeled using immunofluorescence techniques.
• Determination of the rates and extents of enzyme reactions using conjugates of
fluorochromes whose fluorescence changes due to enzymatic activity. An example
is the use of nucleotide coenzymes (NADH and NADPH) where the reduced
forms are fluorescent and the oxidized forms are nonfluorescent. Other synthetic
reagents are capable of releasing a fluorescent dye during enzyme-catalyzed
reactions.
• Study of cell viability and the effects of factors that influence the rate of apoptosis
in cells using a combination of dyes that are permeant and impermeant to the
plasma membrane. A synthetic fluorophore known as JC-1 can be used to analyze
mitochondria membrane potential to monitor cell viability. Numerous other syn-
thetic fluorophores can pass through the membrane of dead cells, but not those
that are living.
• Examination of cell functions, such as endocytosis, exocytosis, signal transduc-
tion, and the generation of transmembrane potentials, using fluorescent dyes.
Although a number of synthetic fluorophores, including quantum dots, have been
developed for monitoring endocytosis and exocytosis, these assays are increas-
ingly being fine-tuned with the application of genetically engineered fluorescent
protein biosensors.

PHYSICAL BASIS OF FLUORESCENCE

Fluorescence is the emission of photons by atoms or molecules whose electrons are

transiently stimulated to a higher excitation state by radiant energy from an outside
source. It is a beautiful manifestation of the interaction of light with matter and forms
the basis for fluorescence microscopy, so we will take a moment to examine the physi-
cal basis of the phenomenon.
When a fluorescent molecule absorbs a photon of the appropriate wavelength, an
electron is excited to a higher energy state and almost immediately collapses back to
its initial ground state. In the process of energy collapse, the molecule can release the
absorbed energy as a fluorescent photon. Since some energy is lost in the process, the
emitted fluorescent photon typically exhibits a lower frequency of vibration and a
longer wavelength than the excitatory photon that was absorbed. The situation is
depicted graphically in what is known as a Jablonski diagram (Fig. 11.2), which shows
a series of increasing energy states as a stack of horizontal lines. Each energy level is
in turn composed of a number of subenergy levels, which do not concern us here. There
are two categories of excited states, characterized by different spin states of the excited
electron—the singlet excited state and the triplet excited state. Most commonly, an
excited electron occupies an excitation level within the singlet excited state (straight
upward pointing arrows), and when it collapses to the ground state, energy can be given
up as fluorescence emission (straight downward pointing arrows). Alternatively, energy
can be given up as heat (internal conversion), in which case no photon is emitted (wavy
downward pointing arrows). When excited above the ground state, there is a probability
that an electron can also enter the triplet excited state through a process called inter-

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 PHYSICAL BASIS OF FLUORESCENCE 203

Figure 11.2
Jablonski diagram showing energy levels occupied by an excited electron within a fluorescent
molecule (chlorophyll a). Chlorophyll a is unique in absorbing blue and red wavelengths of
the visual spectrum. Once excited, energy is released through any of the following three
pathways: Chlorophyll can give off a photon (fluorescence emission, straight downward point-
ing red arrow); it can release vibrational energy as heat without photon emission (internal
conversion, wavy downward pointing purple arrow); or its electron can enter an excited triplet
state (intersystem crossing, diagonal wavy blue arrow), which can make the molecule chemi-
cally reactive. Electrons in the triplet excited state can return to the ground state through
internal conversion (diagonal wavy cyan arrow) or by emission of phosphorescence (red
arrow). Refer to the text for details.

system crossing (diagonal wavy blue arrow). The triplet state is important, because
molecules with electrons in this state are chemically reactive, which can lead to pho-
tobleaching and the production of damaging free radicals (discussed later in this
chapter). During fluorescence, the absorption and re-emission events occur nearly
simultaneously, the interval being only 10−9–10−12 seconds; therefore, fluorescence stops
the moment there is no more exciting incident light. The emission process is called
phosphorescence if the period between excitation and emission is not instantaneous and
lasts fractions of a second to minutes. These processes should not be confused with
bioluminescence, such as that exhibited by firefly luciferase, in which electrons are
excited by chemically driven processes rather than by absorbing external radiation.
Molecules that are capable of fluorescing are called fluorescent molecules, fluores-
cent dyes, or fluorochromes. If a fluorochrome is conjugated to a large macromolecule

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 204 FLUORESCENCE MICROSCOPY

(through a chemical reaction or by simple adsorption), the tagged macromolecule is

said to contain a fluorophore, the chemical moiety capable of producing fluorescence.
Fluorochromes exhibit distinct excitation and emission spectra that depend on their
atomic structure and electron resonance properties. Fluorescent dyes usually contain
several unconjugated double bonds and are usually heterocycles containing nitrogen,
sulfur, and/or oxygen. The absorption and emission spectra for fluorescein-conjugated
immunoglobulin (IgG) are shown in Figure 11.3. Molecules absorb light and re-emit
photons over a spectrum of wavelengths (the excitation spectrum) and exhibit one or
more characteristic excitation maxima. Absorption and excitation spectra are distinct
but usually overlap, sometimes to the extent that they are nearly indistinguishable.
However, for fluorescein and many other dyes, the absorption and excitation spectra
are clearly distinct. The widths and locations of the spectral curves are important, par-
ticularly when selecting two or more fluorophores for labeling different molecules
within the same specimen.
Re-emission of fluorescent light from excited dye molecules likewise occurs over
a broad spectrum of longer wavelengths (the emission spectrum) even when excitation
is performed with a monochromatic source, such as a laser. This is because electrons
occupy excited states for various lengths of time during which they give up varying
amounts of vibrational energy, some of it as heat, resulting in the re-emission of lower
energy, longer-wavelength photons over a spectrum of wavelengths. Because some
energy is lost during the process, the wavelength of a fluorescent photon is usually

Figure 11.3
Normalized absorption and fluorescence emission spectra of fluorescein conjugated to IgG.
Both spectra span a wide range of wavelengths. Fluorescein has an absorption/excitation
peak at 494 nm and looks yellow-green to the eye, but actually fluoresces at wavelengths
ranging from blue to red with a peak at 518 nm. The difference in nanometers between the
excitation and emission maxima is called the Stokes shift. The molar extinction coefficient is
measured at the peak of the absorbance spectrum as indicated in the figure.

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 PROPERTIES OF FLUORESCENT DYES 205

longer than the wavelength of the photon exciting the molecule. It should be noted that
the energy of a photon is given as E = hc/λ, where h is Planck’s constant, c is the speed
of light, and λ is the wavelength. Since the photon energy is reduced during absorption
and re-emission, the wavelength increases. The reader is encouraged to review the
relationships between the energy, frequency, and wavelength of photons described in
Chapter 2.
Finally, the shapes of the spectral curves and peak wavelength of absorption and
emission spectra vary, depending on factors contributing to the chemical environment
of the system, including pH, ionic strength, solvent polarity, O2 concentration, presence
of quenching molecules, and others. This fact explains why the fluorescence of a dye,
such as fluorescein, varies depending on whether it is free in solution or conjugated to
a protein or other macromolecule.

PROPERTIES OF FLUORESCENT DYES

The excitation and emission spectra of fluorescent molecules are commonly observed
to overlap. The difference in wavelength or energy between the excitation and emitted
fluorescent photons is called the Stokes shift. In practice, the Stokes shift is the differ-
ence between the excitation and emission maxima (Fig. 11.3). Depending on the par-
ticular fluorescent molecule, the shift can range from just a few to several hundreds of
nanometers. The Stokes shift for fluorescein is ∼25 nm, while that for porphyrins is
over 200 nm. Dyes exhibiting a large Stokes shift are advantageous in fluorescence
microscopy, because the bands of excitation and fluorescence wavelengths are easier
to isolate using interference filters. Another important criterion for dye selection is the
molar extinction coefficient, which describes the potential of a fluorochrome to absorb
photon quanta, and is given in units of absorbance (optical density) at a reference
wavelength (usually the absorption maximum) under specified conditions. The quantum
efficiency (QE) of fluorescence emission is the fraction of absorbed photon quanta that
is re-emitted by the fluorochrome as fluorescent photons. QE varies greatly between
different fluorochromes and for a single fluorochrome under different conditions. For
soluble fluorescein dye at alkaline pH, the quantum efficiency can be as high as 0.9—an
extremely high value—–but for protein-bound fluorescein at neutral pH, the quantum
efficiency is typically 0.3–0.6.
Other important characteristics of dyes are their resistance to photobleaching, solu-
bility in aqueous media, and chemical stability. Quenching and photobleaching reduce
the amount of fluorescence and are of great practical significance to the microscopist.
Quenching reduces the quantum yield of a fluorochrome without changing its fluores-
cence emission spectrum and is caused by interactions with other molecules, including
other fluorochromes. Conjugation of fluorescein to a protein usually causes a significant
reduction in the quantum yield because of charge-transfer interactions with nearby
aromatic amino acids. Proteins like IgG or albumin that are conjugated with five or
more fluorescein molecules, for example, fluoresce less than when bound to two to
three molecules, because energy is transferred to nonfluorescent fluorescein dimers.
Photobleaching refers to the permanent loss of fluorescence by a dye due to photon-
induced chemical damage and covalent modification. As previously discussed, photo-
bleaching occurs when a dye molecule, excited to one of its electronic singlet states,
transits to a triplet excited state (Fig. 11.2). Molecules in this state are able to undergo
complex reactions with other molecules. Reactions with molecular oxygen permanently

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 206 FLUORESCENCE MICROSCOPY

destroy the fluorochrome and produce singlet oxygen species (free radicals) that can
chemically modify other molecules in the cell. Once the fluorochrome is destroyed, it
usually does not recover. The rate of photobleaching can be diminished by reducing
excitation energy or lowering the oxygen concentration. Methods for reducing oxygen
concentration as a way to protect live cells are described at the end of the chapter.

Demonstration: Fluorescence of Chlorophyll and Fluorescein

The phenomena and principles of fluorescence can be demonstrated using a fil-

tered alcoholic extract of spinach leaves and an aqueous solution of fluorescein.
The procedures for preparing these samples are given in Appendix B.
Although an extract of spinach contains a mixture of chlorophylls a and b,
plus carotene and xanthophyll, most of the fluorescence phenomena are due to
chlorophyll a. The fluorescence spectra of chlorophyll a, the major chlorophyll
component, are shown in Figure 11.4. The figure shows that the absorption/
excitation spectrum is broad and distinctly bimodal, with absorption peaks widely
separated in the blue and red portions of the visible spectrum. Fluorescence emis-
sion at red wavelengths with an emission peak at 670 nm is the same regardless
of whether blue or red wavelengths are used to excite the molecule. Notice too
the very large Stokes shift from 420 to 670 nm—a difference of some 250 nm.
Normally, the chlorophyll and other pigments in intact chloroplasts act as energy

Figure 11.4
Absorption and emission spectra of chlorophyll a. The spectrum for absorption–
excitation (blue curve) is unusual in showing two prominent peaks at ∼420 and ∼660 nm
and a pronounced trough corresponding to green wavelengths of the visible spectrum.
The emission curve is shown as a red curve. It might seem unusual that the major
chlorophyll species of plants does not absorb in the green, where the peak of solar
radiation occurs. This job is performed by other pigments (chlorophyll b, xanthophyll,
and carotene), which transfer captured energy of incident radiation to chlorophylls a
and b, thus providing an efficient design for light absorption across parts of the UV and
much of the visual spectrum.

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 PROPERTIES OF FLUORESCENT DYES 207

transducers and do not fluoresce; rather, the electrons excited by absorbed photons
are transferred to nearby enzyme assemblies (photosystems I and II) for the fixa-
tion of carbon dioxide into carbohydrate. However, when chlorophyll is extracted
into a soluble form in alcohol, no electron transfer is possible, and absorbed
photons are re-emitted as deep red fluorescence.
If the flask of chlorophyll is placed over a bright white light source, a deep
emerald green color is seen. Green wavelengths are observed because red and
blue wavelengths are selectively absorbed. (Refer to subtraction colors in Chapter
2.) Red fluorescence is emitted by the solution, but its presence is masked by the
bright green color of the nonabsorbed illuminating wavelengths. If the flask is
now illuminated with deep blue wavelengths <450 nm or with long-wave ultra-
violet (UV) (invisible) wavelengths from a black light in a darkened room, the
red fluorescence can be easily observed. Chlorophyll demonstrates features
important to fluorescence microscopy: the requirement to selectively isolate
bands of wavelengths corresponding to the excitation and emission maxima, and
the benefits of using molecules that exhibit both a large Stokes shift and high
quantum efficiency.
We can examine a 10 µM solution of fluorescein using the same illuminators.
Fluorescein appears bright yellow in white light (blue wavelengths are efficiently
absorbed, leaving red and green wavelengths, which the eye perceives as yellow).
Brilliant yellow-green fluorescence is observed under excitation illumination
with the black light. The effect of environmental conditions can be demonstrated
with fluorescein by adding a few drops of concentrated (10 N) sodium hydroxide,
which causes a twofold increase in quantum efficiency and therefore a dramatic
increase in fluorescence. If an opaque mask with a slit 5 cm long by 2–5 mm
wide is placed up against the flask on the side facing the observer, and the slit
of bright fluorescence examined in a darkened room while holding a holographic
(sinusoidal) diffraction grating immediately in front of the eye, the full fluores-
cence 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 fluorescein re-
emits light over a broad spectrum of wavelengths and that the yellow-green fluo-
rescence color is not due to a single spectral line or narrow band of yellow-green
wavelengths.

Tables 11.1 and 11.2 and Figure 11.5 catalog important properties of many
valuable fluorescent dyes and fluorescent proteins commonly used in fluorescence
microscopy. New classes of synthetic fluorochromes, such as the Alexa Fluor dyes
(Life Technologies, Carlsbad, CA), ATTO dyes (ATTO-TEC GmbH, Siegen, Germany)
and cyanine dyes (Amersham-Pharmacia, Piscataway, NJ), are especially notable,
because they have very large quantum efficiencies and offer high resistance to photo-
bleaching. GFP, isolated from the jellyfish Aequorea victoria, and its mutated allelic
forms, blue, cyan, and yellow fluorescent protein (BFP, CFP, and YFP), are used to
produce fluorescent chimeric proteins that can be expressed in living cells, tissues,
and whole organisms (Table 11.2). Orange, red, and far red fluorescent proteins that
are useful in fluorescence microscopy have been derived from chromoproteins and

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 208 FLUORESCENCE MICROSCOPY

TABLE 11.1 Properties of Commonly Used Fluorescent Dyesa

Fluorochrome Color Bandb Excitation (nm) Emission (nm) Filter Setc

Acridine orange Cyan 502 525 FITC

Allophycocyanin Red 621/650 661 Cy5
AMCA UV 350 445 DAPI
Alexa Fluor 405 UV 401 421 DAPI
Alexa Fluor 488 Cyan 495 519 FITC
Alexa Fluor 568 Yellow 578 603 TxRed
Alexa Fluor 647 Red 650 665 Cy5
ATTO 488 Cyan 501 523 FITC
ATTO 550 Green 554 576 TRITC
ATTO 594 Yellow 601 627 TxRed
ATTO 740 Far-Red 740 764 Cy7
BODIPY FL Cyan 503 512 FITC
BODIPY TMR Green 542 574 TRITC
Cascade blue UV 400 425 DAPI
Carboxy-SNARF-1 Green 548 587 TRITC
(low pH)
Cy2 Cyan 489 506 FITC
Cy3 Green 548 562 TRITC
Cy5 Red 650 670 Cy5
Cy7 Far-Red 710 805 Cy7
DAPI (bound to DNA) UV 350 470 DAPI
DiIC18 (bound to lipid) Green 549 565 TRITC
DiOC6 Cyan 484 501 FITC
Fluorescein (FITC) Cyan 494 518 FITC
Fluo-3 (with calcium) Cyan 485 503 FITC
FM 1-43 (bound to Cyan 473 578 Special
Lipid)
Fura-2 (with calcium) UV 335 505 Special
Hoechst 33258, 33342 UV 352 461 DAPI
Indo-1 (with calcium) UV 350 405/482 Special
Lissamine-rhodamine B Yellow 575 595 TxRed
Lucifer yellow Blue 425 528 Special
LysoTracker green Cyan 504 511 FITC
LysoTracker red Yellow 577 590 TxRed
MitoTracker red Yellow 581 644 TxRed
Oregon green 488 Cyan 496 524 FITC
Oregon green 514 Green 511 530 FITC
Phycoerythrin-R Green 565 578 TRITC
Propidium iodide Green 520 610 TRITC
SYTOX green Cyan 504 523 FITC
SYTOX orange Green 547 570 TRITC
Tetramethylrhodamine Green 540 578 TRITC

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 PROPERTIES OF FLUORESCENT DYES 209

TABLE 11.1 (Continued )

Fluorochrome Color Bandb Excitation (nm) Emission (nm) Filter Setc

Texas red Yellow 592 610 TxRed

TOTO-1 Green 514 533 FITC
TO-PRO-3 Red 642 661 Cy5
Ultralite T680 Red 656 678 Cy5
YOYO-1 Cyan 491 509 FITC
a
Most values are from Handbook of Fluorescent Probes and Research Chemicals (2010). In most cases,
the solvent used was methanol. Absorption maxima are typically close to the peak excitation wavelength.
b
Excitation band.
c
Recommended filter set.

TABLE 11.2 Physical Properties of Useful Fluorescent Proteins

Excitation Emission
Proteina Colorb (nm) (nm) Brightnessc Photostabilityd Filter Sete

EBFP2 Blue 383 448 18 ++ DAPI

mCerulean Cyan 433 475 17 ++ CFP
mTurquoise Cyan 433 474 25 +++ CFP
mTFP1 Teal 462 492 54 +++ CFP
mEGFP Green 488 507 34 ++++ FITC/GFP
mEmerald Green 487 509 39 ++++ FITC/GFP
mVenus Yellow 515 528 53 ++ FITC/YFP
mCitrine Yellow 516 529 59 ++ FITC/YFP
mKO2 Orange 551 565 40 +++ TRITC
tdTomato Orange 554 581 95 +++ TRITC
TagRFP Orange 555 584 48 ++ TRITC
mApple Orange 568 592 37 +++ TRITC
mCherry Red 587 610 17 +++ TxRed
mKate2 Far-Red 588 633 25 ++ TxRed
mPlum Far-Red 590 649 3.2 +++ TxRed
mNeptune Far-Red 600 650 13 ++++ Cy5
a
Common literature abbreviation.
b
Spectral class.
c
Product of the molar extinction coefficient and the quantum yield (mM × cm)−3.
d
Relative to mEGFP (++++).
e
Recommended filter set.

fluorescent proteins isolated from reef corals and sea anemones. 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 (Tsien et al., 2006)
and other authors in Pawley (2006) and by consulting the Molecular Probes Handbook,
A Guide to Fluorescent Probes and Labeling Technologies (Johnson and Spence, 2010),
which is available in electronic format on the internet ([Link] or
on hard copy through Molecular Probes, Inc. (Eugene, OR; a division of Life Technolo-
gies). The spectra and properties of many fluorescent labels, including cyanine dyes,

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 210 FLUORESCENCE MICROSCOPY

Figure 11.5
Absorption and emission spectra of synthetic dyes and proteins for fluorescence microscopy.
The Alexa series of dyes introduced by Molecular Probes, Inc. (Eugene, OR), and the cyanine
dyes by Amersham International, Inc. (available from Jackson ImmunoResearch Laborato-
ries, Inc, West Grove, PA), are exceptionally photostable, have high quantum efficiency, and
are soluble in aqueous media. The EGFP and mCherry proteins, provided as DNA vectors
by Addgene (Cambridge, MA) and Clontech, Inc. (Palo Alto, CA), respectively, are used to
construct fluorescent protein chimeras that can be observed in cells after transfection with
the engineered vectors (see Table 11.2). This technique avoids the problem of purifying,
tagging, and introducing labeled proteins into cells or having to produce specific antibodies.
Fluorescent protein chimeras are also suitable for studies of protein dynamics in living cells.
For examples and visualization methods, see Sullivan and Kay (1999).

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 AUTOFLUORESCENCE OF ENDOGENOUS MOLECULES 211

are available at the website ([Link] of Jackson Immuno-

Research Laboratories, Inc., and of GFP and other fluorescent proteins at the website
of Clontech Laboratories, Inc. ([Link] Another excellent reference
for spectral information is the website of Omega Optical ([Link]
Websites covering all phases of fluorescence microscopy, including fluorophores
and fluorescent proteins, and advanced microscopy techniques, can be found at Mole-
cular Expressions ([Link] Nikon MicroscopyU ([Link]
[Link]), the Zeiss Microscopy Campus ([Link] the
Hamamatsu Learning Center ([Link] and the Olympus Microscopy
Resource Center ([Link]
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,
respectively; 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,
porphyrins, uncoupled cytochromes, lipofuchsin pigments, serotonin, and catechol-
amines (see Demonstration and Table 11.3). Background fluorescence emission is
greatest when live cells are examined with blue and UV excitation wavelengths. The
strength of endogenous autofluorescence depends on the particular metabolite and
excitation wavelength being employed and also on the cell type. Macrophages, neurons,
and sperm cells exhibit particularly strong autofluorescence. Fixation of cells with
aldehydes in preparation for labeling with fluorescently tagged marker molecules may
also induce unwanted fluorescence, particularly in cell nuclei and organelles. For immu-
nofluorescence studies, aldehyde-induced fluorescence can be diminished by treating
fixed samples for 10 minutes with 20 mM sodium borohydride or ammonium chloride.
Fortunately, autofluorescent signals are usually low in amplitude. Interference from
autofluorescence can sometimes be avoided by simply selecting a longer wavelength
fluorochrome.
Autofluorescence adds to the background signal in a cell and may overlap the signal
of a fluorophore used in a labeling experiment, causing misinterpretation of the distribu-
tion pattern of the fluorophore. After acquiring fluorescence images of labeled speci-
mens, it is therefore important to prepare similar exposures from unlabeled specimens.
If necessary, a camera exposure time can be selected that minimizes the autofluorescent
contribution, but still allows adequate imaging of the labeled experimental material.

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 (a)

(b)

(c)

Figure 11.6
Use of multiple fluorescent dyes for examining cells with mercury illumination. The absorption
and emission spectra of (a) DAPI, (b) fluorescein, and (c) rhodamine are shown. The absorp-
tion spectra of DAPI and rhodamine overlap strong spectral lines of the mercury arc lamp at
366 and 546 nm, respectively. Although the emission spectra of these three dyes partially
overlap, it is possible to examine each dye separately using fluorescence filter sets that are
specific for each dye. Dashed vertical lines indicate registration of wavelengths between plots.

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 FLUORESCENT DYES AND PROTEINS IN FLUORESCENCE MICROSCOPY 213

Demonstration: Fluorescence of Biological

Materials under UV Light

Fluorescent compounds and metabolites are abundant in living cells and tissues.
To become familiar with these signals and recognize them when they occur,
examine the fluorescence of naturally occurring compounds in foodstuffs and
tissue extracts illuminated with a handheld black light in a darkened room. A list
of common foodstuffs and their fluorescence properties is given in Table 11.3.
Instructions for preparing certain extracts are given in Appendix B.

TABLE 11.3 Fluorescence of Naturally Occurring Substances

Specimen Substance Color

Powdered milk Oxidized riboflavin (lumiflavin) Blue

Margarine Fatty acids Blue
Yeast extract Oxidized vitamin B2 (lumiflavin) Blue
Brain extract Catecholamines, serotonin Blue
Yeast on agar plate Vitamin B2 (riboflavin) Green
Liver extract Vitamin B2 other B vitamins Yellow
Carrot extract β-carotene Yellow
Butter, milk Free riboflavin Yellow
Spinach extract Chlorophyll a, b Red
Shells of brown eggs Porphyrins Red

FLUORESCENT DYES AND PROTEINS IN

FLUORESCENCE MICROSCOPY

The history of synthetic fluorescent probes dates back over a century to the late 1800s
when many of the cornerstone dyes for modern histology were developed. Among these
were pararosaniline, methyl violet, and malachite green. Although these dyes were
highly colored and capable of absorbing selected bands of visible light, most were only
weakly fluorescent and would not be useful for the fluorescence microscopes that would
be developed several decades later. However, several synthetic dye classes synthesized
during this period, based on the xanthene and acridine heterocyclic ring systems, proved
to be highly fluorescent and provided a foundation for the development of modern
synthetic fluorescent probes. Most notable among these early fluorescent dyes were the
substituted xanthenes, fluorescein, and rhodamine B.
Fluorophores were introduced to fluorescence microscopy in the early twentieth
century as vital stains for bacteria, protozoa, and trypanosomes, but did not see wide-
spread use until the 1920s when fluorescence microscopy was first used to study dye
binding in fixed tissues and living cells. In the early 1940s, Albert Coons developed a
technique for labeling antibodies with fluorescent dyes, thus giving birth to the field of
immunofluorescence (Coons et al., 1941). Over the past 75 years, advances in immu-
nology and molecular biology have produced a wide spectrum of secondary antibodies

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 214 FLUORESCENCE MICROSCOPY

and provided insight into the molecular design of fluorescent probes targeted at specific
regions within macromolecular complexes.
Fluorescent probe technology and cell biology were dramatically altered by the
discovery of GFP from jellyfish and the development of mutant spectral variants, which
have opened the door to noninvasive fluorescence multicolor investigations of subcel-
lular protein localization, intermolecular interactions, and trafficking using living cell
cultures. More recently, the development of nanometer-sized fluorescent semiconductor
quantum dots has provided a new avenue for research in fluorescence microscopy. All
of these fluorescent probes are discussed below, and examples showing their use in
organelle labeling are shown in Figure 11.7.

Alexa Fluor Dyes

Advances in modern fluorophore technology are exemplified by the Alexa Fluor dyes
introduced by Molecular Probes (Alexa Fluor is a registered trademark of Molecular
Probes). These fluorescent probes exhibit higher quantum yields for more intense fluo-
rescence emission than spectrally similar probes, and have improved features, including
enhanced photostability, absorption spectra matched to common laser lines, pH insen-
sitivity, and a high degree of water solubility. In fact, the resistance to photobleaching
of Alexa Fluor dyes is so substantial that even when subjected to irradiation by high-
intensity laser sources, fluorescence intensity remains stable for relatively long periods
of time in the absence of antifade reagents. This feature enables the water-soluble Alexa
Fluor probes to be readily utilized for both live-cell and tissue section investigations,
as well as in traditional fixed preparations.
Alexa Fluor dyes are available in a broad range of fluorescence excitation and
emission wavelength maxima, ranging from the UV and deep blue to the near-infrared
(IR) regions. Alphanumeric names of the individual dyes are associated with the specific
excitation laser or arc-discharge lamp spectral lines for which the probes are intended.
For example, Alexa Fluor 488 is designed for excitation by the blue 488-nm line of the
argon or krypton-argon ion lasers, while Alexa Fluor 568 is matched to the 568-nm
spectral line of the krypton-argon laser. Several of the Alexa Fluor dyes are specifically
designed for excitation by either the blue diode laser (405 nm), the orange/yellow
helium-neon laser (594 nm), or the red helium-neon laser (633 nm). Other Alexa Fluor
dyes are intended for excitation with traditional mercury arc-discharge lamps in the
visible (Alexa Fluor 546) or UV (Alexa Fluor 350, also useful with high-power argon-
ion lasers), and solid-state red diode lasers (Alexa Fluor 680). Because of the large
number of available excitation and emission wavelengths in the Alexa Fluor series,
multiple labeling experiments can often be conducted exclusively with these dyes.

Cyanine Dyes

The family of cyanine dyes, Cy2, Cy3, Cy5, Cy7, and their derivatives, feature fluo-
rescence excitation and emission profiles that are similar to many of the traditional
dyes, such as fluorescein and tetramethylrhodamine, but with enhanced water solubility,
photostability, and higher quantum yields. Most of the cyanine dyes are more environ-
mentally stable than their traditional counterparts, rendering their fluorescence emission
intensity less sensitive to pH and organic mounting media. In a manner similar to the
Alexa Fluors, the excitation wavelengths of the cyanines are tuned specifically for use

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 FLUORESCENT DYES AND PROTEINS IN FLUORESCENCE MICROSCOPY 215

(a) (b)

(c) (d)

(e) (f)

Figure 11.7
Imaging of synthetic fluorophores and fluorescent proteins in fluorescence microscopy.
(a) Rat thoracic aorta cells stained with Alexa Fluor 350 (actin), Alexa Fluor 568 (tubulin),
and SYTOX Green (DNA). (b) Kangaroo rat cells (PtK2) stained with Cy2 (keratin), Mito-
Tracker Red (mitochondria), and DAPI (nucleus). (c) HeLa cell stained with QDot 525
(tubulin), QDot 585 (Golgi), and QDot 655 (histones). (d) Thirty-micrometer rat brain section
stained with Alexa Fluor 488 (vimentin), Alexa Fluor 568 (GFAP), and DRAQ5 (nucleus).
(e) HeLa cells expressing mTurquoise-H2B (nucleus), mEGFP-Gal-T (Golgi), and mCherry-
pyruvate dehydrogenase (mitochondria). (f) Madin–Darby canine kidney cells stained with
Alexa Fluor 488 (nuclear pores) and Alexa Fluor 568 (ZO-3). Bars = 10 µm (a)–(c), (e), and
(f); and 50 µm (d).

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 216 FLUORESCENCE MICROSCOPY

with common laser and arc-discharge sources, and the fluorescence emission can be
detected with traditional filter combinations.

Fluorescent Environmental Probes

A wide variety of fluorescent probes have been developed to monitor such effects as
localized concentrations of alkali and alkaline earth metals, heavy metals (employed
biochemically as enzyme cofactors), inorganic ions, thiols and sulfides, nitrite, as well
as pH, solvent polarity, and membrane potential. Originally, the experiments in this
arena were focused on changes in the wavelength and/or intensity of absorption and
emission spectra exhibited by fluorophores upon binding calcium ions in order to
measure intracellular flux densities. These probes bind to the target ion with a high
degree of specificity to produce the measured response and are often referred to as
spectrally sensitive indicators. Ionic concentration changes are determined by optical
ratio signal analysis to monitor the association equilibrium constant between the ion
and its host. The concentration values derived from this technique are largely indepen-
dent of instrumental variations and probe concentration fluctuations due to photobleach-
ing, loading parameters, and cell retention.

Organelle Probes

Fluorophores targeted at specific intracellular organelles, such as the mitochondria,

lysosomes, Golgi apparatus, and endoplasmic reticulum, are useful for monitoring a
variety of biological processes in living cells using fluorescence microscopy. Organelle
probes consist of a fluorochrome nucleus attached to a target-specific functional group
that assists in localizing the fluorophore through covalent, electrostatic, hydrophobic,
or similar types of bonds. Many of the fluorescent probes designed for selecting organ-
elles are able to permeate or sequester within the cell membrane and therefore are useful
in living cells, while others must be applied using immunocytochemistry techniques.
In living cells, organelle probes are useful for investigating transport, respiration,
mitosis, apoptosis, protein degradation, acidic compartments, and membrane phenom-
ena. Cell impermeant fluorophore applications include nuclear functions, cytoskeletal
structure, organelle detection, and probes for membrane integrity. In many cases, living
cells that have been labeled with permeant probes can subsequently be fixed and coun-
terstained with additional fluorophores in multicolor labeling experiments.

Quantum Dots

Quantum dots are inorganic semiconductor nanocrystals composed of a cadmium sel-

enide (CdSe) core surrounded by a zinc sulphide (ZnS) shell that exhibit fluorescent
properties due to a phenomenon known as confined exciton emission. A passivation
layer and hydrophilic coating must be applied to quantum dots for biological applica-
tions in fluorescence microscopy, and they must also be conjugated to streptavidin or
antibodies for targeting. The fluorescence emission profile of quantum dots is remark-
ably symmetrical and generally exhibits a large quantum yield, while their broad
absorption profile enables them to be excited over a large wavelength range (tens to
hundreds of nm). The size of the CdSe core dictates the emission spectral profile, with

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 FLUORESCENT DYES AND PROTEINS IN FLUORESCENCE MICROSCOPY 217

smaller cores (∼2 nm) emitting in the blue regions and larger cores (5–7 nm) emitting
in the yellow and red wavelengths. In general, the photostability for quantum dots
dramatically exceeds that of all other known fluorophores. Because they do not penetrate
the cell plasma membrane, targeting remains a problem with quantum dots in live-cell
imaging applications, but conjugates to secondary antibodies are widely used in prepar-
ing samples using immunofluorescence techniques.

Fluorescent Proteins

The discovery and development of naturally occurring fluorescent proteins and their
mutated derivatives have led to a virtual revolution in the investigation of a wide spec-
trum of intracellular processes in living organisms. These biological probes have pro-
vided scientists with the ability to visualize, monitor, and track individual molecules
with high spatial and temporal resolution in both steady-state and kinetic experiments.
Fluorescent proteins have become so useful that three of the pioneering investigators,
Roger Tsien (University of California, San Diego), Martin Chalfie (Columbia Univer-
sity), and Osamu Shimomura (Marine Biological Laboratory) were awarded the 2008
Nobel Prize in chemistry for the discovery, demonstration of expression, and develop-
ment of color analogs for these remarkable genetically encoded fluorescent probes.
A variety of marine organisms have been the source of more than 200 fluorescent
proteins and their analogs, which arm the microscopist with a balanced color palette of
noninvasive biological probes for single, dual, and multispectral fluorescence analysis.
Among the advantages of fluorescent proteins over the traditional organic and new
semiconductor probes described previously is their response to a wider variety of bio-
logical events and signals. Coupled with the ability to specifically target fluorescent
probes in subcellular compartments, the extremely low or absent photodynamic toxicity
and the widespread compatibility with tissues and intact organisms, these biological
macromolecules offer an exciting new frontier in live-cell imaging.
The green fluorescent protein (GFP) was the first isolated from the North Atlantic
jellyfish, Aequorea victoria, and exhibits a high degree of fluorescence without the aid of
additional substrates or coenzymes. In native GFP, the fluorescent nucleus is a tripeptide
derivative of serine, tyrosine, and glycine that autocatalytically forms and only requires
molecular oxygen for activation, but no additional cofactors or enzymes. The GFP gene
can be expressed in many other organisms besides the jellyfish, including mammals, to
yield fully functional analogs that display no adverse biological effects. In fact, fluores-
cent proteins can be genetically fused to virtually any protein in living cells using DNA
cloning technology to target virtually any organelle or cellular structure. Lack of a need
for cell-specific activation cofactors renders the fluorescent proteins much more useful
as generalized probes than other biological macromolecules, such as the phycobilipro-
teins, which require insertion of accessory pigments in order to produce fluorescence.
A broad range of fluorescent protein genetic variants have been developed that
feature fluorescence emission spectral profiles spanning almost the entire visible light
spectrum. These variants have often been given names of common minerals, celestial
bodies, or fruits bearing colors similar to the emission spectral colors, such as emerald,
turquoise, Venus, Neptune, cherry, and plum (Table 11.2). Mutagenesis efforts in the
original jellyfish GFP resulted in new fluorescent probes that range in color from blue to
yellow and are some of the most widely used in vivo reporter molecules in biological
research. Longer wavelength fluorescent proteins, emitting in the orange and red spectral

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 218 FLUORESCENCE MICROSCOPY

regions, have been developed from the marine anemones and reef corals. Still other
species have been mined to produce similar proteins having cyan, green, yellow, orange,
red, and far-red fluorescence emission. Research efforts are ongoing to improve the
brightness and stability of fluorescent proteins, thus improving their overall usefulness.

ARRANGEMENT OF FILTERS AND THE EPI-ILLUMINATOR IN

THE FLUORESCENCE MICROSCOPE

The fluorescence microscope is modified in several important ways in order to obtain

images that are bright and well defined:

• A bright light source, such as a mercury, xenon, or metal halide arc lamp, is
required because only a narrow band of wavelengths, and consequently a small
portion of the illuminator output, is used to excite fluorophores in the specimen.
• For efficient high contrast imaging, both the illuminator and objective are posi-
tioned on the same side of the microscope slide as the specimen. In this arrange-
ment, the lamp and light delivery assembly are called an epi-illuminator, and the
objective functions both as the condenser delivering excitatory light to the speci-
men and as the objective imaging lens, collecting fluorescent light and forming
an image of the fluorescent object in the image plane.
• Fluorescence filter sets containing three essential filters (excitation filter, dichro-
matic mirror, and barrier [or emission] filter) are positioned in the optical path
between the epi-illuminator and the objective. This arrangement is shown in
Figure 11.8.
• High NA, oil immersion objectives made of low-fluorescence glass are used
to maximize light collection and provide the greatest possible resolution and
contrast.

Epi-illumination is made possible by the employment of a dichromatic mirror, which

is mounted together with exciter and barrier filters as a fluorescence filter set in a filter
cube (or optical block) that is positioned along the optical axis. The first component,
the excitation filter (or exciter), selectively transmits a band of short wavelengths (rela-
tive to the fluorescence wavelengths) for exciting a specific fluorophore in the speci-
men. The second component, the dichromatic mirror, reflects the short wavelength
excitation light toward the objective and specimen, while transmitting returning long-
wave fluorescent light toward the detector. The dichromatic mirror also directs any
excitation wavelengths reflected by the specimen back toward the exciter filter and
illuminator. The third component is the emission or barrier filter, which transmits the
band of fluorescence wavelengths while blocking any residual short excitation wave-
lengths. The fluorescent wavelengths then form an image on the eye or camera.

Lamps for Fluorescence Excitation

The excitation source can be a band of wavelengths isolated from a metal halide,
mercury, or xenon arc illuminator with an interference filter, or it can be monochromatic
light from a laser or light-emitting diode (LED), but illuminators must produce photons

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 ARRANGEMENT OF FILTERS AND THE EPI-ILLUMINATOR 219

Figure 11.8
Arrangement of filters in a fluorescence filter cube. The diagram shows the orientation of
filters in a filter cube in an epi-illuminator for an upright microscope. The excitation beam
(yellow line) passes through the exciter and is reflected by the dichromatic mirror and directed
toward the specimen (green line). The return beam of emitted fluorescence wavelengths (red
line) passes through the dichromatic mirror and the emission filter to the eye or camera.
Excitation wavelengths backreflected or scattered at the specimen are again reflected by the
dichromatic mirror back toward the light source. Excitation wavelengths that manage to pass
through the dichromatic mirror are blocked by the barrier (emission) filter.

of the proper wavelength and at a sufficiently high intensity to be efficient. To obtain

a particular wavelength of the desired intensity, it may be necessary to use a certain
illuminator—for example, a mercury arc excites DAPI much more effectively than a
xenon arc, whereas a metal halide illuminator is far more efficient at exciting GFP and
fluorescein than a mercury lamp. Therefore, it is important that the microscopist match
the illuminator to the fluorescent dye in question.
For fluorescence microscopy, 100-W mercury, 75-W xenon, and 150-W metal
halide arc lamps are commonly used. All three lamps produce bright, continuous emis-
sion over the visible spectrum (400–700 nm), but mercury and metal halide are distinct
in containing sharply defined emission lines at 366 (UV), 405, 436, 546, and 578 nm.
It is important to reexamine the spectra and characteristics of arc lamps, which are
presented in Chapter 3. At 546 nm, mercury and xenon lamps are 10–100× brighter
than a 100-W quartz halogen lamp, which is typically used for brightfield microscopy,
but is too weak to excite most fluorophores adequately. The bright emission lines of
the mercury and metal halide arc lamps are useful for exciting certain fluorophores,
such as DAPI and Hoechst dye 33258, Lucifer yellow, rhodamine, Cy3, and Texas red,

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 220 FLUORESCENCE MICROSCOPY

but at other spectral regions, xenon is brighter and may be more useful (fluorescein,
Alexa 488, Flo3, allophycocyanine, and Cy5). Refer to Table 11.1 and Figure 11.5 on
fluorescent dyes and the spectra of xenon and mercury light sources in Chapter 3 to
confirm this for yourself and to find other well-matched dye-illuminator partners.

The Epi-Illuminator

The epi-illuminator consists of the lamp and its collector lens plus a connector tube
fitted with a field stop diaphragm, a relay lens, and slots for additional filters (Fig. 11.9).
It is attached to the lamp at one end and is mounted at its other end to the microscope
in the vicinity of the fluorescence filter cube. The lamp housing should contain a focus-
able collector lens to fill the rear aperture of the objective as required for Koehler
illumination. On most research microscopes, the epi-illuminator is a built-in component
of the microscope body. Apart from proper adjustment and focus of the lamp, little
other manipulation of the illumination pathway is required. Check that the field stop
diaphragm is centered and is opened to provide optimal framing of the specimen. Epi-
illumination is much more efficient than transmitted mode (or diascopic) illumination
because there is much less background in the fluorescence image.
One of the most important adjustments in fluorescence microscopy is the alignment
of the illuminator. Since the amplitude of fluorescence signals depends directly on the
amount of excitation, uneven illumination of the object by a misaligned lamp will result
in bright and dark regions in the fluorescence image. This is especially detrimental for
quantitative work. Lamp alignment is discussed in Chapter 3 and demands attention to
two points: (1) centration of the image of the arc and its reflection on the optical axis,
and (2) spreading of the illumination beam with the lamp collector lens to fill the rear
aperture of the objective evenly and homogeneously.

Figure 11.9
Internal anatomy of a fluorescence microscope epi-illuminator. The unit attaches to an upright
microscope and contains a mercury arc-discharge lamphouse and a collector lens system to
collimate light and direct it into the microscope optical train. Included are a heat filter to
remove IR wavelengths, neutral density filters, aperture and field diaphragms, and a filter
cube turret enabling a choice of up to six filter combinations.

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 ARRANGEMENT OF FILTERS AND THE EPI-ILLUMINATOR 221

Filters

Filters used to isolate bands of wavelengths in fluorescence microscopy include colored

glass filters, thin-film interference filters, or a combination of filter types. The filters
also include longpass or shortpass edge filters and narrow or broad bandpass filters.
The design and action of filters are discussed in Chapter 3; the optical performance of
representative filters used for fluorescence microscopy is shown in Figures 11.10 and
11.11. It is essential to understand the properties and value of these indispensable
components of the fluorescence microscope. You will need to know how to “read” the
absorption and emission spectra of a dye and the transmission profiles of the filters.
This knowledge is required to select the best among several possible filter sets for
examining the fluorescence of a certain fluorophore. You will also need to interpret and
explain differences among fluorescence microscope images obtained using different
filter sets. In addition, you may need to create new filter sets using components of
filter sets already in hand. This will greatly extend the capacity of a limited collection
of filters. As you install and remount filters, remember that interference filters are
usually mounted in a specific orientation with respect to the illuminator and the speci-
men in order to obtain optimal performance (see Chapter 3). The transmission profiles
of filter sets for fluorescence microscopy can be examined at the websites of Chroma,
Inc., ([Link] Semrock, Inc., ([Link] and Omega
Optical, Inc. ([Link] among others (see spectral viewers in
Microscopy Web Resources section).

The Dichromatic Mirror

The dichromatic mirror or beamsplitter is a special longpass filter coated with multiple
layers of dielectric materials similar to those contained in thin-film interference filters, but
specially designed for reflection and transmission at certain boundary wavelengths. The
mirror, which is the key component in a fluorescence microscope epi-illumination system,
is mounted at a 45° angle with respect to the optical axis within a filter cube and faces the
light source. At this angle, the dichromatic mirror reflects short excitation wavelengths at
a 90° angle along the optical axis to the specimen, but transmits long fluorescence wave-
lengths that are collected by the objective and directed to the image plane. The transition
from near total reflection to maximal transmission can be remarkably sharp, occurring
over 20–30 nm, allowing the mirrors to act as precise discriminators of excitation and
fluorescence wavelengths (Fig. 11.10). (For a description of the design and performance
of interference filters, see Chapter 3.) The specifications for a dichromatic mirror assume
a 45° angle between the axis of the incident beam and the plane surface of the mirror.
Dichromatic mirrors should be handled with extreme care, because the exposed dielectric
layers can be scratched and damaged during handling and cleaning. The cleaning proce-
dure is the same as that used for cleaning other optical surfaces (Chapter 4).
Transmission profiles for dichromatic mirrors usually show multiple broad peaks
and troughs that correspond to bands of wavelengths that experience high transmittance/
low reflectance (at peaks) and low transmittance/high reflectance (at troughs) (Fig.
11.11). Filter sets are designed so that the band of excitation wavelengths (high-percent
transmission) from the exciter precisely matches a trough in the dichromatic (low-
percent transmission) so that these wavelengths are reflected to the specimen. Longer
fluorescent wavelengths emitted by the specimen must also match the peak to the right

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 (a)

(b)

Figure 11.10
Transmission profiles of filters in a fluorescein filter set. The figure shows (a) the excitation
and emission spectra of fluorescein and (b) the transmission profiles of three filters belonging
to a high performance filter set (BrightLine® Single-Band FITC, Semrock, Inc., Rochester,
NY). Bandpass excitation and emission filters (blue and red profiles shown in panel b) transmit
bands of light that occupy the peak regions of the respective absorbance and emission
spectra shown in the upper panel. To maintain a distinct separation of these components,
the transmission profiles (gray boxes in panel a) are not exactly centered on the excitation
and emission maxima of the dye. The dichromatic mirror (yellow line) reflects light (100%
reflection corresponds to 0% transmission on the curve) or partially transmits light (90%),
depending on the incident wavelength. The pronounced trough in the transmission profile,
representing a peak of reflectance, is used to reflect the band of excitation wavelengths from
the exciter filter onto the specimen. Boundaries between transmitted and reflected bands of
wavelengths are designed to be as steep as possible to assure complete separation of the
reflected and transmitted wavelengths. The pattern of rapidly rising and falling spikes (termed
ringing), typical of dichromatic mirrors, indicates major improvements in thin-film technology
over the last several years.

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 ARRANGEMENT OF FILTERS AND THE EPI-ILLUMINATOR 223

(a)

(b)

(c)

Figure 11.11
Transmission profiles of a triple-band filter set for DAPI, FITC, and Texas red (BrightLine®
Triple-Band, Semrock, Inc., Rochester, NY). Each of the three filters contains multiple band-
widths that transmit or reflect three distinct bands of wavelengths simultaneously. The eye
or camera sees a multicolor image based on the three dyes. Profiles of the exciter filter
(a, top panel), dichromatic mirror (b, middle panel), and emission filter (c, bottom panel) are
shown. The broad vertical gray band across the drawing distinguishes the spectral regions
of the filters for FITC from those for DAPI and Texas red.

of the trough so that they are transmitted to the barrier filter and the detector. It is
important that the transmission, reflectance, and emission characteristics of the exciter
and dichromatic be closely matched, and that they be appropriate for the absorption
and emission maxima of the dye; otherwise, excitation wavelengths can pass through
the dichromatic and fog the image, or fluorescent wavelengths can be reflected at the

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 224 FLUORESCENCE MICROSCOPY

dichromatic mirror, reducing image brightness. Even when filters and fluorophores are
appropriately matched, performance is usually compromised somewhat if the transmis-
sion profiles of the exciter and dichromatic mirror overlap. When this happens, some
excitation light passes through the dichromatic mirror, reflects off the walls of the filter
cube, and can be partially transmitted by the emission filter because the angle of inci-
dence with that filter is oblique. Transmission of unwanted wavelengths through a filter
set is called bleedthrough, and the amount of bleedthrough for typical filter sets is
generally about 10%. Microscope manufacturers continue to improve fluorescence
optical designs to give higher contrast images.
It is important to recognize that in addition to reflecting the excitation band of
wavelengths, a dichromatic mirror usually reflects bands of wavelengths shorter than
the excitation band. Therefore, you cannot always depend on the dichromatic filter to
block transmission of unwanted short wavelengths, which to a greater or lesser extent
always leak through the exciter filter. It is usually wise to insert an additional UV-
blocking filter into the beam when examining live cells by fluorescence microscopy.

Dichromatic versus Dichroic Mirrors

Mirrors or beamsplitters that reflect or transmit selected bands of wavelengths

are often referred to as dichromatic or dichroic, but the latter term can be confus-
ing because it also refers to a property related to pleochroism, which describes
the differential ability of a substance to absorb light based on the polarization
azimuth of the incident light. Use of the term dichroic is widespread in the
fluorescence filter literature, but here we will refer to fluorescence microscopy
beamsplitters as dichromatic mirrors.

Advances in thin-film coating technology allow for the creation of multiple transmis-
sion peaks and alternating reflection troughs in a single interference filter or dichromatic
mirror. When matched appropriately, two filters and a dichromatic mirror can be com-
bined to create a multiple fluorescence filter set that allows simultaneous excitation and
fluorescence transmission of multiple fluorophores (Fig. 11.11). Multiple-wavelength
filters and dichromatic mirrors are now commonly employed in research-grade micro-
scopes and confocal fluorescence microscope systems (see Chapter 13). Double, triple,
and even quadruple fluorescence filter sets are available for fluorescence microscopy,
although these sets are expensive and suffer somewhat from bleedthrough—that is, the
transmission of fluorescence from one dye through bandwidths intended for other dyes.
The clearest multifluorophore images are obtained by taking separate grayscale pictures
with filter sets optimized for each dye and then combining the images into a single
composite color image.

OBJECTIVES AND SPATIAL RESOLUTION IN

FLUORESCENCE MICROSCOPY

Proper selection of an objective is important, especially for imaging dim fluorescent

specimens. High-NA, oil immersion plan-fluorite lenses and plan-apochromatic objec-

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 CAUSES OF HIGH FLUORESCENCE BACKGROUND 225

tives are ideal, because at NA = 1.3 or 1.4, their light-gathering ability is especially
high. These lenses feature excellent color correction, so different fluorescent wave-
lengths are brought to the same focus in the focal plane. They are also transparent to
UV light—a requirement for examining UV-excitable dyes, such as DAPI, Hoechst,
and AMCA. In addition, they contain low-fluorescence glass—a feature that minimizes
background fluorescence and gives high contrast. Since image brightness (photon flux
per unit area and time) is proportional to NA4/M2, where NA is the numerical aperture
and M is the magnification, a 60 × 1.4 NA plan apochromatic objective is among the
brightest objectives and is very well suited for fluorescence imaging.
The spatial resolution d for two noncoherent fluorescent point objects is the same
as in brightfield microscopy with incoherent light, and is given as d = 0.61λ/NA, where
λ is the mean wavelength of fluorescent light transmitted by the barrier filter. Resolu-
tion, brightness, and other features defining the optical performance of objectives are
described in Chapter 4.

CAUSES OF HIGH FLUORESCENCE BACKGROUND

For research microscopes with properly selected fluorescence filter sets, the amount of
background fluorescence in the image of a specimen containing a single fluorophore is
usually 15–30% of maximum specimen brightness—not 0% as might be expected. An
example of background signal in cells expressing a fluorescent protein is shown in
Figure 11.12. Because background fluorescence is always present, it is important to
take steps to keep the background signal as low as possible.

• Less than ideal performance of filter sets, where transmission and reflectance by
interference filters and the dichromatic mirror are not 100% and where the transi-
tion boundaries between transmission and reflection are not sharply defined, is a
major contributor to background signal. These problems are compounded when
poorly performing filters are used together in a single filter set.
• Specimen preparation must include complete neutralization of unreacted aldehyde
groups and blocking of remaining reactive sites to minimize nonspecific binding
of the fluorescent probe. It is also necessary to completely remove unbound fluo-
rophore by thorough rinsing. However, even if these precautions are followed,
background fluorescence from unbound antibody can be high for a mounted cov-
erslip containing labeled cultured cells. This is because the labeling reaction is an
equilibrium between bound and free states of the antibody. Even with an equilib-
rium dissociation constant of 1 nM and a modest concentration of 106 antigen
binding sites per cell, a significant fraction of antibody would be expected to dis-
sociate and be free in the mounting medium.
• Use of lipid-based transfection reagents to introduce DNA vectors into living cells
often increases autofluorescence and background signal when the cells are
observed in fluorescence microscopy. In addition, fluorescent protein expression
levels vary widely in transient transfection so that poorly expressing cells that are
not bright enough to image increase the background when they are adjacent to
cells that express well. After culturing the cells for a few days posttransfection,
autofluorescence usually drops to acceptable levels.

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 (a)

(b)

(c)

Figure 11.12
Comparison of specimen and background fluorescence in a typical fluorescence image.
(a) Live HeLa cells were transiently transfected with mEmerald (a GFP derivative) fused to
annexin A4, a protein involved in trafficking of vesicles and membrane organization. Upon
induction of calcium by addition of ionomycin to the culture medium, the annexin fusion trans-
locates to the plasma and nuclear membranes as illustrated in panel a. Three adjacent cells in
the upper left (yellow asterisk) and right (red asterisk), as well as the lower right-hand side of
the image (green asterisk), express the fusion at much lower levels and thus generate a high
level of background. The horizontal blue and red lines represent rows of pixels whose numeric
values are shown in panels b and c. (b) Intensity profile of the pixel values under the blue line
in panel a, with regions corresponding to the asterisks marked. Note that the background is not
black at any point, but shows a minimum intensity that is ∼25% of that at the center of the bright
cells. (c) Intensity profile of the pixel values under the red line in panel a with the region corre-
sponding to the asterisk marked. This is typical of most transient transfections, as well as cells
stained with synthetic dyes or labeled using immunofluorescence techniques. The causes of
background fluorescence are discussed in the text. Bar = 20 µm.

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 THE PROBLEM OF BLEEDTHROUGH WITH MULTIPLY STAINED SPECIMENS 227

• Reflections and scattering in the optical pathway cause rays to enter a filter at an
oblique angle, reducing the filter ’s transmission/reflection efficiency. One site of
concern is the rear wall of the filter cube, where excitatory rays that are partially
transmitted by the dichromatic mirror are reflected and are transmitted by the
emission filter because they are not incident at an angle perpendicular to the plane
of the emission filter. In recent Zeiss designs where the back wall of the filter cube
is removed, reflections at this location are removed, and image contrast is improved
by 15–20%. Other filter cube manufacturers incorporate light-absorbing coatings
on the interior of optical blocks to reduce or eliminate scattered light.
• Dust, fingerprints, and scratches on filters and lens elements scatter significant
amounts of light, resulting in an increase in background signal and reduced con-
trast. Cleaning filters and optical surfaces significantly reduces this problem.
Interference filters also deteriorate gradually over time due to handling and the
presence of water vapor and chemicals in the air. Blemishes in old interference
filters in the form of microscopic scaling, pinholes, and scratches can also scatter
significant amounts of light.
• Fluorescence from other sources, including the glass in certain objectives, immer-
sion oil, plastic tissue culture dishes, and autofluorescence from the specimen
itself contribute to the background. Most microscope manufacturers make objec-
tives of low fluorescence glass and provide low fluorescence immersion oil for
fluorescence microscopy. The new low-fluorescence immersion oils available
from microscope manufacturers and independent sources, such as Cargill, Inc.
(Cedar Grove, NJ), increase contrast significantly and must be employed.

THE PROBLEM OF BLEEDTHROUGH WITH MULTIPLY

STAINED SPECIMENS

Another problem in imaging double-stained specimens is bleedthrough, the crossover

of fluorescence signal from one fluorophore through the filter set of the other fluoro-
phore. A common example is double labeling with fluorescein and rhodamine using
separate filter sets to capture different images of each of the two fluorophores.
Bleedthrough can be reduced, but is never completely eliminated, because:

• The excitation spectra of two or more fluorophores are broad and overlap to
a significant extent (Figs. 11.5 and 11.6). Thus, the excitation of fluorescein
at 490 nm also causes rhodamine to fluoresce. The solution is to choose fluoro-
phores with well-separated excitation spectra so that the excitation peak for one
fluorophore is many wavelengths shorter than the excitation peak for the second
fluorophore.
• The fluorescence spectra of the fluorophores may overlap, allowing fluorescence
from the shorter-wavelength fluorophore 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
fluorophore; a longpass barrier filter is usually used for the longer wavelength
fluorophore.
• The fluorescence emission of one fluorophore (fluorescein) may stimulate a second
longer-wavelength dye (rhodamine) to fluoresce. Selecting well-separated dyes

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 228 FLUORESCENCE MICROSCOPY

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 fluorophores
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
second 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 or metal halide arc lamp frequently forget that rho-
damine 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 bleedthrough
generally remains about 10–15%. In experiments involving double labeling, you should
always examine single-stained specimens using the filter set for the other fluorophore
to assure that the amount of bleedthrough is minimal. In many cases, the main cause
of bleedthrough 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.

QUENCHING, BLINKING, AND PHOTOBLEACHING

The consequences of quenching, photobleaching, and molecular blinking occur in

practically all forms of fluorescence microscopy, and result in an effective reduction in
the levels of emission. The phenomena are distinct in that quenching and blinking are
often reversible, whereas photobleaching is not. Quenching arises from a variety of
competing processes that induce nonradiative relaxation (without photon emission)
of excited state electrons to the ground state. Because these nonradiative transition
pathways compete with the fluorescence relaxation, they usually dramatically lower
or completely eliminate emission. A wide variety of simple elements and compounds
behave as quenching agents, including oxygen, halogens, amines, some biological poly-
mers, and many organic molecules.
Blinking is a phenomenon that occurs during continuous excitation of a fluorescent
molecule where emission transitions between “on” and “off” states somewhat like
twinkling stars in the night sky. This was predicted in 1913 by Niels Bohr in a quantum
mechanical explanation of intensity fluctuations on short timescales. However, experi-
ments have determined that fluorophores can remain in the off state for intermittent
periods ranging from seconds to minutes. The mechanism underlying fluorescence
blinking is not well understood, but it is thought to occur by a transition from the singlet
excited state to a dark triplet state that occurs on a timescale much longer than fluores-
cence emission. In most cases, during routine imaging in fluorescence microscopy,
blinking goes unnoticed, but it can become a problem (or solution) when examining
specimens using superresolution techniques as discussed in Chapter 15. Blinking can
be regulated to some extent by the addition of oxidizing and/or reducing agents into
the imaging medium.
In contrast to quenching and blinking, photobleaching (also termed fading) occurs
when a fluorophore permanently loses the ability to fluoresce due to photon-induced

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 QUENCHING, BLINKING, AND PHOTOBLEACHING 229

chemical damage and covalent modification. Upon transition from an excited singlet
state to the excited triplet state, fluorophores may interact with another molecule to
produce irreversible covalent modifications leading to the destruction of fluorescence.
The triplet state is relatively long-lived with respect to the singlet state, thus allowing
excited molecules a much longer timeframe to undergo chemical reactions with com-
ponents in the environment. The average number of excitation and emission cycles that
occur for a particular fluorophore before photobleaching is dependent upon the molecu-
lar structure and the local environment. Some fluorophores bleach quickly after emitting
only a few photons (Fig. 11.13), while others that are more robust can undergo thou-
sands or even millions of cycles before bleaching.
An important class of photobleaching events is called photodynamic, meaning they
involve the interaction of the fluorophore with a combination of light and oxygen.
Reactions between fluorophores and molecular oxygen permanently destroy fluores-
cence and yield a free radical singlet oxygen species that can chemically modify other
molecules in living cells. This serves as the basis for an advanced imaging technique

(a) (b) (c)

(d) (e) (f)

Figure 11.13
Photobleaching (fading) observed in a series of digital images captured at different time points
for a multiply-stained culture of Indian Muntjac deerskin fibroblast cells. The nuclei were
stained with DAPI (blue fluorescence), while the mitochondria and actin cytoskeleton were
stained with MitoTracker Red (red fluorescence) and an Alexa Fluor phalloidin derivative
(Alexa Fluor 488; green fluorescence), respectively. Time points were taken in 2-minute
intervals using a fluorescence filter combination with bandwidths tuned to excite the three
fluorophores simultaneously while also recording the combined emission signals. Note that
all three fluorophores have a relatively high intensity in panel a, but the DAPI (blue) intensity
starts to drop rapidly at 2 minutes and is almost completely gone at 8 minutes. The mito-
chondrial and actin stains are more resistant to photobleaching, but the intensity of both drops
over the course of the timed sequence (10 minutes). (a) t = 0. (b) t = 2 m. (c) t = 4 m. (d)
t = 6 m. (e) t = 8 m. (f) t = 10 m. Bar = 20 µm.

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 230 FLUORESCENCE MICROSCOPY

called chromophore-assisted laser inactivation (CALI). The amount of photobleaching

due to photodynamic events is a function of the molecular oxygen concentration and
the proximal distance between the fluorophore, oxygen molecules, and other cellular
components. Photobleaching can be reduced by limiting the exposure time of fluoro-
phores to illumination or by lowering the excitation energy. However, these techniques
also reduce the measurable fluorescence signal. Perhaps the best protection against
photobleaching is to limit exposure of the fluorophore to intense illumination (using
neutral density filters) coupled with the judicious use of commercially available anti-
fade reagents (discussed below) that can be added to the mounting solution or cell
culture medium.
Under certain circumstances, the photobleaching effect can also be utilized to
obtain specific information that would not otherwise be available. For example, in
FRAP experiments (see Chapter 12), fluorophores within a target region are intention-
ally bleached with excessive levels of irradiation. As new fluorophore molecules diffuse
into the bleached region of the specimen (recovery), the fluorescence emission intensity
is monitored to determine the lateral diffusion rates of the target fluorophore. In this
manner, the translational mobility of fluorescently labeled molecules can be ascertained
within a very small region of a single cell.

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
(1O2), superoxide (O•− 2 ), hydroxyl radical (OH•), various peroxides (ROOR’), hydro-
peroxides (ROOH), and others. They react with oxidizable metabolites and components
in the cell, such as the pyridine nucleotides in nucleic acids, several amino acids, glu-
tathione, lipids, and ascorbate. Among the immediate effects is damage to membrane
lipids and proteins, including ion pumps, channels, and gates, which leads to loss of
ion balance, loss of transmembrane potential, and rapid cell death. We discussed the
consequences of phototoxicity on cell shape and cell behavioral responses in Chapter
3, and will cover live-cell imaging in detail in Chapter 16. Briefly, cells cease move-
ment; intracellular organelle trafficking ceases; cells round up and form vacuoles and
eventually lyse. Excitable cells, such as neurons and free-living amoebae, are among
the most sensitive to light exposure in the microscope. Just 3 seconds of continuous
exposure to the blue excitation wavelengths from a mercury lamp filtered by a standard
fluorescein filter set is sufficient to cause Acanthamoeba and Dictyostelium amoebae
to stop moving and round up. The requirement to minimize the exposure of living cells
to light can place extreme demands on photography, because short exposures reduce
the signal-to-noise ratio and result in grainy images. Control of the exposed dose is
especially difficult during the acquisition of time-lapse sequences. The following
approaches reduce the effects of phototoxicity:

• Additional UV and IR cutoff filters should be inserted near the illuminator, since
the majority of the spectral output of metal halide, mercury, and xenon lamps

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 EXAMINING FLUORESCENT MOLECULES IN LIVING CELLS 231

occurs in the UV and IR regions of the spectrum, and because most fluorescence
filter sets are not completely effective in removing these unwanted wavelengths.
• Minimize exposure to light to allow time for dissipation and degradation of free
radicals. Phenol red-free basal salt solution is recommended to reduce photon
absorption.
• Addition of millimolar concentrations of anti-free radical reagents to live cells
and in vivo preparations of purified cell organelles and filaments minimizes free
radical damage, because the reagents readily react with free radicals and help spare
endogenous molecules. Sodium ascorbate, reduced glutathione, or Trolox (a
water-soluble form of vitamin E) at 10 mM concentrations are effective, particu-
larly for in vivo systems.
• To retard the rate of free radical formation, the concentration of dissolved oxygen
can be reduced to ∼4% of the saturating value for buffer in room air by adding
oxygen-depleting enzymes to the medium. Low oxygen concentration is usually
not damaging to tissue culture cells—in fact, cells exist at similarly low values of
oxygen tension in body tissues. One effective commercial product is Oxyrase
(Oxyrase, Inc., Mansfield, OH), a preparation of respiratory particles from Esch-
erichia coli membranes. Alternatively, an oxygen-scavenging system can be con-
structed based on a combination of catalase, glucose oxidase, and D-glucose. Cells
in well chambers open to the air can be covered with a layer of embryo-grade
mineral oil, such as that available from Sigma Chemical Company (St. Louis,
MO), to prevent resaturation of the medium with oxygen.

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