Chapter 5
Visualizing cells and the CSK
1. Visible Light microscopy
Ordinary microscopy trans-illuminates the specimen (light shining from above the
specimen into the objective lens), whose image contrast is based on differences in optical
density, i.e. light absorbance, within the specimen. Improved images can be obtained
through phase contrast microscopy, where contrast is based on differences in refractive
index, i.e. the optical path of light as it travels through the specimen. Phase contrast
gives better definition of cell borders, allowing easier micromanipulation of the cells.
Another type of microscope useful for cytomechanics is differential interference contrast
(DIC) that registers the phase shift of light through the specimen. DIC microscopy is
much more accurate for metrological measurements, compared with conventional
microscopy, as illustrated below (from Fung). In the left panel of Figure 5.1 are phase
contrast images of RBCs (transverse and sagittal views) in which the focus was slightly
changed from top to bottom. The center panel gives the best view of its biconcave
structure, but with slight focus changes, halo effects around the cell perimeter
significantly change the cell appearance and apparent size. The 2 DIC images, however,
are also taken at different focus adjustments, however the apparent size does not change
and measurement would be more reliable.
Figure 5.1 Comparison of phase contrast with
differential interference micrscopy. At left are
views of an RBC at 3 slightly different focal
adjustments. Border definition is difficult. At
right, DIC images show a clear border,
independent of focal adjustment..
2. Fluorescent Labeling
Fluorescent probes enable researchers to detect particular components of complex
biomolecular assemblies, including live cells. Most compounds, ranging from minerals
to proteins, can be detected or labeled with a fluorescent dye. Calcium, for example, can
be detected quantitatively in its ionic form using dyes whose light output is proportional
to [Ca++]. Proteins can be specifically labeled with an antibody carrying a fluorescent
dye. The basic plan is schematized below. To look at a specific protein, cells are labeled
with a fluorescent dye that is conjugated with an antibody for that protein. This process is
sometimes called ‘decorating’ the cell with a dye. Standard techniques and commercial
dyes for many different CSK components are available (i.e. , Molecular Probes, Eugene,
OR). Once labeled, the protein can be visualized by exciting the dye with a short
wavelength light, usually in the ultraviolet range. The dye will then emit (fluoresce) light
at a longer wavelength that can be quantified and turned into an image. The mixture of
reflected and emitted fluorescent light is focused onto a photodetector (photomultiplier)
via a dichroic mirror (beam splitter). The reflected light is deviated by the dichroic mirror
while the emitted fluorescent light passes through in the direction of the photomultiplier
(PM). The PM can quantify the number of photons, and relate it to concentration of
proteins. Cameras can also produce images such as those shown in below:
Structure by
immunofluorescence
Photo
Antibody-labelled
specimen
PMT
Filter
Dichroic
mirror
Short 
Figure 5.2
Multiple proteins can be imaged simultaneously, as shown in the cell below that was
decorated with Phalloidin, staining F actin green, and with Texas red, staining G actin.
Specific antibodies for both F and G actin were attached to those dyes. Note that the
exact colorization of different proteins was determined by computer imaging software
and thus represents ‘false’ colors. It should be appreciated that the only light seen in this
image has come from either F or G actin- all other components of the cell are dark, due to
the optics of fluorescent microscopes.
Figure 5.3 Fibroblast
Since the image was obtained from an ordinary fluorescent microscope, the light
collected from the proteins represents most or all of the light coming from the entire cell.
In other words, while the 2-D resolution is excellent, the 3-D resolution is not, since the
cell has a thickness. The consequence of this is that the red coming from the G actin in
the nucleus appears more diffuse relative to that coming from F actin. This is because the
nucleus is thicker than the rest of cell, and the relative abundance of G actin emits a
saturating amount of red light, diffusing its appearance. The nucleus may actually have a
lower proportion of G actin for its volume than is indicated. Higher resolution
microscopy can be obtained using con-focal optics, as described below.
Dyes that are sensitive to cellular salts, such as Na+, K+ and Ca++ can be introduced into
the cell to indicate their respective concentrations. Ca++ is perhaps the most important
small-molecule regulator of cell mechanics, and its concentration can be imaged in
several ways.. Certain events, such as electrical depolarization, or mechanical stimuli,
can release Ca from internal stores, as exemplified below. These image sequences (read
left to right) show a temporal wave of Ca++ after an egg cell has been perturbed
mechanically at its upper right quadrant (arrow). The experiment started at top left. The
wave is indicated by the spectrum of colors that represent calibrated concentrations of
Ca++, according to the bar at lower right. Note that the color-coding is done by the
computer based on photon intensity, not on color, since Ca++ produces only one color.
Thus red represents the highest [Ca++], and blue the lowest (background). These images,
taken in near real-time show that a Ca++ wave results from the poke. Fluorescent dyes for
Ca++ include Fura-2 and Indo.
Begin
Calibration bar
Figure 5.4 Ca Wave
To see the effect of a sudden rise in Ca++ into a cell, it can be introduced into the cell
while trapped in a cage compound loaded with Ca++, and then release it all of a sudden
have been devised to see its instantaneous effects on cells. One such is the ‘caged
compound’ technique, where an organic compound is structured to hold Ca++ in a cage
until it is needed. This procedure is analogous to placing a Trojan horse into the cell.
The cage releases upon deliverance of a pulse of high-energy light. An example of a
caged release of Ca++ in a nerve cell is shown below, where green is the color of the Ca
dye.
Figure 5.5 Caged Ca release
Physical Basis of Immunofluorescence
(Adapted From http://www.probes.com/handbook/sections/0001.html)
Fluorescence is the result of a three-stage process that occurs in certain molecules
(generally polyaromatic hydrocarbons or heterocycles) called fluorophores or fluorescent
dyes. A fluorescent probe is a fluorophore designed to localize within a specific region of
a biological specimen or to respond to a specific stimulus. The process responsible for the
fluorescence of fluorescent probes and other fluorophores is illustrated by the simple
electronic-state diagram (Jablonski diagram) shown in Figure 1.
Stage 1: Excitation
A photon of energy h EX is supplied by an external source such as an incandescent lamp
or a laser and absorbed by the fluorophore, creating an excited electronic singlet state
(S1'). This process distinguishes fluorescence from chemiluminescence, in which the
excited state is populated by a chemical reaction.
Stage 2: Excited-State Lifetime
The excited state exists for a finite time (typically 1–10 nanoseconds). During this time,
the fluorophore undergoes conformational changes and is also subject to a multitude of
possible interactions with its molecular environment. These processes have two important
consequences. First, the energy of S1' is partially dissipated, yielding a relaxed singlet
excited state (S1) from which fluorescence emission originates. Second, not all the
molecules initially excited by absorption (Stage 1) return to the ground state (S0) by
fluorescence emission. Other processes such as collisional quenching, fluorescence
resonance energy transfer (FRET) and intersystem crossing (see below) may also
depopulate S1. The fluorescence quantum yield, which is the ratio of the number of
fluorescence photons emitted (Stage 3) to the number of photons absorbed (Stage 1), is a
measure of the relative extent to which these processes occur.
Stage 3: Fluorescence Emission
A photon of energy h EM is emitted, returning the fluorophore to its ground state S0. Due
to energy dissipation during the excited-state lifetime, the energy of this photon is lower,
and therefore of longer wavelength, than the excitation photon h EX. The difference in
energy or wavelength represented by (h EX – h EM) is called the Stokes shift. The Stokes
shift is fundamental to the sensitivity of fluorescence techniques because it allows
emission photons to be detected against a low background, isolated from excitation
photons. In contrast, absorption spectrophotometry requires measurement of transmitted
light relative to high incident light levels at the same wavelength.
Fluorescence Spectra
The entire fluorescence process is cyclical. Unless the fluorophore is irreversibly
destroyed in the excited state (an important phenomenon known as photobleaching, see
below), the same fluorophore can be repeatedly excited and detected. The fact that a
single fluorophore can generate many thousands of detectable photons is fundamental to
the high sensitivity of fluorescence detection techniques. For polyatomic molecules in
solution, the discrete electronic transitions represented by h EX and h EM in Figure 1 are
replaced by rather broad energy spectra called the fluorescence excitation spectrum and
fluorescence emission spectrum, respectively. The bandwidths of these spectra are
parameters of particular importance for applications in which two or more different
fluorophores are simultaneously detected (see below). With few exceptions, the
fluorescence excitation spectrum of a single fluorophore species in dilute solution is
identical to its absorption spectrum. Under the same conditions, the fluorescence
emission spectrum is independent of the excitation wavelength, due to the partial
dissipation of excitation energy during the excited-state lifetime, as illustrated in Figure
1. The emission intensity is proportional to the amplitude of the fluorescence excitation
spectrum at the excitation wavelength (Figure 2).
Fluorescence Instrumentation
Four essential elements of fluorescence detection systems can be identified from the
preceding discussion: 1) an excitation source, 2) a fluorophore, 3) wavelength filters to
isolate emission photons from excitation photons and 4) a detector that registers emission
photons and produces a recordable output, usually as an electrical signal or a
photographic image. Regardless of the application, compatibility of these four elements is
essential for optimizing fluorescence detection. Fluorescence instruments are primarily
of four types, each providing distinctly different information:

Spectrofluorometers and microplate readers measure the average properties of bulk (µL to
mL) samples.

Fluorescence microscopes resolve fluorescence as a function of spatial coordinates in two or
three dimensions for microscopic objects (less than ~0.1 mm diameter).

Fluorescence scanners, including microarray readers, resolve fluorescence as a function of spatial
coordinates in two dimensions for macroscopic objects such as electrophoresis gels, blots and
chromatograms.

Flow cytometers measure fluorescence per cell in a flowing stream, allowing subpopulations
within a large sample to be identified and quantitated.
Other types of instrumentation that use fluorescence detection include capillary
electrophoresis apparatus, DNA sequencers and microfluidic devices. Each type of
instrument produces different measurement artifacts and makes different demands on the
fluorescent probe. For example, although photobleaching is often a significant problem in
fluorescence microscopy, it is not a major impediment in flow cytometry or DNA
sequencers because the dwell time of individual cells or DNA molecules in the excitation
beam is short.
Fluorescence Signals
Fluorescence intensity is quantitatively dependent on the same parameters as absorbance
— defined by the Beer–Lambert law as the linear relationship between absorbance and
concentration of an absorbing species. It is the product of the molar extinction
coefficient, optical path length and solute concentration — as well as on the fluorescence
quantum yield of the dye and the excitation source intensity and fluorescence collection
efficiency of the instrument.
The general Beer-Lambert law applies to dilute solutions or suspensions as:
A=
-dependent molar
-1
-1
absorptivity coefficient with units of M cm , b is the path length, and c is the analyte
concentration. In, fluorescence intensity is linearly proportional to these parameters.
When sample absorbance exceeds about 0.05 in a 1 cm pathlength, the relationship
becomes nonlinear and measurements may be distorted by artifacts such as selfabsorption and the inner-filter effect.
Because fluorescence quantitation is dependent
on the instrument, fluorescent reference standards are essential for calibrating
measurements made at different times or using different instrument configurations. To
meet these requirements, Molecular Probes offers high-precision fluorescent microsphere
reference standards for fluorescence microscopy and flow cytometry and a set of readymade fluorescent standard solutions for spectrofluorometry
A spectrofluorometer is extremely flexible, providing continuous ranges of excitation and
emission wavelengths. Laser-scanning microscopes and flow cytometers, however,
require probes that are excitable at a single fixed wavelength. In contemporary
instruments, the excitation source is usually the 488 nm spectral line of the argon-ion
laser. As shown in Figure 3, separation of the fluorescence emission signal (S1) from
Rayleigh-scattered (diffraction) excitation light (EX) is facilitated by a large fluorescence
Stokes shift (i.e., separation of A1 and E1). Biological samples labeled with fluorescent
probes typically contain more than one fluorescent species, making signal-isolation issues
more complex. Additional optical signals, represented in Figure 3 as S2, may be due to
background fluorescence or to a second fluorescent probe.
Background Fluorescence
Fluorescence detection sensitivity is severely compromised by background signals, which
may originate from endogenous sample constituents (referred to as autofluorescence) or
from unbound or nonspecifically bound probes (referred to as reagent background).
Detection of autofluorescence can be minimized either by selecting filters that reduce the
transmission of E2 relative to E1 or by selecting probes that absorb and emit at longer
wavelengths. Although narrowing the fluorescence detection bandwidth increases the
resolution of E1 and E2, it also compromises the overall fluorescence intensity detected.
Signal distortion caused by autofluorescence of cells, tissues and biological fluids is most
readily minimized by using probes that can be excited at >500 nm. Furthermore, at longer
wavelengths, light scattering by dense media such as tissues is much reduced, resulting in
greater penetration of the excitation light.
Multicolor Labeling Experiments
A multicolor labeling experiment entails the deliberate introduction of two or more
probes to simultaneously monitor different biochemical functions. This technique has
major applications in flow cytometry,
DNA sequencing,
fluorescence in situ
hybridization
and fluorescence microscopy.
Signal isolation and data analysis are
facilitated by maximizing the spectral separation of the multiple emissions (E1 and E2 in
Figure 3). Consequently, fluorophores with narrow spectral bandwidths, such as
Molecular Probes' Alexa Fluor dyes and BODIPY dyes, are particularly useful in
multicolor applications.
An ideal combination of dyes for multicolor labeling would
exhibit strong absorption at a coincident excitation wavelength and well-separated
emission spectra (Figure 3). Unfortunately, it is not easy to find single dyes with the
requisite combination of a large extinction coefficient for absorption and a large Stokes
shift
Ratiometric Measurements
In some cases, for example the Ca2+ indicators fura-2 and indo-1 and the pH indicators
BCECF and SNARF, the free and ion-bound forms of fluorescent ion indicators have
different emission or excitation spectra. With this type of indicator, the ratio of the optical
signals (S1 and S2 in Figure 3) can be used to monitor the association equilibrium and to
calculate ion concentrations. Ratiometric measurements eliminate distortions of data
caused by photobleaching and variations in probe loading and retention, as well as by
instrumental factors such as illumination stability. .
Comparing Different Dyes
Fluorophores currently used as fluorescent probes offer sufficient permutations of
wavelength range, Stokes shift and spectral bandwidth to meet requirements imposed by
instrumentation (e.g., 488 nm excitation), while allowing flexibility in the design of
multicolor labeling experiments (Figure 4). The fluorescence output of a given dye
depends on the efficiency with which it absorbs and emits photons, and its ability to
undergo repeated excitation/emission cycles. Absorption and emission efficiencies are
most usefully quantified in terms of the molar extinction coefficient ( ) for absorption
and the quantum yield (QY) for fluorescence. Both are constants under specific
environmental conditions. The value of is specified at a single wavelength (usually the
absorption maximum), whereas QY is a measure of the total photon emission over the
entire fluorescence spectral profile. Fluorescence intensity per dye molecule is
proportional to the product of and QY. The range of these parameters among
fluorophores of current practical importance is approximately 5000 to 200,000 cm-1M-1
for and 0.05 to 1.0 for QY. Phycobiliproteins such as R-phycoerythrin have multiple
fluorophores on each protein and consequently have much larger extinction coefficients
(on the order of 2 106 cm-1M-1) than low molecular weight fluorophores.
Photobleaching
Under high-intensity illumination conditions, the irreversible destruction or
photobleaching of the excited fluorophore becomes the factor limiting fluorescence
detectability. The multiple photochemical reaction pathways responsible for
photobleaching of fluorescein have been investigated and described in considerable
detail.
Some pathways include reactions between adjacent dye molecules, making the
process considerably more complex in labeled biological specimens than in dilute
solutions of free dye. In all cases, photobleaching originates from the triplet excited state,
which is created from the singlet state (S1, Figure 1) via an excited-state process called
intersystem crossing.
The most effective remedy for photobleaching is to maximize detection sensitivity, which
allows the excitation intensity to be reduced. Detection sensitivity is enhanced by lowlight detection devices such as CCD cameras, as well as by high–numerical aperture
objectives and the widest bandpass emission filters compatible with satisfactory signal
isolation. Alternatively, a less photolabile fluorophore may be substituted in the
experiment. Molecular Probes' Alexa Fluor 488 dye is an important fluorescein substitute
that provides significantly greater photostability than fluorescein , ), yet is
compatible with standard fluorescein optical filters. Antifade reagents can also be applied
to reduce photobleaching; however, they are usually incompatible with live cells. In
general, it is difficult to predict the necessity for and effectiveness of such
countermeasures because photobleaching rates are dependent to some extent on the
INCLUDEPICTURE "http://www.probes.com/images/ref.gif" \*
MERGEFORMATINET
Signal Amplification
The most straightforward way to enhance fluorescence signals is to increase the number
of fluorophores available for detection. Fluorescent signals can be amplified using 1)
avidin–biotin or antibody–hapten secondary detection techniques, 2) enzyme-labeled
secondary detection reagents in conjunction with fluorogenic substrates
or 3) probes
that contain multiple fluorophores such as phycobiliproteins and fluorescent
microspheres.
Simply increasing the probe concentration can be counterproductive and often produces
marked changes in the probe's chemical and optical characteristics. It is important to note
that the effective intracellular concentration of probes loaded by bulk permeabilization
methods is usually much higher (>10-fold) than the extracellular incubation
concentration. Also, increased labeling of proteins or membranes ultimately leads to
precipitation of the protein or gross changes in membrane permeability. Antibodies
labeled with more than four to six fluorophores per protein may exhibit reduced
specificity and reduced binding affinity. Furthermore, at high degrees of substitution, the
extra fluorescence obtained per added fluorophore typically decreases due to selfquenching.
5.3 Video tracking
Myocytes isolated directly from hearts are log shaped, as shown below. Their speed and
magnitude of contraction can be monitored with a video camera and imaging software
that tracks motion of the edges. Records from such an imaging set-up are shown below.
Figure 4.14 Myocyte contraction
Contraction Measurement
contracting
30 m
resting
TTP
90%
R
0.5
sec
Resting
contraction
Max relaxation
velocity
Max shortening
velocity
3. Scanning Microscopy
Laser Confocal Microscopy (Adapted from Kees van der Wulp)
Imaging of thin optical slices, and 3-D reconstructions of cells and components can be
done with scanning microscopy. One type is Laser scanning confocal microscopy
(LSCM). Here the laser light beam scans the specimen that has been decorated with a
fluorescent label. A confocal aperture (pinhole) is placed in front of the photodetector,
such that the fluorescent light (not the reflected light!) from points on the specimen that
are not within the focal plane (the so called out-of-focus light) where the laser beam was
focused will be largely obstructed by the pinhole. In this way, out-of-focus information
(both above and below the focal plane) is greatly reduced. This becomes especially
important when dealing with thick specimens. The spot that is focused on the center of
the pinhole is often referred to as the "confocal spot."
A 2-D image of a small partial volume of the specimen centered around the focal plane
(referred to as an optical section) is generated by performing a raster sweep of the
specimen at that focal plane. As the laser scans across the specimen, the analog light
signal, detected by the photomultiplier, is converted into a digital signal, contributing to a
pixel-based image displayed on a computer monitor attached to the LSCM. The relative
intensity of the fluorescent light, emitted from the laser-hit point, corresponds to the
intensity of the resulting pixel in the image (typically 8-bit greyscale). The plane of focus
(Z-plane) is selected by a computer-controlled fine-stepping motor which moves the
microscope stage up and down. Typical focus motors can adjust the focal plane in as little
as 0.1 micron increments. A 3-D reconstruction of a specimen can be generated by
stacking 2-D optical sections collected in series.
The general setup of an entire LSCM system is shown below. It should be noted that
most laser scanning confocal microscopes consist of a confocal unit attached to a
conventional fluorescence microscope.
Electron Beam Scanning
An older scanning technique uses an electron beam instead of a laser, and hence produces
much higher resolution images, while sacrificing some simplicity. A scanning electron
micrograph of ECM below shows strands of collagen (yellow) and elastin (blue).
Image Enhancement
After an image has been acquired it may be preprocessed to improve image quality. The
preprocessing usually involves application of image filters (mathematical algorithms
implimented in software) to the entire data set to remove noise and artifacts, smooth or
sharpen the images, or to correct for problems with contrast and/or brightness. While
these filters are generally performed as preprocessing steps, they can also be carried out
after a 3-D model has been reconstructed from the image. Median and Gaussian filters
have the general affect of smoothing images. These are used to eliminate noise and
background artifacts and to smooth sharp edges, but also tend to remove some of the
detail in small objects.
Sharpening filters can be used to emphazise details in the image stack, but also have the
effect of highlighting noise and other small artifacts. The application of sharpening filters
is most useful when the image consists of fine structural components of a specimen, or
when edge enhancement is desired.
The contrast and brightness of the image can be adjusted to enhance perception of the
sampled specimen. This is usually done by changing the ramping of the grey scale values
for the dataset. Histogram equilization can be used to improve contrast by a non-linear
mapping of the grey levels in an image. This technique is most commonly used when the
grey levels are concentrated in a small portion of the range of possible values.
It is important to realise that the application of filters to the data set can ultimately affect
quantitative measurements of 3-D reconstructions produced from it. As such, the
application of filters in some instances are only used for display purposes, and
quantitative measurements are made on the unprocessed data.
Segmentation refers to the process of extracting the desired object (or objects) of interest
from the background in an image or data volume. There are a variety of techniques that
are used to do this, ranging from the simple (such as thresholding and masking) to the
complex (such as edge/boundary detection, region growing and clustering algorithms.)
Segmentation can be aided through manual intervention or handled automatically through
software algorithms. Examples of simple forms of segmentation that can be used with
confocal data include thresholding and masking.
Thresholding involves limiting the intensity values within an individual image or the
entire image stack to a certain bounded range (or ranges). For example, since each pixel
in an 8-bit greyscale confocal image (with values 0 [black] to 255 [white]) corresponds to
fluorescence intensity at a point within the specimen, the pixels with lower values
represent areas with lower fluorescence while the pixels with higher values represent
brighter regions. It may be decided that all pixels below a certain value do not contribute
significantly to the object(s) of interest and hence can be eliminated. This can be done by
scanning the image(s) one pixel at a time, and keeping that pixel if it is above the selected
intensity value, or setting it to 0 (black) if it is below that value. In a similar manner,
thresholding can also be used to eliminate non-consecutive ranges of intensities while
preserving the regions containing the intensities of interest.
Masking is a procedure whereby an enclosed region(s) of an image (or of the image
stack) are defined for processing. This can be done either by manually tracing around the
regions of interest (e.g. with a mouse in a graphics application) or by an automated
routine. An easy (and useful) application of this is to use a 2-D stacked projection of an
image to define the image mask. The stacked projection of the image stack is a single
image that represents the sum of all of the images in the image stack (these images can
usually be provided automatically from software supplied with the LSCM.) If the object
of interest has a closed, continuous surface (such as that of a neuron) the stacked
projection defines the absolute boundaries of the object in 2-D. A mask can be formed by
either manually tracing around the boundaries of the object(s) of interest in the stacked
projection or by absolute thresholding (making all intensities above a certain value white
and all below this value black.) The mask can now be applied to the entire image stack,
such that regions falling within the mask selection area are preserved, whereas areas
outside this region are eliminated (e.g. set to 0 [black].) After the mask has been applied,
thresholding and image filtering methods can be used to aid in removing the remaining
undesired regions.
4. Flow Cytometry
Some applications in cytomechanics require high throughput analysis of cells and their
content. FC can analyze aspects of cell structure and mechanical makeup at the rate of
10,000 cells per minute.
Figure 5.11 shows scatter plots for ECs
exposed to isotonic and 50% hypotonic
saline for 1 minute and more than an
hour. The scatter plots show a dot for
each cell event, with coordinates
representing forward and side scatter of
light as labeled. Approximately 10,000
cells are represented in each series. The
side and forward scatter results seen at
one minute for the isotonic control and
50% hypotonic controls were
essentially unchanged when retested
after more than an hour incubation in
either set of control conditions.
Hypotonic saline exposure for one
minute increased side scatter.
Figure 5.11: Testing effects of drugs on
mechanical response of endothelial
cells.
5. Exercises
1. How can the excitation light be separated from fluorescence in a microscope?
2. From what you know of physiology, what mechanical role could a Ca++ wave play in
the cell?
3. Explain how confocal optics can “see” through thick objects.
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Fluorescence Microscopy
Allan, V., Ed., Protein Localization by Fluorescence Microscopy: A Practical Approach,
Oxford University Press (1999).
Andreeff, M. and Pinkel, D., Eds., Introduction to Fluorescence In Situ Hybridization:
Principles and Clinical Applications, John Wiley and Sons (1999).
Conn, P.M., Ed., Confocal Microscopy (Methods in Enzymology, Volume 307), Academic
Press (1999).
Denk, W. and Svoboda, K., "Photon upmanship: why multiphoton imaging is more than a
gimmick," Neuron 18, 351–357 (1997).
Diaspro, A., Ed., Confocal and Two-Photon Microscopy: Foundations, Applications and
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Goldman, R.D. and Spector, D.L., Eds., Live Cell Imaging: A Laboratory Manual, Cold
Spring Harbor Laboratory Press (2004).
Herman, B., Fluorescence Microscopy, Second Edition, BIOS Scientific Publishers
(1998).
Inoué, S. and Spring, K.R., Video Microscopy, Second Edition, Plenum Publishing
(1997).
Matsumoto, B., Ed., Cell Biological Applications of Confocal Microscopy, Second
Edition (Methods in Cell Biology, Volume 70), Academic Press (2003).
Michalet, X., Kapanidis, A.N., Laurence, T., Pinaud, F., Doose, S., Pflughoefft, M. and
Weiss S., "The power and prospects of fluorescence microscopies and spectroscopies,"
Annu Rev Biophys Biomolec Struct 32, 161–182 (2003).
Murphy, D.B., Fundamentals of Light Microscopy and Electronic Imaging, John Wiley
and Sons (2001). Available from Molecular Probes (F24840, Section 23.6).
Pawley, J.B., Ed., Handbook of Biological Confocal Microscopy, Second Edition, Plenum
Publishing (1995).
Paddock, S., Ed., Confocal Microscopy (Methods in Molecular Biology, Volume 122),
Humana Press (1998). Available from Molecular Probes (C14946, Section 23.6).
Periasamy, A., Ed., Methods in Cellular Imaging, Oxford University Press (2001).
Rizzuto, R., and Fasolato, C., Eds., Imaging Living Cells, Springer-Verlag (1999).
Sheppard, C.J.R. and Shotton, D.M., Confocal Laser Scanning Microscopy, BIOS
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Stevens, J.K., Mills, L.R. and Trogadis, J.E., Eds., Three-Dimensional Confocal
Microscopy: Volume Investigation of Biological Systems, Academic Press (1994).
Taylor, D.L. and Wang, Y.L., Eds., Fluorescence Microscopy of Living Cells in Culture,
Parts A and B (Methods in Cell Biology, Volumes 29 and 30), Academic Press (1989).
Toomre, D. and Manstein, D.J., "Lighting up the cell surface with evanescent wave
microscopy," Trends Cell Biol 11, 298–303 (2001).
Tsien, R.Y., " Imagining imaging's future," Nat Rev Mol Cell Biol 4, SS16–SS21 (2003).
Wang, X.F. and Herman, B., Eds., Fluorescence Imaging Spectroscopy and Microscopy,
John Wiley and Sons (1996).
Yuste, R., Lanni, F. and Konnerth, A., Imaging Neurons: A Laboratory Manual, Cold
Spring Harbor Laboratory Press (2000).
Flow Cytometry
Darzynkiewicz, Z., Crissman, H.A. and Robinson, J.P., Eds., Cytometry, Third Edition
Parts A and B (Methods in Cell Biology, Volumes 63 and 64), Academic Press (2001).
Davey, H.M. and Kell, D.B., "Flow cytometry and cell sorting of heterogeneous
microbial populations: the importance of single-cell analyses," Microbiological Rev 60,
641–696 (1996).
Gilman-Sachs, A., "Flow cytometry," Anal Chem 66, 700A–707A (1994).
Givan, A.L., Flow Cytometry: First Principles, Second Edition, John Wiley and Sons
(2001).
Herzenberg, L.A., Parks, D., Sahaf, B., Perez, O., Roederer, M. and Herzenberg, L.A.,
"The history and future of the fluorescence activated cell sorter and flow cytometry: a
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Jaroszeski, M.J. and Heller, R., Eds., Flow Cytometry Protocols (Methods in Molecular
Biology, Volume 91), Humana Press (1997).
Lloyd, D., Ed., Flow Cytometry in Microbiology, Springer-Verlag (1993).
Melamed, M.R., Lindmo, T. and Mendelsohn, M.L., Eds., Flow Cytometry and Sorting,
Second Edition, Wiley-Liss (1990).
Ormerod, M.G., Ed., Flow Cytometry: A Practical Approach, Third Edition, Oxford
University Press (2000).
Robinson, J.P., Ed., Current Protocols in Cytometry, John Wiley and Sons (1997).
Shapiro, H.M., "Optical measurement in cytometry: light scattering, extinction,
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Shapiro, H.M., Practical Flow Cytometry, Fourth Edition, Wiley-Liss (2003).
Weaver, J.L., "Introduction to flow cytometry," Methods 21, 199–201 (2000). This
journal issue also contains 10 review articles on various flow cytometry applications.
Other Fluorescence Measurement Techniques
Goldberg, M.C., Ed., Luminescence Applications in Biological, Chemical, Environmental
and Hydrological Sciences (ACS Symposium Series 383), American Chemical Society
(1989).
Gore, M., Ed., Spectrophotometry and Spectrofluorimetry: A Practical Approach, Second
Edition, Oxford University Press (2000).
Hemmilä, I.A., Applications of Fluorescence in Immunoassays, John Wiley and Sons
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Patton, W.F., "A thousand points of light: the application of fluorescence detection
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1123–1144 (2000).
Rampal, J.B., Ed., DNA Arrays: Methods and Protocols (Methods in Molecular Biology,
Volume 170), Humana Press (2001). Available from Molecular Probes (D24835,
Section 23.6).
Schena, M., Ed., DNA Microarrays: A Practical Approach, Oxford University Press
(1999).
Schena, M., Ed., Microarray Biochip Technology, BioTechniques Press (2000).
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