Review Article
Advances in Microscopy Techniques
Daniel B. Schmolze, BS; Clive Standley, PhD; Kevin E. Fogarty, MS; Andrew H. Fischer, MD
NofContext.—Advances in microscopy enable visualization
a broad range of new morphologic features.
Objective.—To review and illustrate advances in mi-
croscopy with relevance to pathologists.
Data Sources.—Literature review and new observations.
Results.—Fluorescence microscopy enables multiantigen
detection; allows novel optical-sectioning techniques, with
some advantages compared to paraffin sectioning; and
permits live-cell imaging. Live-cell imaging allows pathol-
ogists to move from a period when all diagnostic expertise
was reliant on interpreting static images to a period when
cellular dynamics can play a role in diagnosis. New
techniques have bypassed by about 100-fold what had
FLUORESCENCE MICROSCOPY
In traditional transmitted light microscopy, it is the
removal of light from the image that provides information.
There is only so much light that can be removed to provide
more information. In fluorescence microscopy, a virtually
limitless addition of light to the image provides informa-
tion. The spectra of the light from fluorescent microscopy
can be separated (reviewed in Levenson et al1), giving
fluorescence microscopy many ‘‘dimensions’’ for convey-
ing information. It is a straightforward process to digitally
invert a fluorescent image (to have a white background)
and to ‘‘pseudocolor’’ fluorescent nucleic acid and protein
stains (Figure 1, A and B) to closely resemble a standard
hematoxylin-eosin (H&E) section (Figure 1, C).
A possible application for this approach would be to
perform multiple simultaneous immunofluorescent label-
ings on 1 section, while displaying a digital H&E image.
With conventional light microscopy, a practical limit of
about 3 to 4 colors can be usefully resolved, including any
needed counterstain (eg, hematoxylin). Thus, with con-
ventional transmitted microscopy, 2 or possibly 3 color
immunohistochemical labels could be used, with only
hematoxylin as a counterstain. However, colocalized
antigens are difficult to separate in transmitted light
microscopy. On the other hand, if immunofluorescence is
used, at least 3 colocalized antigens can be separately
Accepted for publication April 13, 2010.
From the University of Massachusetts Medical School, Worcester (Mr
Schmolze); and the Departments of Physiology (Dr Standley and Mr
Fogarty) and Pathology (Dr Fischer), University of Massachusetts
Medical School, Worcester.
The authors have no relevant financial interest in the products or
companies described in this article.
Reprints: Andrew H. Fischer, MD, Department of Pathology, University
of Massachusetts Medical School, Biotech Three, 1 Innovation Dr,
Worcester, MA 01605 (e-mail: andrew.fischer@umassmemorial.org).
Arch Pathol Lab Med—Vol 135, February 2011
long been believed to be a limit to the resolution of light
microscopy. Fluorescence resonance energy transfer
(FRET) appears capable of visualizing diagnostically rele-
vant molecular events in living or fixed cells that are
immeasurable by other molecular techniques. We describe
applications of 2-photon microscopy, FRET, structured
illumination, and the subdiffraction techniques of near-
field microscopy, photoactivated localization microscopy,
stochastic optical reconstruction microscopy, and stimu-
lated emission depletion microscopy.
Conclusion.—New microscopy techniques present oppor-
tunities for pathologists to develop improved diagnostic tests.
(Arch Pathol Lab Med. 2011;135:255–263)
detected; at the same time, a pseudo-H&E image of the
same sample can be displayed by using 2 additional colors
in the spectrum. In a possible application, fine-needle
aspirations of lymph nodes could be immunofluores-
cently stained with multiple antibodies, to provide the
information afforded by flow cytometry, but could still be
viewed digitally with standard, light microscope–like
morphology. Several commercial platforms exist for these
manipulations, as well as for extracting spectral informa-
tion and classifying objects (reviewed in Levenson et al1).
LIVE-CELL IMAGING
Transmitted light microscopy requires use of dyes to
visualize nuclear detail; however, no dyes are known to be
suitable for live-cell imaging with transmitted light
microscopy. Phase contrast and differential interference
contrast microscopy techniques cannot display subcellu-
lar morphology, particularly within thick tissue samples
(Figure 2, B). Fluorescent dyes capable of penetrating
viable cells are available for staining nuclei or particular
organelles, as are a wide variety of fluorescently labeled
proteins that can be expressed in living cells through use
of viral vectors (Figure 2, A and C). The fluorescently
stained tissue can subsequently be fixed and convention-
ally stained (Figure 2, D and E).
All of a pathologist’s criteria for cancer diagnosis are
still based on static images. If experience at other levels of
biology is a guide, it will be much easier to learn about the
abnormal physiology of pathologic tissue if dynamic
images are studied. The dynamics of cancer cells will most
likely provide a fundamentally new set of diagnostic
features. The nuclear lamina irregularity, diagnostic of
papillary thyroid carcinoma, appears to be due to a
dynamic deformation of the nuclear lamina during
interphase,2 and this dynamic property appears funda-
mentally different from the irregularity that develops
Advances in Microscopy Techniques—Schmolze et al 255
Figure 1. Fluorescence microscopy can provide the same information as conventional hematoxylin-eosin (H&E) microscopy. A conventional
paraffin section of an ampullary villous adenoma was deparaffinized and rehydrated to 50% alcohol, then simultaneously stained with 49,6-
diamidino-2-phenylindole (DAPI) (A) and eosin (B) without washing out any excess dye. The image was inverted and ‘‘pseudocolored’’ to match the
colors of an H&E section by using Photoshop (C). We have found that [Ru(bpy)3]Cl2 can be used like eosin and has a useful narrower emission
spectrum (original magnification 3100).

during a period of days in neutrophils, or possibly other
benign cells that show nuclear irregularity in static
images. Live-cell imaging in animal models has demon-
strated a direct correlation between tumor cell migration
and seeding of the bloodstream.3 Further, dynamic studies
have shown an interaction between tumor cells and
macrophages in the process of intravasation of tumor
cells into vessels.3 These interactions could not be directly
predicted based on static images, and they point toward
limitless future diagnostic applications of the dynamic
properties of tumor cells.
THE DIFFRACTION LIMIT TO RESOLUTION IN
CONVENTIONAL MICROSCOPY
It has been known since the 19th century that the wave
nature of light imposes limits on optical resolution. The

Figure 2. Transmitted light microscopy of thick tissues does not permit visualization of cell structure, whereas fluorescent proteins expressed in
living cells are easy to resolve. Living normal human thyroid tissue was visualized after being in organ culture for 18 hours (A–C) or after being fixed
at 36 hours (D and E). Green fluorescent protein (GFP) bearing replication-incompetent adenovirus was added at the initiation of the organ culture.
The GFP signal is shown in (A). The GFP in this example is unconjugated and therefore diffuses throughout infected cells. Green fluorescent protein
fused to particular proteins, for example, lamins, permits visualization of subcellular detail (not shown). Barely discernable thyroid follicles are
evident by differential interference contrast microscopy in B. C shows the overlay of A and B. Histologically and cytologically, the tissue appears
normal after 36 hours (D) and GFP can be detected by immunohistochemistry in the paraffin-embedded tissue section (E) (original
magnifications 3200).

256 Arch Pathol Lab Med—Vol 135, February 2011 Advances in Microscopy Techniques—Schmolze et al

effect occurs as light waves cause interference on their
way through the optical system and is known as the
diffraction limit. The practical consequence of this effect is
that a point source of light will produce a fuzzy circle
when viewed through a microscope. This characteristic
pattern is known as an Airy disk (Figure 3, A) and consists
of a central bright dot surrounded by increasingly dimmer
circles of light. The corresponding intensity plot is called
the point spread function, or PSF (Figure 3, B).
The shape of the PSF determines resolution. If the PSF is
very narrow or the separation between the points is wide,
then 2 PSFs will add up in such a way that the peaks can
still have a trough and the 2 images can be resolved. Ernst
Abbe addressed the physics and mathematics of the PSF
in the 19th century4 and defined an equation that yields
the smallest distance x over which 2 objects can be
resolved:
x~l=2n sin a, ð1Þ
where l is the wavelength of light, n is the refractive index
of the medium and a is half the angular aperture of the
lens. As shown in Figure 4, the angular aperture deter-
mines how much of the wave front can be captured by the
lens. A material of high refractive index (for example
immersion oil, see Figure 4) will bend more light into the
field of view of the objective lens, acting like an increase in
a to improve resolution. The Abbe equation shows that
resolution increases as the wavelength decreases. The
higher resolution of an electron microscope (less than
1 nm) relates to the very short wavelength of electrons.
Compared to new microscopy techniques (described
below), whose resolution is starting to approach that of
electron microscopy, the technique of electron microscopy
does not easily yield 3-dimensional information; more-
over, immunolocalization by electron microscopy is
difficult and live-cell imaging for structural dynamics is
impossible.
The denominator of equation (1) is known as the
numerical aperture (NA) of the lens, so the equation can
be rewritten as:
x~l=2NA ð2Þ
With high refractive index media and high NA lenses,
the Abbe limit is roughly 200 nm (0.2 mm) in the X-Y plane.
For objects above and below the plane, the resolution is
Arch Pathol Lab Med—Vol 135, February 2011
Figure 3. Airy disk and point spread func-
tion. Because of diffraction, a microscope
produces a blurry Airy disk (A) as the image of
a point source of light. The surface plot of this
disk defines the point spread function (B),
whose shape in turn determines resolution.
defined by a different PSF, x 5 l/(n sin2a), and the
practical Abbe limit is about 500 nm. Until the 1990s, the
Abbe limit was widely thought of as an absolute barrier to
resolution by light microscopy.
DIFFRACTION-LIMITED MICROSCOPY TECHNIQUES
Before discussing newer optical methods that achieve
molecular-level resolution, it is worth looking at some
useful techniques that remain bound by the Abbe limit.
Confocal Microscopy
In standard epifluorescence microscopy, used in most
laboratories for immunofluorescence of renal and skin
biopsies, or fluorescence in situ hybridization of individ-
ual chromosome probes, no mechanism exists to restrict
out-of-focus light from being imaged. At high magnifica-
tions or with thick sections, blurry light from outside the
focal plane contributes to the image, significantly reducing
contrast and perceived resolution. This effect occurs in a
different, less obtrusive manner in transmitted light
microscopy. A laser scanning confocal microscope focuses
exciting light onto a small area of the specimen, and it
images the resulting emitted light through a pinhole that
only allows light from the focal plane to pass through
(Figure 5). As a result, an ‘‘optical section’’ of the
specimen at the focal plane is obtained by scanning the
focused spot across the specimen. By changing the focal
plane, such sections can be imaged at different depths in
the specimen, and a 3-D representation can be recon-
structed.
Confocal microscopy was first described in the 1950s by
Minsky5 and is now a relatively mature technology with
many commercial implementations. The price and com-
plexity have steadily decreased, and recently a ‘‘bench-
top’’ model has been released.6 Most designs use a set of
mirrors to scan a laser across the specimen. Although
suitable for some live-cell imaging applications, an
alternative ‘‘spinning disk’’ design collects the image
faster, allowing for multiple images per second. The
spinning disk design uses a set of concentrically arranged
pinholes in a spinning disk, effectively allowing multiple
confocal scans to take place simultaneously. Unfortunate-
ly, a relatively powerful illumination source is usually
needed in all confocal designs, since the pinhole passes
only a small amount of light, and this can be damaging to
live cells.
Advances in Microscopy Techniques—Schmolze et al 257
Figure 4. Concept of numerical aperture.
Both a large value of a (half the angular
aperture of the lens) and a high refractive
index (n) will serve to increase the amount of
light from the specimen that is captured by
the lens.
Figure 5. How a confocal microscope captures emitted light from
only 1 plane. The dichroic mirror serves to reflect the exciting light
while allowing the emitted fluorescence to pass through. The pinhole
blocks light from outside the focal plane.
258 Arch Pathol Lab Med—Vol 135, February 2011
Two-Photon Microscopy
In the usual case of fluorescence emission, a fluorophore
that absorbs a single photon of light at a certain
wavelength moves to a higher energy state. When the
fluorophore returns to its ground state, it emits a photon
with a longer wavelength than that of the photon
absorbed. Instead of exciting the fluorophore with 1
photon, 2 photons with half the energy (twice the
wavelength) can achieve the same excitation of the
fluorophore.
A 2-photon microscope operates by using this princi-
ple.7 It uses a pulsed infrared laser that is focused to a spot
in the specimen. This laser requires a high instantaneous
intensity at each pulse, since the absorption of 2 photons
simultaneously is a rare event, but the average intensity is
low. The illumination intensity falls off rapidly above and
below the focal point, and since the chance for absorption
of 2 photons increases with the square of the intensity, the
probability of absorption falls steeply (roughly to the sixth
power) above and below the focal plane. Thus, only
fluorophores in a thin volume are excited. When this spot
is scanned across the specimen, only light from this thin
excitation plane is collected and an optical section is
produced (Figure 6).
Whereas, in a confocal microscope, fluorophores out-
side the focal plane are excited but prevented from being
imaged by a pinhole, in a 2-photon microscope, absorp-
tion is confined to a small volume in the focal plane. The
absorption plane can be as thin as about half a micron.
Outside this plane, the sample is exposed only to a
relatively mild infrared beam with a low average
intensity. Thus, photobleaching and phototoxicity is
restricted to the plane of focus, and the sample as a whole
is exposed to much less potentially damaging radiation,
making the technique well suited to live-cell imaging.
A second advantage of 2-photon imaging is that the
infrared excitation source penetrates more deeply into
Advances in Microscopy Techniques—Schmolze et al
Figure 6. Two-photon microscopy. A pinhole is not needed, since the pulsed laser achieves the necessary high intensity to simultaneously excite
fluorophores with 2 photons at 1 plane.
tissue than the visible or ultraviolet light needed for
confocal or conventional epifluorescence microscopy.
Although the emitted light is still scattered by tissue, it
appears feasible to image to a depth of about 200 mm into
the tissue and still display diagnostic cellular-level
morphology with the 2-photon technique. Confocal
optical sectioning is reasonably crisp only to a depth of
about 100 mm in whole tissue.
The concept of 2-photon microscopy was first described
in 1931.8 Costs and complexity have significantly declined
in recent years, but microscopes remain expensive (largely
owing to the cost of the pulsed laser). Nevertheless, 2-
photon imaging is being explored for various clinical
applications, for example in phototherapy for cancer and
macular degeneration.9,10
Two-photon imaging can allow optical sections to be
cut, analogously to a paraffin section, on unfixed (living)
or fixed unembedded tissues (Figure 7). Fluorescent dyes
are available that can be taken up by living cells to display
nuclear morphology (eg, 49,6-diamidino-2-phenylindole
[DAPI]). The thickness of the optical section can be as thin
as about 1 mm, comparing favorably to a paraffin section.
Compared to confocal microscopy, it should be possible to
cut such optical sections more deeply into the tissue
because of the higher penetration of the exciting infrared
light. The advantage of optical sectioning compared to
paraffin sectioning is the decreased technical steps
required for imaging (there is no paraffinization or
physical sectioning, and the staining itself can take place
in a liquid fixative or tissue culture media without the
need for washing out the fluorescent dyes). Another major
Arch Pathol Lab Med—Vol 135, February 2011
advantage is that the plane of imaging can be precisely
chosen without wasting tissue. Applications would
appear to be particularly useful for small biopsies in
which a tangential optical section on the various sides of
the biopsy specimen could allow diagnosis. Diagnostic
features for many common problems (eg, determining the
presence of invasion in breast biopsy specimens) typically
span a diameter of only 200 mm.11
4Pi Microscopy
The Abbe limit refers to resolution in the X-Y plane.
Resolution in the Z plane is much lower for all microscope
designs previously described. Confocal and 2-photon
microscopy are limited to about 500 nm of resolution in
the Z axis. The Abbe equation shows that an objective lens
with a large angular aperture will produce a higher-
resolution image than one with a smaller angular
aperture. 4Pi microscopy places 2 identical objective
lenses on either side of the specimen, thereby effectively
doubling the angular aperture and thus the resolution in
the Z plane of the 2 lenses, to about 200 nm (Figure 8).12 4Pi
microscopy remains diffraction limited but can be paired
with subdiffraction techniques such as stimulated emis-
sion depletion, discussed below.
Spatially Modulated Illumination Microscopy
When 2 densely patterned images are combined, an
interference effect called a Moiré pattern can result. This
effect is often undesirable (for example, showing up in
scanned magazine images). The Moiré effect is exploited
in spatially modulated illumination (SMI) by using a
Advances in Microscopy Techniques—Schmolze et al 259
Figure 7. Demonstration of 2-photon microscopy optical sectioning
of ovarian cancer in a liquid fixative. Two-photon image, about 5 mm in
thickness, of a cluster of ovarian cancer cells from ascites, stained with
49,6-diamidino-2-phenylindole (DAPI) and eosin in CytoRich Red
fixative (Thermo Scientific, Hudson, New Hampshire), without washing
out any excess dye. Pseudo hematoxylin-eosin staining was accom-
plished as in Figure 1 (original magnification, approximately 31000).
pattern of fine lines as the illumination source.13,14 When
this illumination pattern (Figure 9, B) interferes with the
detail emitted from or transmitted by the fine structure of
the sample (Figure 9, A), a new pattern results (Figure 9,
C).
The Moiré pattern is always more coarsely patterned
than the sample, and so it may be resolvable even if the
fine structure of the sample is not. The Moiré pattern does
not necessarily actually resemble the sample, but by
collecting a series of images in which the illumination
pattern has been slightly altered (eg, by simply shifting its
position), the underlying structure can be computed. The
interference effect only occurs at the focal plane, and as the
illumination pattern is changed, the out-of-focus light
remains invariant and can thus be subtracted from the
final image. In this way, efficient optical sectioning is
possible. Although the technique requires the collection of
multiple images, the computation can be performed
quickly, enabling live-cell imaging in conjunction with
fast cameras. Spatially modulated illumination is com-
mercially available as an add-on to most epifluorescence
microscopes at a cost that is considerably cheaper than
that of confocal microscopy.15
Spatially modulated illumination is limited by diffrac-
tion in that there is a limit to the detail of the illumination
pattern. Like 4Pi microscopy, SMI can be combined with
other methods to yield further resolution enhancement.
One technique merges SMI and stimulated emission
depletion to yield subdiffraction imaging with a method
termed nonlinear structured illumination.16
SUBDIFFRACTION TECHNIQUES
With lenses of the largest possible angular aperture, in
media with the highest possible refractive index, the Abbe
260 Arch Pathol Lab Med—Vol 135, February 2011
Figure 8. 4Pi microscopy principle. Two lenses are placed on
opposing sides of the specimen, thereby effectively doubling the
angular aperture and the resolution.
equation limits optical resolution to about 200 nm. For a
long time, this limit was thought to be unsurpassable, but
recently several microscopy methods have been devel-
oped that achieve far higher resolution. In fact, the
diffraction limit was never a ‘‘hard’’ limit, and several
caveats exist that point the way toward subdiffraction
imaging. First, equation (2) only applies in the so-called far
field, where the light rays are allowed to travel a certain
distance before being imaged (roughly, longer than the
wavelength of light). This is because very close to a source
(in the ‘‘near field’’) the action of so-called evanescent
nonpropagating waves predominates, and these waves
are not accounted for by the usual rules of interference.17
Secondly, molecular interactions between different fluo-
rophores can be visualized. Finally, the Abbe equation
only applies if 2 objects with an identical interaction with
light are simultaneously visualized. If there can be a
distinction between the 2 objects in the spectra of their
emitting photons, or their temporal release of photons, the
Abbe limit does not apply, and in fact there is no theoretic
limit to light microscopy resolution, even in living cells!
Near-Field Scanning Optical Microscope
Microscopy techniques have been developed that
exploit all of these caveats to break the diffraction limit.
The near-field scanning optical microscope scans a tiny
probe across the surface of a specimen at distances over
which near-field effects come into play. The resolution is
limited only by the size of the probe, which can be as small
as 10 nm.18 A similar diffraction-limited technique called
total internal reflection fluorescence microscopy generates
a near-field wave at the surface of a specimen.19 This wave
then excites fluorophores over a very narrow distance
(Figure 10). Both techniques have been successfully used
to visualize dynamic events, in the range of 10 nm,
occurring at the plasma membrane of living cells.
Advances in Microscopy Techniques—Schmolze et al
Figure 9. The Moiré effect in structured illumination microscopy. A sample containing fine detail (A) when illuminated with a regularly patterned
image (B) gives rise to a coarsely patterned Moiré image (C). The original fine detail can be computed through collection of several Moiré images. An
important effect is that the out-of-plane component of the image is able to be computationally subtracted. Reprinted with permission from
Gustafsson.15 Copyright 2005, National Academy of Sciences, United States.
Fluorescence/Förster Resonance Energy Transfer
Fluorescence/Förster resonance energy transfer (FRET)
exploits a different physical principle to obtain high
spatial precision.20 Two fluorophores interact in propor-
tion to the sixth power of their intermolecular separation.
Within a few diameters of fluorescent molecules, one
excited molecule (the donor) can transfer its excited state
to a different nonexcited fluorescent molecule (the
acceptor). No emission of light is involved in this transfer,
and the acceptor will proceed to fluoresce at its charac-
teristic emission wavelength. The degree of separation of
the 2 fluorochromes (in the range of 1 nm) can be
estimated from the decrease in donor fluorescence and a
Figure 10. Near-field microscopy with the total internal reflection
fluorescence microscope. The exciting light is angled such that it is
completely reflected when it reaches the sample. A near-field wave is
generated, which excites fluorophores in a very narrow range. The
resulting fluorescence is imaged as usual.
Arch Pathol Lab Med—Vol 135, February 2011
corresponding increase in acceptor fluorescence. Donor
and acceptor probes can be attached to 2 different
molecules of interest, or to 2 different domains of a single
protein, making FRET a useful tool for measuring protein-
protein interactions or conformational changes at the
molecular level (Figure 11). Fluorescence/Förster reso-
nance energy transfer is used in a nonmorphologic context
as a read-out for human papilloma virus (HPV) detection
in the Third Wave Technologies of Hologic Inc (Bedford,
Massachusetts). In this case, 2 adjacent FRET fluorophores
on a nucleic acid probe become separated through the
action of a specific nuclease that cleaves a site between
them.
Fluorescence/Förster resonance energy transfer also
has morphologic applications in anatomic pathology, with
the potential to characterize diagnostic molecular events
that are invisible to existing light microscopy or existing
nonmorphologic molecular assays. An important example
is provided by the signaling pathway from receptor
tyrosine kinases to RAS, RAF, and, ultimately, MAPK.
There is strong evidence for activation of this pathway
(particularly at points upstream and downstream of RAS)
in papillary thyroid carcinomas.21–23 Paradoxically, activa-
tion of RAS is common in follicular-type thyroid tumors, a
tumor with distinctly different morphologic and clinical
features.23 Immunohistochemistry and Western blotting
have not been able to distinguish papillary thyroid
carcinomas with tyrosine kinase or BRAF activations from
follicular-type tumors or normal thyroid.23,24 The solution
to the paradox of how the same signaling pathway to
MAPK can cause 2 different responses (apparently in the
same cell of origin) could be the existence of differences in
the precise temporal and/or spatial aspects of signaling
(reviewed in Murphy and Blenis26). The power of FRET is
its ability to combine cellular-level localization with
dynamic information about molecular interactions, and
FRET applications are beginning to dissect out the precise
spatial and temporal involvement of events in the MAPK
pathway that accounts for paradoxes such as this.27,28 By
analogy to the findings in PC12 neuroblastoma cells, one
may expect papillary thyroid carcinomas to have a
sustained low-level activation of MAPK, whereas follicu-
Advances in Microscopy Techniques—Schmolze et al 261

Figure 11. Fluorescence resonance energy transfer. When 2 fluoro-
chromes remain separated by more than a few molecular distances, the
exciting light causes only the first fluorochrome to fluoresce. As the
distance is decreased, the excited state is transferred to the ‘‘acceptor’’
molecule, which proceeds to fluoresce at a different wavelength.
lar-type tumors with RAS mutations would be expected to
have a transient high level activation of MAPK.26 It is
conceivable that FRET-based assays could find use some
day in ‘‘diagnosis’’ of specific oncogenic signals or
molecular interactions that are otherwise ‘‘invisible’’ by
mutational analysis or immunohistochemistry. Fluores-
cence/Förster resonance energy transfer applications on
viable cells provide dynamic, temporal information and
would typically require some in vitro manipulations (eg,
adenoviral-induced expression of green fluorescent pro-
tein fusions, see Figure 2), but static FRET can also be
performed on fixed material after labeling with informa-
tive FRET pairs.29
Photoactivated Localization Microscopy and Stochastic
Optical Reconstruction Microscopy
Even if a point source of light is converted by an optical
system into a fuzzy Airy disk, repeated sampling of an
individual source of light can statistically show the
location of the center of its Airy disk. The statistical
sampling defines localization precision, a concept distinct
from optical resolution. Localization precision is not
limited by the Abbe limit, and
statistical
pffiffiffiffi sampling
improves resolution by a factor of 1 N, where N is the
number of sampled photons. Photoactivated localization
microscopy30 and stochastic optical reconstruction micros-
copy31 are related applications of this concept. Within a
large population of fluorophores (Figure 12, A), a low-
intensity laser excitation first ‘‘activates’’ rare fluoro-
phores to become susceptible to a second excitation laser
pulse (Figure 12, B and C). The first pulse does not
actually induce fluorescence; it merely brings the fluo-
rophore to a state where it can become repeatedly excited
262 Arch Pathol Lab Med—Vol 135, February 2011
Figure 12. Photoactivated localization microscopy and stochastic
optical reconstruction microscopy. Within a large population of
fluorophores (A), an initial pulse of light (B) causes a sparse subset of
the fluorophores to become ‘‘activated’’ (C). A second pulse of light
then causes the activated fluorophores to actually fluoresce (D). The
process is repeated until the location of every fluorophore in the sample
is individually recorded.
to fluoresce. The first pulse is weak enough to activate
only about 1 fluorophore molecule in the area of a laser
excitation (an area about 300 nm in diameter). By
following the first low-intensity ‘‘activation’’ pulse with
a second excitation pulse (Figure 12, D), individual
molecules within the 300-nm area fluoresce a population
of photons. The population of photons scatters according
to a point spread function, and the center of the function
can be estimated. The process is repeated through
successive 300-nm areas until the location of every
fluorescent molecule in the sample is multiply sampled.
A high-resolution image is then computed. The process
can take many hours to complete, and is thus not yet
suitable for studying cellular dynamics, but resolution of
about 15 nm has been achieved.31
Stimulated Emission Depletion
Stimulated emission depletion32 is notable in that a
commercial product has recently been announced.33
Stimulated emission depletion makes use of 2 separate
geometries and wavelengths of pulsed light. The first
pulse of light is focused to the smallest possible spot
(about 300 nm in diameter) and excites all of the
fluorochromes within this spot, just like a conventional
confocal microscope. A second pulse of light is focused
into a donut-shaped image, with the hole of the donut
centered on the first pulse (Figure 13). The second pulse
has a wavelength that quenches fluorescence induced by
the first pulse. The subdiffraction resolution of stimulated
emission depletion is due to nonlinear optical effects34 that
come into play as the intensity of the quenching beam
increases. The end result is a point-by-point scan through
the sample at a much higher resolution than conventional
Advances in Microscopy Techniques—Schmolze et al

techniques—about 20 nm. Stimulated emission depletion
can be applied to green fluorescent proteins and their
derivatives, enabling live-cell imaging, but it is currently
too slow for visualizing live-cell dynamics.
IMPLICATIONS FOR PATHOLOGY
At the finest levels of biology it is easy to appreciate
how a change in protein structure affects the function of a
protein, and it is easy at the tissue level or organ level to
appreciate how changes in structure lead to changes in
physiology. At the cellular level, the relation between
structure and function is less obvious. For example, it is
not obvious why nuclear structure should differ between
papillary thyroid carcinoma and follicular-type thyroid
tumors, or why nucleolar enlargement would be charac-
teristic of prostatic intraepithelial neoplasia. The cellular
level is the bottleneck for understanding mechanisms of
disease. We can know the gene that is responsible for
certain diseases (eg, Huntington disease) and we can
know the tissue-level structural changes that result from
the gene, but precise understanding of disease mecha-
nisms is lost at the cellular level. Advances in imaging at
the cellular to molecular level, especially techniques that
allow dynamic imaging of living cells, should enable
pathologists to play a central role in uncovering the
elusive structure-function relationships that underlie and
define diseases.
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