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C
Denis Semwogerere
Eric R. Weeks
Emory University, Atlanta, Georgia, U.S.A.
excited states
absorb
high
energy Fig. 1 Mechanism of fluorescence. The horizon-
emit lower
photon tal lines indicate quantum energy levels of the
energy photon
molecule. A fluorescent dye molecule is raised to
an excited energy state by a high-energy photon.
It loses a little energy to other molecules and
drops to a lower excited state. It loses the rest
of the energy by emitting light of a lower energy.
ground state (View this art in color at www.dekker.com.)
and it is also possible to use more than one type of light from the fluorescing specimen passes through both
fluorophore.[6] Thus, by switching the excitation light the objective and the dichroic mirror. This particular
different parts of the specimen can be distinguished. type of fluorescence microscopy, in which the objective
used by the illuminating light is also used by the fluor-
escing light in conjunction with a dichroic mirror, is
called epifluorescence. In the case of reflected light
FLUORESCENCE MICROSCOPY microscopy, a beamsplitter is used in place of the
dichroic mirror.
In conventional fluorescence microscopy a dyed speci-
men is illuminated with light of an appropriate wave-
length and an image is formed from the resulting
fluorescent light. In Fig. 2 the excitation light is blue CONFOCAL MICROSCOPY
and the emitted light is green. The microscope uses a
dichroic mirror (also called a ‘‘dichromatic mirror’’) To understand confocal microscopy it is instructive to
that reflects light shorter than a certain wavelength imagine a pair of lenses that focuses light from the
but transmits light of longer wavelength. Thus, the light focal point of one lens to the focal point of the other.
from the main source is reflected and passes through the This is illustrated by the dark blue rays in Fig. 3. The
objective to the sample, while the longer-wavelength light blue rays represent light from another point in
filters
dichroic mirror
the specimen, which is not at the focal point of the Because the focal point of the objective lens forms an
left-hand-side lens. (Note that the colors of the rays image where the pinhole=screen is, those two points
are purely for purposes of distinguishing the two are known as ‘‘conjugate points’’ (or alternatively, the
sets—they do not represent different wavelengths of specimen plane and the pinhole=screen are conjugate
light.) Clearly, the image of the light blue point is not planes). The pinhole is conjugate to the focal point of
at the same location as the image of the dark blue the lens, hence the name ‘‘confocal’’ pinhole.
point. (Recall from introductory optics that points
do not need to be at the focal point of the lens for
the system of lenses to form an image.) HOW DOES A CONFOCAL MICROSCOPE WORK?
In confocal microscopy, the aim is to see only the
image of the dark blue point.[1] Accordingly, if a screen The confocal microscope incorporates the ideas of
with a pinhole is placed at the other side of the lens sys- point-by-point illumination of the specimen and rejec-
tem, then all of the light from the dark point will pass tion of out-of-focus light.
through the pinhole.a Note that at the location of the One drawback with imaging a point onto the speci-
screen the light blue point is out of focus. Moreover, men is that there are fewer emitted photons to collect
most of the light will get blocked by the screen, resulting at any given instant. Thus, to avoid building a noisy
in an image of the light blue point that is significantly image each point must be illuminated for a long time
attenuated compared to the image of the dark blue point. to collect enough light to make an accurate measure-
To further reduce the amount of light emanating ment.[1] In turn, this increases the length of time needed
from ‘‘light blue’’ points, the confocal microscope setup to create a point-by-point image. The solution is to use
minimizes how much of the specimen is illuminated. a light source of very high intensity, which Minsky did
Normally, in fluorescence microscopy the entire field of with a zirconium arc lamp. The modern choice is a
view of the specimen is completely illuminated, making laser light source, which has the additional benefit of
the whole region fluoresce at the same time. Of course, being available in a wide range of wavelengths.
the highest intensity of the excitation light is at the focal In Fig. 4A the laser provides the intense blue
point of the lens, but the other parts of the specimen do excitation light. The light reflects off a dichroic mirror,
get some of this light and they do fluoresce. Thus, light which directs it to an assembly of vertically and hori-
at a ‘‘dark blue’’ point may include light that has been zontally scanning mirrors. These motor-driven mirrors
scattered from other ‘‘light blue’’ points, thereby obscur- scan the laser across the specimen. Recall that
ing its fluorescence. To reduce this effect the confocal Minsky’s invention kept the optics stationary and
microscope focuses a point of light at the in-focus dark instead scanned the specimen by moving the stage back
blue point by imaging a pinhole aperture placed in front and forth in the vertical and horizontal directions. As
of the light source.[1] Thus, the only regions that are illu- awkward (and slow) as that method seems it does have
minated are a cone of light above and below the focal among others the following two major advantages:[7]
(dark blue) point (Fig. 9A).
Together the confocal microscope’s two pinholes sig- The specimen is everywhere illuminated axially,
nificantly reduce the background haze that is typical of a rather than at different angles as in the case of the
conventional fluorescence image, as shown in Fig. 5. scanning mirror configuration, thereby avoiding
optical aberrations. Thus, the entire field of view
is illuminated uniformly.
The field of view can be made larger than that of the
a
Actually not all the light from the focal point reaches the pinhole. static objective by controlling the amplitude of the
Some of it is reflected and absorbed by the optics in between. stage movements.
4 Confocal Microscopy
rotating
mirrors screen with
laser pinhole
detector (PMT)
} microscope
fluorescent
specimen
Fig. 4 Basic setup of a confocal microscope. Light from the laser is scanned across the specimen by the scanning mirrors. Opti-
cal sectioning occurs as the light passes through a pinhole on its way to the detector. (View this art in color at www.dekker.com.)
In Fig. 4 the dye in the specimen is excited by the number of factors, the most important of which will
laser light and fluoresces. The fluorescent (green) light be discussed below.
is descanned by the same mirrors that are used to scan The image created by the confocal microscope is of
the excitation light (blue) from the laser and then a thin planar region of the specimen—an effect referred
passes through the dichroic mirror. Thereafter, it is to as optical sectioning. Out-of-plane unfocused light
focused onto the pinhole. The light that makes it has been rejected, resulting in a sharper, better-
through the pinhole is measured by a detector such resolved image. Fig. 5 shows an image created with
as a photomultiplier tube. and without optical sectioning.
In confocal microscopy, there is never a complete
image of the specimen because at any instant only
one point is observed. Thus, for visualization the detec- THREE-DIMENSIONAL VISUALIZATION
tor is attached to a computer, which builds up the
image one pixel at a time. For a 512 512-pixel The ability of a confocal microscope to create sharp
image this is typically done at a frame rate of 0.1– optical sections makes it possible to build 3D rendi-
30 Hz. The large range in frame rates depends on a tions of the specimen. Data gathered from a series of
optical sections imaged at short and regular intervals The Airy disk limits the maximum resolution that can
along the optical axis are used to create the 3D recon- be attained with the confocal microscope—the best reso-
struction. Software can combine the 2D images to lution is typically about 200 nm. Ideally, the image of a
create a 3D rendition. Representing 3D information point would just be a single intense point right at
in a meaningful way out of 2D data is nontrivial, radius ¼ 0. However, the finite size of the Airy disk sets
and a number of different schemes have been devel- the scale for which details can be resolved. According to
oped. Fig. 6 shows a 3D reconstruction, from slices the Rayleigh criterion, the minimum separation between
of a suspension of 2 mm diameter colloidal particles two Airy disks for which they are distinguishable is equal
using ‘‘alpha blending’’—a technique that combines to their radius. This corresponds to the maximum of one
images by first making each of their individual pixels Airy disk superimposed on the minimum of the other.
less or more transparent according to a computed Resolution along the optical axis is also limited by
weight called the ‘‘alpha’’ value.[8] The result is a 3D- diffraction effects. As in the lateral direction there is
like structure. a periodic, but elliptical distribution of intensity in
the shape of an Airy disk.[10]
A B
0.3
0.25
0.2
Intensity
0.15
0.1
0.05
0
–10 –5 0 5 10
Radius
Fig. 7 Airy disk similar to that of an image of a very small particle. (A) The image is ‘‘overexposed’’ and in reverse color to
allow distinction of the faint secondary peak. (B) A graph of the intensity change with radius.
arriving at the detector. In practical terms this can be For living specimens it should not significantly alter
done by averaging data from many frames—which the dynamics of the organism; and an extra considera-
has the drawback of slowing down the effective frame tion is the effect of the specimen on the fluorophore—
rate of the microscope. Or, it can be done by increasing its chemical environment can affect the position of
the intensity of the fluorescence signal. the peaks of the excitation and emission spectra.[14]
Fluorescence can be increased by dyeing the speci-
men with a larger concentration of fluorophore mole-
cules or by raising the intensity of the excitation Photobleaching
light. However, each of these methods increases excita-
tion up to some limit. For high fluorophore concentra- A major problem with fluorophores is that they
tions, the individual molecules can quench each fade (irreversibly) when exposed to excitation light
other.[5] They may also reduce the amount of fluores- (Fig. 8). Although this process is not completely under-
cence deep inside the specimen—fluorophores nearest stood, it is believed in some instances to occur when
the light source can absorb enough light to signifi- fluorophore molecules react with oxygen and=or oxy-
cantly reduce the portion available to the rest of the gen radicals and become nonfluorescent.[13,15] The
specimen.[3] Increasing fluorescence by increasing the reaction can take place after a fluorophore molecule
excitation light intensity leads eventually to saturation transitions from the singlet excited state to the triplet
of the fluorophore. Higher intensities drive a larger excited state. Although the fraction of fluorophores
fraction of fluorophore molecules into excited states, that transitions to the triplet state is small, its lifetime
which in turn leads to a smaller fraction of ground is typically much longer than that of the singlet state.
state molecules. Ultimately, the rate at which fluoro- This can lead to a significant triplet state fluorophore
phores are excited matches their decay rate, causing population and thus to significant photobleaching.[5]
ground state depopulation. Fluorescence then ceases Several strategies have been developed to reduce the
to increase with excitation intensity.[13] rate of photobleaching.[5,13] One method is to simply
reduce the amount of oxygen that would react with
the triplet excited states. This can be done by displa-
Fluorophores cing it using a different gas.[5] Another method is by
the use of free-radical scavengers to reduce the oxygen
Among the most important aspects of fluorescence radicals. Shortening the long lifetime of the triplet
confocal microscopy is the choice of fluorophore. It is excited state has also been shown to be effective.[16]
typically influenced by several factors. The fluorophore Other ways include using a high numerical aperture
should tag the correct part of the specimen. It must be lens to collect more fluorescence light and thus use less
sensitive enough for the given excitation wavelength. excitation light.[17] Also, keeping the magnification as
Confocal Microscopy 7
Multicolor Fluorescence
Fig. 8 Dyed suspension of densely packed polymethyl-
To distinguish between small features such as proteins
methacrylate beads with significant photobleaching. The rec-
tangular region near the center faded after about 30 sec of within a cell it is useful to tag them with different fluor-
exposure to excitation light. ophores and image them as separate colors. There are
two ways to do this: in one method fluorophores are
selected to correspond with the wavelengths of a multi-
low as is permissible spreads the excitation light over a line laser and in the other their response to the same
larger area, thereby reducing the local intensity. excitation wavelength causes emission at different
While photobleaching makes fluorescence micro- wavelengths.[20] In both cases the resulting emission is
scopy more difficult, it is not always undesirable. One separated with appropriate filters and directed to dif-
technique that takes advantage of it is fluorescence ferent detectors. However, there can be cross talk
photobleaching recovery (FPR) or fluorescence recovery between channels of the emitted light.[14] For most of
after photobleaching (FRAP). It involves exposing a the commonly used fluorophores there is usually some
small region of the specimen to a short and intense laser overlap between their emission spectra, making perfect
beam, which destroys the local fluorescence, and then channel separation impossible by filtering alone. To
observing as the fluorescence is recovered by transport first order this can be corrected by determining the
of other fluorophore molecules from the surrounding level of overlap of emission of each individual fluoro-
region. Quantities such as the diffusion coefficient of phore into the channels of the other fluorophores
the dyed structures can then be determined.[18] and subtracting it out mathematically.
using confocal microscopy there is a significant noni- 100 mm. In two-photon microscopy all fluorescent
maged portion of the specimen that has been subjected light is from the focal volume and is thus acceptable.
to photobleaching and photodamage. Two-photon Plus it is much more likely to reach the detector
microscopy cleverly limits fluorescence to the focal because it does not have to pass through a pinhole.[26]
volume while also reducing the average intensity of This arrangement allows imaging deeper than
the excitation light.[25] The lower amount of photo- 400 mm.[25]
bleaching and photodamage makes it particularly desir- Why not avoid the confocal microscope altogether
able for biological and medical imaging. in favor of a two-photon microscope? The main draw-
Two-photon microscopy is based on a novel method back with two-photon microscopy is the challenges
of exciting fluorophore molecules. In confocal micro- associated with the type of laser it needs. Ultrashort
scopy, a single high-energy photon excites a fluoro- pulse lasers are still very expensive compared with
phore molecule while it takes two lower-energy the continuous wave lasers used in confocal micro-
photons absorbed simultaneously to achieve the same scopy. They are also typically more demanding to set
result in two-photon microscopy. By itself a single up.[27] For example, they have to be on the same opti-
photon cannot be absorbed because it does not have cal table as the microscope because of difficulties in
enough energy to excite a fluorophore molecule and maintaining the pulse characteristics through an opti-
thus causes no fluorescence. It must reach the fluoro- cal fiber. They need a stronger power supply and much
phore molecule within approximately 1016 sec of the more cooling—usually water pumped through it at
arrival of another photon so that together they reach several gallons per minute.
the energy threshold for excitation. Not surprisingly,
this simultaneous absorption of light is a very low-
probability event at moderate light intensities. To get CONCLUSIONS
a sense of how rare it is, a molecule of rhodamine B
will absorb a single photon approximately once a sec- A confocal microscope provides a significant imaging
ond in bright sunlight, but will absorb a two-photon improvement over conventional microscopes. It creates
pair once every 10 million years.[26] Because the prob- sharper, more detailed 2D images, and allows collec-
ability of absorption is proportional to the square of tion of data in three dimensions. In biological applica-
the instantaneous light intensity, lasers that generate tions it is especially useful for measuring dynamic
very short (<1012 sec) but intense pulses are processes. A number of designs have been developed
used.[26,27] Such lasers can be operated at low average to achieve video-rate confocal microscopy, which
power—typically, five orders of magnitude lower than enables the capture of short-timescale dynamics.
for confocal microscopy—thereby causing less photo-
damage.[25]
How does the above help in microscopy? The key is FURTHER READING
that because two-photon excitation events are unlikely
at low laser intensities, fluorescence only occurs within For detailed discussions about most aspects of confo-
the focal volume of the objective where the intensity is cal microscopy the book cited in Ref.[6] is an excellent
highest (Fig. 9B). The hourglass-shaped region of source. Ref.[25] cites a book that along with providing
fluorescence characteristic of one-photon excitation insight and detail about confocal microscopy also gives
fluorescence confocal microscopy is instead a small plenty of information on two-photon microscopy.
volume at the focus, which needs no detector pinhole
for optical sectioning.[25,26]
A major advantage of two-photon microscopy is ACKNOWLEDGMENT
that it is possible to image deeper into a specimen than
with confocal microscopy. The reason is that without The authors would like to acknowledge the support
the pinhole in front of the detector more of the fluores- from the National Science Foundation under Grant
cence signal is collected. This is important because as DMR-0239109.
the observation depth within the specimen is increased,
fluorescent light from the focal volume becomes more
likely to be scattered. In confocal microscopy, the pin- REFERENCES
hole will reject that scattered light if its trajectory
makes it appear not to originate from the focal plane. 1. Minsky, M. Memoir on inventing the confocal
To make matters worse, the pinhole may accept out- microscope. Scanning 1988, 10, 128–138.
of-plane fluorescence light that is scattered and appears 2. Wilson, T.; Carlini, A.R. Three dimensional ima-
to come from the focal plane. Together the two effects ging in confocal imaging systems with finite-sized
significantly reduce the image contrast at depths of detectors. J. Microsc. 1988, 141, 51–66.
10 Confocal Microscopy
3. Sheppard, C.J.R.; Shotton, D.M. Image formation 16. Song, L.; Varma, C.A.; Verhoeven, J.W.; Tanke,
in the confocal laser scanning microscope. In H.J. Influence of the triplet excited state on the
Confocal Laser Scanning Microscopy; Springer- photobleaching kinetics of fluorescein in micro-
Verlag, New York Inc.: New York, 1997; 15–31. scopy. Biophys. J. 1996, 70, 2959–2968.
4. Guilbault, G. General aspects of luminescence 17. Lemasters, J.J. Confocal microscopy of single living
spectroscopy. In Practical Fluorescence, 2nd cells. In Fluorescence Imaging Spectroscopy and
Ed.; Guilbault, G., Ed.; Marcel Dekker, Inc.: Microscopy; Wang, X.F., Herman, B., Eds.; John
New York, 1990; 1–40. Wiley & Sons, Inc.: New York, 1996; 157–177.
5. Atkins, P. Spectroscopy 2: electronic transitions. 18. Axelrod, D.; Koppel, D.E.; Schlessinger, J.;
In Physical Chemistry, 5th Ed.; W. H. Freeman Webb, W.W. Mobility measurement by analysis
and Company: New York, 1994; 590–622. of fluorescence photobleaching recovery kinetics.
6. Tsien, R.Y.; Waggoner, A. Fluorophores for con- Biophys. J. 1976, 16, 1055–1069.
focal microscopy. In Handbook of Biological 19. Teraski, M.; Dailey, M.E. Confocal microscopy
Confocal Microscopy, 2nd Ed.; Pawley, J.B., of living cells. In Handbook of Biological Con-
Ed.; Plenum Press: New York, 1995; 267–280. focal Microscopy, 2nd Ed.; Pawley, J.B., Ed.;
7. Sheppard, C.J.R.; Shotton, D.M. Introduction. In Plenum Press: New York, 1995; 327–346.
Confocal Laser Scanning Microscopy; Springer- 20. Stelzer, E.H. The intermediate optical system of
Verlag New York Inc.: New York, 1997; 1–13. laser-scanning confocal microscopes. In Hand-
8. Porter, T.; Duff, T. Compositing digital images. book of Biological Confocal Microscopy, 2nd
Comput. Graphics 1984, 18 (3), 253–259. Ed.; Pawley, J.B., Ed.; Plenum Press: New York,
9. Inoué, S.; Spring, K.R. Microscope image forma- 1995; 139–154.
tion. In Video Microscopy the Fundamentals; 21. Tsien, R.Y.; Bacskai, B.J. Video-rate confocal
Plenum Press: New York, 1997; 13–118. microscopy. In Handbook of Biological Confocal
10. Keller, H.E. Objective lenses for confocal micro- Microscopy, 2nd Ed.; Pawley, J.B., Ed.; Plenum
scopy. In Handbook of Biological Confocal Press: New York, 1995; 459–478.
Microscopy, 2nd Ed.; Pawley, J.B., Ed.; Plenum 22. Iyer, V.; Losavio, B.E.; Saggau, P. Compensation
Press: New York, 1995; 111–126. of spatial and temporal dispersion for acousto-
11. Wilson, T. The role of the pinhole in confocal optic multiphoton laser-scanning microscopy. J.
imaging system. In Handbook of Biological Con- Biom. Opt. 2003, 8 (3), 460–471.
focal Microscopy, 2nd Ed.; Pawley, J.B., Ed.; 23. Xiao, G.Q.; Corle, T.R.; Kino, G.S. Real-time
Plenum Press: New York, 1995; 167–182. confocal scanning optical microscope. Appl.
12. Pawley, J. Fundamental limits in confocal micro- Phys. Lett. 1988, 53 (8), 716–718.
scopy. In Handbook of Biological Confocal 24. Kino, G.S. Intermediate optics in Nipkow disk
Microscopy, 2nd Ed.; Pawley, J.B., Ed.; Plenum microscopes. In Handbook of Biological Confo-
Press: New York, 1995; 19–38. cal Microscopy, 2nd Ed.; Pawley, J.B., Ed.; Ple-
13. Becker, P.L. Quantitative fluorescence measure- num Press: New York, 1995; 155–165.
ments. In Fluorescence Imaging Spectroscopy 25. Diaspro, A.; Sheppard, C. Two-photon micro-
and Microscopy; Wang, X.F., Herman, B., Eds.; scopy: basic principles and architectures. In Con-
John Wiley & Sons, Inc.: New York, 1996; 1–29. focal and Two-Photon Microscopy Foundations,
14. Sheppard, C.J.R.; Shotton, D.M. Confocal fluor- Applications and Advances; Diaspro, A., Ed.;
escence microscopy. In Confocal Laser Scanning Wiley-Liss, Inc.: New York, 2002; 39–73.
Microscopy; Springer-Verlag New York Inc.: 26. Denk, W.; Svoboda, K. Photon upmanship: why
New York, 1997; 61–70. multiphoton imaging is more than a gimmick.
15. Chen, H.; Swedlow, J.R.; Grote, M.; Sedat, J.W.; Neuron 1997, 18, 351–357.
Agard, D.A. The collection, processing, and dis- 27. Denk, W.; Piston, D.W.; Webb, W.W. Two-
play of digital three-dimensional images of biolo- photon molecular excitation in laser-scanning
gical specimens. In Handbook of Biological microscopy. In Handbook of Biological Confocal
Confocal Microscopy, 2nd Ed.; Pawley, J.B., Microscopy, 2nd Ed.; Pawley, J.B., Ed.; Plenum
Ed.; Plenum Press: New York, 1995; 197–210. Press: New York, 1995; 445–458.