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Optical Microscopy
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1. INTRODUCTION
Light
Microscopy studies the enlargement of the image of objects
Figure 1. Light crossing the interface separating two transparent
too small to be properly seen by the unaided eye. Microscopy media of refractive index n1 and n2, respectively (n2 4 n1 9). The
accomplishes its task making use of the radiations emitted, angle a1 between the light ray and the normal to the interface is
absorbed, transmitted, or reflected by the specimen to be called angle of incidence. The angle a2 is called angle of refraction.
observed. The nature of the radiation specifies the type of (This figure is available in full color at http://www.mrw.
microscopy: electron microscopy, x-ray microscopy, acoustic interscience.wiley.com/ebe.)
microscopy, etc. The visible part of electromagnetic spec-
trum is the type of radiation used by optical microscopy. Methylene iodide 1.74
Rough magnifying glasses were used in ancient times, Diamond 2.42
but the evolution of modern microscopes started in the
seventeenth century. Although the first compound micro- According to this effect, the light crossing a thin lens
scope was built by Hans and Zacharias Janssen in 1595, passes from air into glass and then from glass into air,
Antoni van Leeuwenhoek (1632–1723) managed to make being refracted twice. Rays entering a lens parallel to the
lenses so good to achieve the amazing magnification of optical axis emerge from it along a path intersecting the
about 300x in their very simple microscopes. Thanks to focal point on the opposite side. Thin lenses have a focal
the suggestions of the scientist Robert Hook (1635–1702) point at every side, and the focal length f is the distance
around 1670, the instrument maker Christopher Cock in between the focal points and the lens. If o and i are the
London built a very successful compound microscope. distances from the lens of object and image respectively,
With this instrument, Hook was able to observe the cells. then 1/o þ 1/i ¼ 1/f, and the magnification m is i/o (Fig. 2).
The Hook’s microscope can be regarded as the father of A thin lens divides the space in an incident- and in a re-
modern instruments. fracted-light emispace. The values of o, i, m, and f can be
positive ( þ ) or negative ( ) according to the following
2. THIN LENSES conventions:
resolving power of a lens is a quantitative measure of this immersion media having a different refractive index, like
ability: points closer than the limit of resolution cannot be water (n ¼ 1.33), glycerine (n ¼ 1.47), or oil (n ¼ 1.52).
distinguished as separate points. Ernst Abbe (1840–1905),
in 1873 first fixed the value of the minimal distance d be-
tween two adjacent points allowing them to be perceived 4. OPTICAL ABERRATIONS
as separated:
d ¼ l=2n sin a; Deviations from perfect image formation by optical sys-
tems are called aberrations. Unfortunately, thin lenses
where l is the wavelength of light, a is one-half the angu- suffer from a variety of optical defects leading to distorted
lar aperture of the lens, and n is the refractive index of the representations of the object, with the main final result of
medium between the object and the lens. a rather severe loss of details from the observed specimen.
At present, the smallest linear separation of two object Chromatic aberration is a very common optical defect
points for which they may be resolved by an objective is related to the dependence of the refractive index of every
fixed by the Rayleigh criterion: material on the wavelength of light. The lens deviates the
different color components of white light at different re-
fraction angles, a phenomenon called dispersion, bringing
d ¼ 1:22ðl=2NAÞ;
them to focus at different points (Fig. 5). This aberration
gives rise to the formation of colored fringes surrounding
where NA is the numerical aperture of the objective. Both the image. Manufacturers reduce or eliminate these prob-
the Abbe criteria and the Rayleigh criteria are very similar, lems by assembling objectives with a group of lenses hav-
being the numerical aperture related to the imaging me- ing different dispersion properties (achromatic and
dium by NA ¼ nsina (Fig. 4). The maximal value of sina is 1 apochromatic objectives).
(a ¼ 901), therefore the theoretical maximal numerical ap- Spherical aberration and field curvature are artifacts
erture of an objective in air (n ¼ 1) is NA ¼ 1. As a high NA both caused by the spherical curvature of lens surfaces.
is an essential requirement for high resolution, immersion Spherical aberration occurs because light rays leaving
optics have been developed. Biological specimens can be the object at different distances from the optical axis are
imaged at a very short distance from the objective through focalized at different distances along the axis when they are
i2
O1 i1 O2
Obj1
F′1 F2 F′2
Figure 3. The simple microscope. A microscope uses a F1 im1 obj2
very short focal length objective lens to form a greatly
enlarged image. This image is then viewed with a
short focal length eyepiece used as a simple magnifier. im2
According to this assumption, the resulting magnifica-
tion M is M ¼ m1 m2 ¼ i1 / o1 i2 / o2 ¼ i1 i2 /o1 o2.
In modern microscopes, the image is formed at infinity
to minimize eyestrain. (This figure is available in full
color at http://www.mrw.interscience.wiley.com/ebe.)
OPTICAL MICROSCOPY 3
structively and destructively at the eyepiece image plane, ting lasers are now used in modern fluorescence microscopy
showing details of the specimen having a different thick- tecniques, as confocal laser-scanning microscopy and
ness as a separation between different colors. The overall multi-photon excitation microscopy. In the most common
effect is a pseudo-3-D image of the biological specimen. configuration (epifluorescence microscope), the excitation
wavelength, selected by an excitation filter, is reflected by a
suitable dichroic mirror (dichromatic beam splitter), reach-
6. FLUORESCENCE MICROSCOPY ing the specimen through the objective. A careful regulation
of excitation intensity helps to avoid extinction of the flu-
Fluorescence is the emission of radiation in the visible orophore emission (photobleaching). The fluorescence light
range of the electromagnetic spectrum, by some materials emitted by the fluorescent dyes is collected back by the
stimulated by the absorption of another electromagnetic objective, transmitted by the dichroic mirror, selected from
radiation having higher energy. Materials experiencing undesired wavelengths by an emission filter (stop filter),
fluorescence are called fluorophores or fluorochromes and and finally focalized at the eyepiece image plane (Fig. 7).
are characterized by an excitation and an emission spec- Thanks to the very fast growth of the number and type
trum (Fig. 6). Fluorescent dyes can be used to stain tissues of fluorescent dyes, fluorescence microscopy is currently
and cells to highlight interesting details. the most used microscopic technique for biomedical appli-
The fluorescence microscope is a modification of the cations. Fluorescent dyes may be roughly divided in mor-
compound optical microscope, arranged to admit light of a phological dyes, mainly addressed to evidence structural
selected wavelength to the stained specimen and to image it details of biological structures, and functional dyes,
through the light emitted by the fluorescent dyes. The light mainly addressed to reveal or measure physiological pro-
source of a fluorescent microscope is a gas-vapor arc lamp, cesses in tissues and cells in vivo. Modern fluorescence
using either a discrete (Mercury) or a continuous (Xenon) microscopes allow one to stain cells with many types of
light-emission spectrum. Continuous- or pulsed-light-emit- fluorescent dyes simultaneously.
HO O O
7. ADVANCED TECHNIQUES IN FLUORESCENCE
MICROSCOPY
O Eyepiece
Arc Emission
lamp filter
Epifluorescence
box
Dichroic
Emission spectrum
Excitation spectrum
Excitation mirror
filter
Objective
Specimen
Figure 7. Schematic drawing of a conventional epifluorescence
400 500 600 microscope. The excitation filter selects the wanted wavelength
Wavelength (nm) from the different color components of the light generated by the
Figure 6. An example of a fluorescent molecule. In the upper gas-vapor arc lamp. Light, reflected by an appropriate dichroic
part of the figure, the structure of Fluorescein (C20H12O5), is mirror, hits the fluorescent specimen passing through the objec-
shown a molecule that exhibits intense yellow-green fluorescence tive. Light emitted by the fluorescent dyes is collected back by the
in highly diluted solutions and is used, conjugated to antibodies, objective, transmitted by the dichroic mirror, selected from unde-
in biology and in medicine for diagnostic purposes. The normal- sired wavelengths by the emission filter, and finally focalized at
ized fluorescence excitation and emission spectra are shown in the eyepiece image plane. In most epifluorescence microscopes,
the lower part of the figure. The light absorption properties of a different combinations of excitation filters, dichroic mirrors, and
molecule are shaped by both the number of aromatic rings and the emission filters are pre-assembled in an epifluorescence box.
number of double bonds. (This figure is available in full color at (This figure is available in full color at http://www.mrw.
http://www.mrw.interscience.wiley.com/ebe.) interscience.wiley.com/ebe.)
OPTICAL MICROSCOPY 5
have been further strengthened and improved with the Donor Acceptor
coming of confocal laser-scanning microscopy and multi- hex hem hex hem
photon excitation microscopy.
> 100 Å
7.1. Fluorescence Resonance Energy Transfer (FRET)
Fluorescence resonance energy transfer is a quantum-me-
chanical process described over 50 years ago by Theodor
Förster (1). FRET is a distance-dependent dipole-dipole
interaction between neighboring fluorophores, consisting
in a nonradiative transfer of the excitation energy of the
donor molecule to the ground state of an acceptor flu-
orophore. In the presence of FRET, excitation of the donor Donor Acceptor
molecule will produce both quenching of the donor fluo- hex FRET hem
rescence and emission of light at the acceptor wavelength.
FRET efficiency depends on the inverse sixth power of the 10÷100Å
donor-acceptor distance, making it suitable for investigat-
ing biological phenomena over distances on the order of
the size of a typical protein. In a certain sense, FRET
imaging may override the resolution limits of optical
microscopy in applications like structure, conformational
shifts, binding, colocalization, interactions between pro-
Figure 8. Fluorescence resonance energy transfer. The upper
teins, nucleic acids, and other biological macromolecules.
part of the figure shows the excitation and emission spectra of
Three main conditions are required for an optimal en- donor and acceptor molecules. Donor emission spectrum and ac-
ergy transfer. (1) Donor emission spectrum and acceptor ceptor excitation spectrum are partially overlapped (dashed
absorption spectrum should substantially overlap. The com- area). When the intermolecular distance is larger than 100 Å,
mon spectral area, spectral overlap integral, must be at the fluorescence properties of both molecules are not affected. As
least 30% of the total. (2) The distance between donor and shown in the lower part of the figure, for intermolecular distances
acceptor molecules should typically range from 10 to 100 Å. shorter than 100 Å, the excitation energy of donor is transferred
(3) The donor and acceptor transition dipoles must be in the to the acceptor. The donor decays to the ground state through a
proper orientation with respect to one another (Fig. 8). nonradiative energy loss and the excited acceptor through fluo-
rescence emission. The common spectral area (spectral overlap
integral), shown in black in the figure, must be at least 30% of the
7.2. Fluorescence Lifetime Imaging (FLIM) total. The donor and acceptor transition dipoles (white arrows)
must be in the proper orientation with respect to one another.
In the absence of molecular interactions, every fluorescent (This figure is available in full color at http://www.mrw.
molecule is characterized by a unique duration of the ex- interscience.wiley.com/ebe.)
cited state. Typical fluorescence decays are exponentials
with a time-constant in the order of 10 9 s. Fluorescence the fluorescence and from the reduced emission amplitude
lifetime is a peculiar property of a fluorophore that, unlike modulated at the same frequency of excitation.
fluorescence emission intensity, is independent of photo- The lifetime of fluorescence emission is environmen-
bleaching, excitation light intensity, and optical path tally sensitive. Imaging using fluorescence lifetimes may
length in biological specimens. In FLIM, lifetime, rather provide information about functional properties of biolog-
than intensity, of the fluorescence emission is measured ical samples under investigation. Modifications induced by
and spatially mapped in tissues and cells. the environment in fluorescence lifetimes of fluorophores
Fluorescence lifetime may be measured according to used as remote sensors allow one to measure ion concen-
the frequency-domain technique or the time-domain tech- trations (mainly oxygen, hydrogen, and calcium), hydro-
nique (Fig. 9). phobic properties, binding, and interactions among
In the time-domain technique, the fluorophore is ex- macromolecules in living cells.
cited by a very short pulse of light and the time-course of
the emission intensity is monitored and recorded. Excita-
7.3. Fluorescence Recovery After Photobleaching (FRAP)
tion pulses are usually generated by solid-state lasers or
by mode-locked lasers with a pulse duration of a few hun- Fluorescence recovery after photobleaching is a technique
dred picoseconds. The fluorescence image can be obtained suitable for investigating the mobility of fluorescent mole-
by gated special detectors making use of image-intensifi- cules in living cells. Thanks to the introduction of green
cation techniques. The pseudocolor representation of im- fluorescent proteins and other cell-labeling techniques,
ages at all pixels in the field of view is related to the FRAP, born in the late 1970s to study viscosity of biologi-
fluorescence lifetime across the biological specimen. cal membranes and diffusional properties of molecules in
In the frequency-domain technique, the specimen is lipid bilayers, is now a very popular modern imaging tech-
continuously excited with a sinusoidally modulated laser nique. In a FRAP experiment, a brief intense light pulse
light (1–200 megahertz). The fluorescence lifetime can be from a laser is used to irreversibly bleach fluorophores
measured from the phase shift between the excitation and in a limited volume of a biological sample. If fluorescent
6 OPTICAL MICROSCOPY
Fluorescence emission
Φ
Figure 9. Fluorescence lifetime imaging. In the time-domain
7.4. Total Internal Reflection Fluorescence Microscopy
technique, fluorophores are excited at t ¼ 0 by a very short light
pulse (green area). The gradual decay of fluorescence intensity ðTIRFMÞ
with time (red curve) can be fitted by a single-exponential func- According to Snell law, a light beam propagating from a
tion, according to I(t) ¼ I(0) exp ( t/t), where t is the fluorescence medium of higher index of refraction n1 to a medium of
lifetime. In the frequency-domain technique, fluorophores are
lower index of refraction n2 does not undergo refraction if
continuously excited with a sinusoidally modulated light (green
curve). The emitted fluorescence (red curve) shows a similar
y1 4 yc ¼ sin 1 (n2 / n1). yc is called critical angle, and for
waveform, but is modulated in amplitude and phase-shifted of incident angles 4 yc the light beam is totally reflected. In a
an angle F from the excitation curve. Both amplitude modulation biological specimen, light crosses the interface between the
and phase-shift depend on the fluorescence emission lifetime t. glass coverslip and the aqueous medium being refracted
(This figure is available in full color at http://www.mrw. and reflected as a function of the incident angle yg. The
interscience.wiley.com/ebe.) critical angle is yc(g-w) ¼ sin 1 (nw / ng), where nw D 1.33
Fluorescence emission from the bleached region (A.U.)
and ng D 1.52. If yg 4yc(g-w), light is totally reflected from microscope. They can be roughly divided in two main
the glass/water interface, rather than passing through. groups. The first group makes use of an added prism to di-
Although the light is totally reflected, a portion of the rect a laser beam toward the glass/water interface with an
incident energy, named evanescent wave, penetrates angle wider than the critical angle. The second group uses
through the interface and propagates in the aqueous me- immersion objectives with a very high numerical aperture
dium. The intensity of this electromagnetic field decays to both direct illumination to the interface at supercritical
very rapidly with the distance z from the interface, angles and capture the emitted fluorescence.
according to I(z) ¼ I(0)e z/d, where d is a factor depen-
dent on the beam wavelength, the refraction indices and 7.4.1. Detection of Microscopic Images. For several de-
the incident angle. The evanescent wave propagates in the cades, detection of microscopic images has been committed
aqueous medium only for less than 200 nanometers, to ordinary photographic cameras using emulsion-based
however, having the same wavelength of the incident films as storage medium. Thanks to the rapid improve-
light, it is able to excite fluorophores in a very thin region ment of photographic technology, in the last twenty years
of the biological specimen facing the interface. electronic imaging has gradually replaced conventional
This phenomenon is the physical foundation of the total photomicrography. Initially, cameras were video rate cam-
internal reflection fluorescence microscopy (Fig. 11), a tech- eras. This type of camera makes use of light detectors
nique mainly used to observe fluorescence of molecules based on vacuum-tube technology and generates an ana-
placed very near to the cell surface, and allowing to study, log output signal that conforms to the industry standards
for instance, the cell-substrate contact, the adsorption of RS-170, PAL, RGB, NTSC, etc. The electrical signal is
macromolecules to surfaces, and different cell-membrane usually recorded into analog signal storage devices as
processes. The main advantage of this technique is a result video recorders; however, to be sent to a computer for im-
of the exponential decay of the evanescent wave, which age analysis, it must be digitized by a frame grabber. Ac-
prevents the excitation of fluorescence from outside the tually, vacuum-tube-based detectors (photomultipliers)
focal plane, which allows one to focus on an optical section are used only in laser-scanning microscopy techniques.
about five times thinner than optical sections achieved In recent years, video rate cameras have been replaced
with confocal microscopy, by far improving both the signal- by cameras using a solid-state detector whose output is
to-noise ratio and the spatial resolution. already a digital signal. Solid-state detectors are two-
Many different optical configurations have been designed dimensional arrays of photodiodes mainly manufactured
to realize TIRFM with a conventional epifluorescence according to CCD (charge-coupled device) or CMOS
(complimentary metal-oxide semiconductor) technology.
High-level performance is expected from scientific-
grade digital cameras, but the device properties heavily
Distance from interface