10. lecture
Laser-aided Microscopy
The images of modern high-resolution microscopes are constructed by computers and the lasers play
determining role in the digital microscopy. Nowadays diode lasers, fiber lasers and solid state lasers
are the most important light sources in modern confocal imaging instruments and have taken the part
of formerly used gas lasers almost completely. Special attention should be paid to the diode lasers
which offer high beam quality, high power stability, relatively low cost and show direct modulation
capabilities with superior performance to any laser microscopes. Below, some selected microscopic
techniques will be surveyed concentrating on the role of the laser and on potential biophysical
applications.
Laser Scanning Microscope (LSM). In scanning laser microscopy, the images are assembled by
pixel information via scanning point by point and line by line. In addition, the confocal laser
scanning microscope (CLSM) allows for the collection of light exclusively from a single plane (Fig.
10.1 and see for more details in Lecture 8). The microscope is equipped typically by Argon laser,
He-Ne laser (633 nm and 561 nm) and/or diode laser (405 nm) for scanned images and by thermal
light sources (mercury and halogen) lamps for transmission images.
Micropatterning of DNA-tagged Vesicles. LSM can be used to check the quality of micropatterned
nanoobjects such as vesicles. Figure 10.2 shows the schematic image of the multistep surface
modification used for intact vesicles immobilization via DNA: the vesicle is anchored by complex
structure of DNA molecules to a firm surface which can be repeated several times on a microplate.
The fluorescent microscopy image of surface-immobilized DNA-tagged vesicles can be visualized in
the small squares and can be used for several generic/biochemical tests.
Fluorescence recovery after photobleaching (FRAP) is a special technique for studies of mobility of
fluorophores in the membranes or in the cytoplasm of the cells. The laser beam is focused on the
region of interest in the view field of the microscope and its intensity is bursted (transiently brought
to high value) to photobleach the fluorophores in the focal region. The laser intensity is then
decreased to allow continuous monitoring of the emission of fluorescence from the same region.
After the photobleaching, the intensity of the observed fluorescence increases gradually as the
unbleached probes diffuse into the bleached area. Finally, the original fluorescence level measured
before the flash will be re-established. The kinetics of the recovery is complex as the probes are not
freely diffusing species in the cytoplasm or in the membranes of the cell but are exposed to
interactions with the local environment (cytoscaleton, membrane constituents etc.). The evaluation
of the kinetics can be used to determine the diffusion coefficients in situ.
Figure 10.3 shows the result of a typical FRAP experiment with green fluorescence protein
(GFP) inside the cell of E. coli bacterium. The steps to carry out the experiment are the following: 1)
Express the fluorescent protein GFP. 2) Use a laser to “photo-bleach” the fluorescent protein in part
of a single bacterial cell. The laser flash permanently (irreversibly) destroys the fluorescence of
proteins in the target area. 3) Measure the recovery of the intensity of fluorescence as the intact
proteins diffuse into the region which was photo-bleached earlier. From the kinetics, one can
determine the diffusion coefficient of the GFP: Dcytoplasm = 7.7 µm2/s (= 7.7 · 10-8 m2/s) which value
is 11-fold less than the diffusion coefficient in water: Dwater = 87 µm2/s. The observed slow
translational diffusion is probably due to the crowding effect resulting from the very high protein
concentration in the bacterial cytoplasm (200 -300 mg/ml).
Fluorescence-Lifetime Imaging Microscopy (FLIM). Fluorescence microscopy is based on the
known affinities of fluorescence probes for specific biomolecules in the cell. For example,
fluorophores such as DAPI and Hoechst have affinity for nucleic acids and are primarily nuclear
stains. Probes such as rhodamine 123 have affinity for mitochondria. The image of fluorescence
intensity can give valuable qualitative information but restricts the quantitative determination
because the local concentrations of the fluorophores within cells are not known. The probes undergo
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rapid diffusion and photobleaching and can be easily washed out of the cells. Also, the fluorescence
quantum yields of probes can change due to the local environment. Imaging based on the lifetimes of
the fluorophoes avoids these problems.
Advantages of lifetime imaging. 1) The fluorescence-lifetime imaging microscopy provides
measurements that are independent of the probe concentration. While the intensity of the
fluorescence of the fluorophore depends on its concentration, the lifetime is mostly independent of
its concentration. 2) The FLIM can be used for imaging of ions and biomolecules whose proximity,
and conditions result in a change of the lifetime of the fluorophore. There are a larger number of
probes that display changes in lifetime in response to ions or to the environment, without displaying
spectral shifts. For example, there are several long-wavelength probes for sodium and calcium ions.
Probes for chloride are based on collisional quenching controlled by the Stern-Volmer expression,
which decreases the lifetimes but does not cause a spectral shift. 3) Intracellular measurements of
Förster resonance energy transfer (FRET) using the donor lifetimes can be more reliable than
intensity measurements of the donor (D) and/or acceptor (A) fluorescence. The complex decay of the
donor fluorescence reflects the time-dependent population of D–A pairs. Those donors with nearby
acceptors decay more rapidly, and donors more distant from acceptors decay more slowly.
If the donors and acceptors are randomly distributed in the three dimensional solutions, the
(Förster’s) theory is relatively simple. Following Dirac δ-function excitation, the intensity decay of
the donor is given by
 t
t 
F DA t   F (0)  exp 
 2
(10.1)



D 
 D
with γ = A/A0, where A is the acceptor concentration and A0 is the critical concentration that can be
expressed also by the Förster radius. A0 represents the acceptor concentration that results in 76%
energy transfer between D and A. The fluorescence intensity (relative steady-state quantum yield) of
the donor is given by
F DA
 1    exp( ) 2 1  erf ( )
FD
where „erf” is the „error function”: erf ( ) 
2

(10.2)

 exp(  x
2
)dx .
0
In cells, the situation is much more difficult. For illustration, let’s assume two proteins that
are labeled each with a donor or acceptor and associate with each other in response to a stimulus. In
principle, the extent of association can be determined from the decrease of the intensity of the donor
fluorescence (see Eq. 10.2). However, since the local donor concentration is not known, the extent of
quenching cannot be determined from the donor intensity alone. The intensity of the donor
fluorescence in the absence of acceptor (FD) must also be known. Such control measurements are
difficult to perform when using cells. We would not have similar difficulties if the changes of donor
fluorescence lifetime due to energy transfer to the acceptor is considered.
The concept of FLIM is an optical analogue of the widely used magnetic resonance imaging
(MRI). In MRI, the proton relaxation times determined by the local chemical composition of the
tissue are measured at each location in the patient. The numerical value of the relaxation time is used
to calculate the contrast in the image which is not based on proton concentration. The contrast in
FLIM is determined by similar principles. The local environment determines the lifetime of the
fluorescence, which is then used to calculate an image that is independent of the probe concentration.
Principle of operation: 1) Phase modulation. The measurement of fluorescence lifetimes from the
phase-sensitive intensities is illustrated in Figure 10.4. The cuvettes containing different
fluorophores are illuminated with light modulated at 80 MHz. The phase angle Θ and modulation m
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of the emissions are different because of the different lifetimes and are related to the observed
intensity
 1

I obs  k  c 1  mdet  m  cos(   det ) ,
 2

(10.3)
where k is a constant, c is the concentration of the fluorophore, mdet and m are the modulation of the
detector and the sample, respectively and Θdet and Θ are the phase angle of the detector and the
sample, respectively. The phase angles and modulations at each point (region) of the sample are
determined by fitting the phase-sensitive intensities to a cosine function and are then used to
calculate the lifetime image (see lecture 7).
The heart of the set-up is the frequency-modulated image intensifier. The gain or voltage
across the intensifier is modulated at the same frequency as the frequency of the modulated
excitation, and the signals are phase locked, therefore there is no drift between them. The image
intensifier contains a phosphor screen where the brightness is proportional to the intensity which is
recorded by a CCD camera. This measurement results in a constant image from each cuvette
(position of the sample).
2) Pulsed illumination. Time-correlated single-photon counting (TCSPC) is frequently employed
because fluctuations in source intensity and photoelectron amplitudes are ignored and the time
resolution can be upwards of 4 ps (Fig. 10.5). 16~64 multichannel photomultiplier systems have
been commercially available, whereas the recent CMOS single-photon avalanche diode (SPAD)TCSPC FLIM systems can offer additional reliable and low-cost options.
Gating method uses pulsed excitation. Before the pulse reaches the sample, some of the light
is reflected by a dichroic mirror and gets detected by a photodiode that activates a delay generator
controlling a gated optical intensifier (GOI) that sits in front of the CCD detector. The GOI only
allows for detection for the fraction of time when it is open after the delay. Thus, with an adjustable
delay generator, one is able to collect fluorescence emission after multiple delay times encompassing
the time range of the fluorescence decay of the sample.
Applications. Imaging of calcium concentration. The measurement of calcium concentration in the
cell requires a probe that displays calcium-dependent lifetimes. Among others, Quin-2 has proved to
be an adequate fluorophore. After proper calibration, the phase and modulation sensitive images can
be converted to a calcium concentration sensitive image in Cos cells labeled with Quin-2 (Fig. 10.6).
The calcium concentration appears higher in the periphery of the cells than near the center. The use
of FLIM allows the determination of the calcium concentrations without knowledge of the local
probe concentrations within the cell.
Lifetime Imaging of Cellular Biomolecules. FLIM can yield excellent images showing the locations
of different biomolecules within cells. Figure 10.7 shows fluorescence intensity and lifetime images
of bovine artery endothelial cells stained with three fluorophores. Nucleic acids were stained with
DAPI, F-actin was stained with Bodipy FLphallacidin, and the mitochondria were stained with MitoTracker Red CMX Ros. The intensity image shows regions of the cell with different brightness but
does not distinguish between the three fluorophores. TCSPC measurements in each region of the cell
showed that DAPI decayed with a lifetime of 1.1 ns, Bodipy-FL displayed a lifetime of 2.5 ns, and
MitoTracker CMX Ros displayed a lifetime of 1.8 ns. These lifetimes were used to assign
pseudocolors to each of the fluorophores. The lifetime image clearly resolved the locations of the
probes based on their lifetimes.
FRET FLIM imaging of protein kinase C activation. The FRET measurements using FLIM can
provide a method to discriminate between the states/environments of the fluorophore. In contrast to
intensity-based FRET measurements, the FLIM-based FRET measurements are insensitive to the
concentration of the fluorophores and can thus filter out artifacts introduced by variations in the
concentration and emission intensity across the sample. Figure 10.8 shows the use of FLIM to study
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protein phosphorylation in response to stimuli. The Cos7 cells express protein kinase C (PKC)
labeled by Green-Fluorescence-Proteins (GFP). The intensity images show the locations of GFPlabeled PKC. This protein is energized by phosphorylation (phosphor is bound to the protein) upon
activation by the tumor-producing substance phorbol myristoyl acetate (PMA). Some cells were
microinjected with Cy3.5-labeled IgG that is specific for the phosphorylated epitope of PKC.
Binding of the labeled antibody to phospholylated PKC is expected to reduce the lifetime of the GFP
label by resonance energy transfer to the acceptor Cy3.5. The lifetime images of the three cells in the
view field of the microscope are shown at various times after start of the phosphorylation by PMA.
All the cells were treated with PMA, and only the central cell in the picture was injected with Cy3.5labeled IgG specific for phosphorylated PKC. Initially the GFP lifetimes are the same in all the cells
and constant within the cells. At later times, the GFP lifetime in the central cell decreases because of
energy transfer from excited GFP (donor) to the Cy3.5-labeled antibody (acceptor).
Atomic Force Microscopy (AFM). The AFM, in contrast to many microscopes (e.g. scanning
transmission microscope, STM) has the advantage of imaging almost any type of surface, including
polymers, ceramics, composites, glass, and biological samples. One of the key elements of the AFM
is a laser beam deflection- and detection system. The laser beam is reflected from the back of the
reflective AFM lever onto a position-sensitive detector consisting of two closely spaced photodiodes
whose output signal is collected by a differential amplifier (Fig. 10.9). The angular displacement of
the cantilever results in one photodiode collecting more light than the other photodiode, producing
an output signal (the difference between the photodiode signals normalized by their sum) which is
proportional to the deflection of the cantilever. Thermal noise limited cantilever deflections (<10
nm) can be detected. The long beam path (several centimeters) amplifies the changes of the angle of
the beam. AFM tips and cantilevers are microfabricated from Si or Si3N4. Typical tip radius is from
a few to tens of nm.
Imaging modes. The primary modes of operation for an AFM are static (contact) mode and dynamic
(non-contact) mode. In static mode, the cantilever is "dragged" across the surface of the sample and
the contours of the surface are measured directly using the deflection of the cantilever (Fig. 10.10).
In the dynamic mode, the cantilever is externally oscillated at or close to its fundamental resonance
frequency (Fig. 10.11). The amplitude and phase of the oscillation and the resonance frequency are
modified by interaction forces between the tip and the sample. These changes in oscillation with
respect to the external reference oscillation provide information about the sample's characteristics.
Probing biomolecules by AFM. The AFM can probe the differences in shape between many types of
biological molecules, such as single-stranded, double-stranded and triple-stranded DNA, and protein
channels in membranes. The AFM can also track some biological processes, such as enzymes
breaking down DNA, or fibrin clots growing. The AFM tracks these processes even though the
molecules are in aqueous solutions and are much smaller than the wavelength of light. Figure 10.12
demonstrates DNA being transcribed by the enzyme RNA polymerase. The enzyme binds to the
DNA and starts to move along the DNA after arrival of the NTP molecules. Note that the DNA
continually wiggles around. During the motion, the RNA polymerase uses the NTPs to make RNA
until it comes to the end of the DNA and falls off.
Take-home messages. Lasers have become unavoidable parts of modern microscopes to track
molecular details of life processes in the microworld. Confocal laser scanning-, fluorescence lifetime
imaging- and atomic force microscopes are robust methods for molecular imaging. Right after their
first construction, the instrumentation was highly complex and expensive (with dedicated personals)
but is becoming presently much more simpler and less expensive. These sophisticated microscopes
can be created using modest modifications of already existing instruments. The use of these
microscopes and the range of their applications are likely to expand greatly in the near future.
Home works.
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1. The donor intrinsic fluorescence lifetime is τf = 2 ns (no acceptor is present). What will be the
actual (observed) lifetime of the donor as a function of acceptor concentration? The Förster radius is
R0 = 3 nm.
2. Compare the sensitivity of the radioactivity with that of fluorescence detection!
(a) 32P has a halftime of 14.3 days. How many disintegration per second will you obtain from
1,000 atoms of 32P? What is the maximum number of counts you can get?
(b) Fluorescein has a molar absorption coefficient of 70,000 1/(M·cm) at 485 nm and a quantum
yield for fluorescence of 0.93. What is its molecular absorption coefficient (cross section) in
cm2 per molecule?
(c) 1,000 molecules of fluorescein are irradiated with an argon laser which has an intensity of 2
mW/cm2 at 485 nm. How many fluorescence photons per second will be emitted?
(d) Discuss the relative merits of detecting small number of molecules by radioactivity and by
fluorescence. What other factors besides counting rate may be important?
3. A swimmer enters a gloomier world on diving to greater depths. Given that the mean molar
absorption coefficient of sea water in the visible region is 6.2·10-5 1/(M·cm), calculate the depth at
which a divier will experience (a) half the surface intensity of light, (b) one-tenth the surface
intensity.
4. What conclusion can be drawn form a FRAP experiment if the fluorescence does not recover
completely?
References
Lakowicz JR (2006) Principles of Fluorescence Spectroscopy, Third Edition, Springer
Science+Business Media, LLC
Bastiaens PIH, Squire A. 1999. Fluorescence lifetime imaging microscopy: spatial resolution of
biochemical processes in the cell. Trends Cell Biol 9:48–52.
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Fig. 10.1 Schematic drawing of the
light path in confocal laser scanning
microscope.
Fig. 10.2 Schematic image of the
multistep surface modification used
for intact vesicles immobilization via
DNA (left). Fluorescence microscopy
image of surface-immobilized DNAtagged vesicles in the squares (right).
Fig. 10.3. Time-dependent variation
of the fluorescence intensity of green
fluorescence protein (GFP) before
and after photobleaching inside the
bacterium E. coli.
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Fig. 10.4. Schematic diagram of an
phase modulation FLIM experiment.
The "object" consists of a row of four
cuvettes each with a different
fluorophore and fluorescence
lifetime. The cuvettes are illuminated
with intensity-modulated light from
pulsed laser. The modulation
frequency was 49.53 MHz and the
(arbitrary) phase angle of the incident
light was θ1 = 241.3o. The emission is
detected with a phase-sensitive image
intensifier, which is imaged onto a
CCD camera. DMSS, 4dimethylamino-ωmethylsulfonyltrans-styrene; 9-CA,
9-cyanoanthracene; POPOP, p-bis[2(5-phenyloxazazolyl)]benzene.
Fig. 10.5. Scheme of a
TCSPC based FLIM
arrangement.
Abbreviations: APD:
avalanche photodiodes and
MCP-PMT: multichannel
plate photomultiplier tube.
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Fig. 10.6. Fluorescence imaging
of Cos cells labeled with Quin-2
using phase modulation
technique (45.53 MHz). The
Calcium concentration image
was constructed from intensity
(modulation) and phase angle
images (not shown) after
calibration.
Fig. 10.7. Fluorescnece intensity
(left) and lifetime image (right) of
bovine artery endothelial cells. The
nuclei were stained with DAPI for
DNA (blue), F-actin was stained
with Bodipy FL-phallacidin (red),
and the mitochondria were stained
with MitoTracker Red CMX Ros
(green). Two-photon excitation of
800 nm. Taken from Lakowitz 2006.
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Fig. 10.8. Activation of
lipid/calcium-dependent protein
kinase C (PKC) in Cos7 cells.
The top panels show the
intensity images of GFP-tagged
(Green Fluorescence Proteintagged) PKC. All the cells were
treated with phorbol myristoyl
acetate (PMA). The lower panels
show the GFP-lifetime images.
The central cell was injected
with Cy3.5-IgG specific for the
phosphorylated epitope of PKC.
Taken from Bastiaens and
Squire (1999).
Fig. 10.9. Laser beam deflection and
detection system of the atomic force
microscope.
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Fig. 10.10. Beam deflection system
of the atomic force microscopy using
a laser and quadrant photodector to
measure the tip position above the
sample.
Fig. 10.11. Non-contact mode of
operation of AFM. The detector
electronics measures the amplitude
and frequency of the oscillation of
the tip above the surface and the
feedback loop assures constant
frequency and amplitude of the
oscillation.
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Fig. 10.12. Transcription of DNA by the enzyme RNA polymerase.The enzyme (white spot) binds
to the DNA (thin line) After the NTP molecules arrive in the third picture on the top row, the
enzyme starts to move along the DNA . As the enzyme moves along the DNA, it uses the NTPs to
make RNA (not visible) until it comes to the end of the DNA and falls off in the bottom row of
pictures.
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