Lasers Play a Bigger Role in Biomedical Applications
Laser diversity is key to widespread penetration in the fast-growing biomedical market.
The laser is now well-established as a critically enabling tool in biomedical applications
that range from research and clinical work at the molecular level to direct human
applications for medical conditions such as strokes.
These techniques employ a wide variety of laser
technologies, from humble helium-neon and air-cooled ion lasers to cutting-edge
solid-state and ultrafast lasers. Applications also include cell sorting, multiphoton
imaging, matrix-assisted laser desorption ionization time-of-flight (Maldi-Tof)
for protein analysis, fluorescence analysis in proteomics and genomics, and a novel
treatment for ischemic strokes.
Three-dimensional microscopy provides a powerful
method for probing cellular structure, physiology and biochemistry. The most widely
used three-dimensional imaging technology is the scanning confocal laser microscope.
In this type of instrument, a laser
beam is directed through an objective and focused to a small spot in the sample
that has been stained with dyes (fluorophores) specific to target molecules or a
structure of interest. Fluorescence induced in the beam waist is imaged in a confocal
arrangement onto an aperture before reaching a low-noise detector. This aperture
serves to block any light that does not originate from the focus of the laser.
A two-dimensional (X-Y) image is built
up by rastering the focused laser spot in the X-Y plane, detecting the position-dependent
sample fluorescence. The third dimension can be added by stepping the microscope
stage in the Z direction.
The choice for this instrument is the
air-cooled argon-ion laser, which delivers a few tens of milliwatts of multiline
output power in the blue/green wavelength region between 457 and 514 nm, or the
air-cooled argon/krypton mixed-gas ion laser, which delivers additional output at
568 nm (yellow). Multiline output is critical because it allows selection of the
wavelength that offers the highest excitation cross section for the multiple fluorescent
labels that are employed in cell imaging. Air-cooled ion lasers provide this flexibility
with high reliability, the requisite TEM00 beam output and low cost of ownership.
Frequently, the use of an additional red diode laser or a HeNe laser further enhances
the wavelength selectivity.
Photobleaching and low penetration
depth, caused by the high scattering of the visible light, limit the utility of
confocal microscopy in certain applications, particularly with living cells and/or
thicker tissue samples. In addition, out-of-focus emission that reaches the detector
can reduce the signal-to-noise ratio of the data.
These limitations are virtually eliminated in
multiphoton imaging, a technique that relies on the fact that an ultrafast (femtosecond
or picosecond) laser of modest average power (up to 2 W) can deliver peak powers
of several tens of kilowatts. The laser beam is tightly focused within the sample
so that it induces two-photon or even three-photon absorption at the beam waist
— resulting in fluorescence that is collected in a way similar to that used
in scanning confocal laser microscopy. These effects do not occur elsewhere along
the beam, thus providing Z-axis discrimination and eliminating the need for any
Because the amount of laser energy
that is actually absorbed is relatively low, multiphoton imaging is much more compatible
with live-cell studies than is scanning confocal laser microscopy. In addition,
penetration depth into tissue is increased because of the lower scattering of the
infrared excitation light. This technique also delivers an inherently high signal-to-noise
ratio because the laser and detection wavelengths are widely separated. Multiphoton
imaging is a large market for ultrafast lasers and was a major factor leading to
the development of fully automated “one box” wavelength-tunable ultrafast
lasers (Figure 1).
Figure 1. A multiphoton excited
image such as this bovine pulmonary endothelial cell relies on an ultrafast laser.
Courtesy of H. Aaron, University of California, Berkeley.
Since the original two- and three-photon
excitation of fluorophores, many other nonlinear optical effects have been used
to produce 3-D maps and images, including coherent anti-Stokes Raman scattering,
more familiar to some as CARS, and harmonic generation at internal cellular membranes
and interfaces. One of the newest techniques combines fluorescence resonance energy
transfer, or FRET, with either confocal or multiphoton imaging. In FRET, one chemical
species absorbs the laser light. This energy can be re-emitted as fluorescence or
transferred to another type of molecule that can fluoresce at a different wavelength.
In the pharmaceutical industry, high-throughput
screening is key to accelerating and reducing the cost of drug discovery. The effects
of many test compounds (drugs) must be screened on cellular samples and multiple
separate genome/protein pathways. At the primary screening stage, it may be necessary
to test 2 million to 3 million compounds against a single target. Automation and
miniaturization are critically important to enable this kind of work. A whole industry
now supports this effort with gene array chips and microwell plate assays. These
allow hundreds to thousands of experiments to be carried out within a single plate
or microscope slide. Examples include fast assays on the effects of test drugs
on specific gene products or on the viability and vitality of whole cells.
At present, photonics offers the only
feasible way of quickly interrogating these experiments. Depending on the application,
there is quite a diversity of plate and array readers that rely on sample fluorescence.
These analyzers tend to use either lamp excitation and CCD detection or laser-induced
fluorescence with 488-nm light. One example of the latter is the Acumen Explorer
developed by Acumen Bioscience of Melbourn, UK. The device employs an air-cooled
Spectra-Physics ion laser delivering 25 mW at 488 nm. A novel optical train enables
this instrument to raster a diffraction-limited laser spot at up to submicron resolution
over the whole well within a microplate. In a recently published study, the company
claims this instrument can screen 100,000 to 1 million compounds a week (Figure
Figure 2. High-resolution
fluorescence imaging captured neurites growing on human neuroblastoma cells stimulated
by retinoic acid. The spatial resolution is 2 μm in the Y direction and 0.5
μm in the X direction. Courtesy of Acumen Bioscience.
Another important application in protein analysis
relies on ultraviolet pulses from a nitrogen laser. A time-of-flight mass spectrometer
measures molecular masses by ionizing the molecules and accelerating them toward
a detector using an electric field. The flight time is proportional to the ion’s
charge-to-mass ratio. A challenge using this simple technique with proteins is in
creating large gasphase ions from nonvolatile materials. One of the most successful
methods is Maldi-Tof. In this technique, a solution of the protein to be analyzed
is mixed with a material such as 2,5 dihydrobenzoic acid, which absorbs ultraviolet
light. The solvent is then removed, leaving the protein dispersed through a matrix
of light-absorbing material. When the matrix is irradiated with pulses of laser
light, a small amount is vaporized and ionized, creating free protein ions. These
ions are accelerated into the time-of-flight instrument using an electric field
(Figure 3). Maldi also is used in conjunction with other types of mass spectrometry.
An instrument introduced by Thermo Finnigan of San Jose, Calif., even operates at
atmospheric pressure in a technique called AP Maldi.
Figure 3. In Maldi-Tof, a short laser pulse
releases charged proteins from the surface of a matrix. The masses of these ions
are computed from their time of flight after acceleration by an electric field.
Nitrogen lasers have owned this application
for several reasons. First, they offer a low-cost source of ultraviolet (337 nm)
laser pulses with sufficient pulse energy (typically 50 μJ) to drive the Maldi
process. Just as importantly, the short pulse duration (typically 4 ns) enables
high-resolution time-of-flight measurements.
Manufacturers have developed high-repetition-rate
(100 Hz) versions of these lasers specifically to support this application with
high data rates. Consequently, until recently, data transfer and analysis was the
rate-determining factor in this technique. But with faster computers, there has
been a growing demand to increase the laser repetition rate. Because 100 Hz is the
practical limit of the nitrogen laser at these pulse energies, Spectra-Physics has
developed a frequency-doubled Nd:YLF laser that offers pulse repetition rates of
1 kHz and beyond at a wavelength of 349 nm, well within the spectral bandwidth for
this application. Dubbed Triton, this new all-solid-state laser offers lifetimes
that are expected to exceed 10,000 hours.
In flow cytometry, cells are made to pass in single
file, either in a liquid stream or in discrete droplets, through a laser interaction
zone. The cells are pretreated with fluorescent markers that preferentially adhere
to various cell types. The resulting fluorescence is separated and detected using
one or more photomultiplier tubes, each with a specific optical bandpass filter.
The intensity and/or spectral composition of the resulting fluorescence allows the
cells to be counted, analyzed and/or sorted. In sorting, a small static charge is
applied to the cells so that they can be deflected into receptacles depending on
their fluorescent signal. Flow cytometry is widely used in research and clinical
applications, and even in agriculture and animal husbandry. Examples include counting
bacteria in milk, sperm-sex selection for dairy cattle breeding and sorting fetal
cells from maternal blood samples.
The laser is a critical component in
flow cytometry. It enables a large light flux to be coupled into a small area, producing
sufficient fluorescence intensity for instant analysis of single cells. Today the
most common laser in these instruments is the air-cooled, 488-nm argon-ion. Many
instruments also incorporate a red HeNe or diode laser, and high-end research instruments
frequently include more at other wavelengths, such as an ultraviolet water-cooled
ion laser, to enable multiparameter studies.
Air-cooled ion lasers are simple, low-cost
sources of blue light with high reliability. Several manufacturers are at various
stages of development of alternative solid-state 488-nm lasers that offer smaller
package size and lower power consumption. A number of approaches have already reached
the market — however, at a much higher cost. In the area of ultraviolet excitation,
the equation is somewhat different. Specifically, the existing source of choice
— the large-frame water-cooled ion laser — is costly to operate and
offers limited longevity. A new alternative is the frequency-tripled quasicontinuous-wave
all-solid-state laser. Although these lasers are pulsed, the repetition rate is
high enough (80 MHz) that the laser appears continuous-wave in the context of cytometry,
which samples up to 50,000 cells per second.
Recent research by scientists at the
Laboratory for Cell Analysis at the University of California, San Francisco, and
colleagues at BD Biosciences in San Jose, Calif., confirmed the suitability of
these lasers in a live-cell study that was presented at the 2002 annual meeting
of the International Society of Analytical Cytology in San Diego.
Q-switched lasers with high pulse energies
are also finding applications in biomedicine. Endovasix Inc. of Belmont, Calif.,
is involved in Food and Drug Administration trials to show the safety and efficacy
of a treatment for ischemic strokes caused by a small blood clot lodged in a cerebral
artery. Their system uses a fiber optic catheter to deliver 532-nm light from a
frequency-doubled, Q-switched Nd:YAG laser.
Rapid heating, cooling
Operating at 5 kHz, with an average power of around
3 W, the laser emulsifies the clot by cavitation, the rapid heating and cooling
caused by the laser pulses. Besides the simplicity and reliability of this technology,
the 532-nm laser is preferred because it results in maximum differential absorption
between the clot and the blood vessel walls, minimizing the possibility of damage
to the surrounding tissue (Figure 4).
Figure 4. In a system currently
undergoing FDA trials, Q-switched laser pulses at 532 nm are delivered through a
novel fiber optic catheter to emulsify the blood clot associated with an ischemic
stroke. Courtesy of Endovasix.
In conclusion, biomedical applications
use a wide range of laser technologies, including gas, solid-state and ultrafast
lasers. Each source occupies a unique area in the price/performance continuum. Thus,
the diversity of laser technology ensures continuing health in the vitally important
Meet the author
Arnd Krueger is director of marketing at Spectra-Physics in Mountain View, Calif.
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