Lasers Play a Bigger Role in Biomedical Applications

Arnd Krueger

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.

Confocal microscopy

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.

Multiphoton imaging

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 detection aperture.

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 2).

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.

Maldi-Tof

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.

Flow cytometry

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 biomedical market.

Meet the author

Arnd Krueger is director of marketing at Spectra-Physics in Mountain View, Calif.