Scientific lasers are in the foreground as an applications market but, in the lab, they must deliver the functionality, reliability and ease of use to remain in the background as tools. Laser manufacturers take note: A nonscientific survey indicates that scientists want a highly tunable turnkey laser with configurable pulse widths and repetition rates. It should be easily mode-locked, have good shot-to-shot and long-term stability, be ready to use with the flip of a switch and cost little more than an optical microscope. “Yes, I want a laser that puts out all wavelengths and that operates from continuous-wave to attosecond pulses and that costs next to nothing,” said Richard Zare, director and eponym of the Zarelab at Stanford University in Stanford, Calif. And yes, he was speaking tongue-in-cheek, but he was much more sincere when he said, “Without lasers, most of my research wouldn’t happen.” That applies to research beyond Stanford, too, limited though lasers are when compared with Zare’s ideal. In fact, scientific laser markets fared well while other revenue streams were drying up for technology vendors. Several suppliers reported that sales of these devices had remained strong while other laser products had declined. Coherent Inc. in Santa Clara, Calif., reported that its scientific laser sales actually increased 20 percent amid an otherwise sluggish laser market. Driving those sales were academic researchers as well as analytical instrument manufacturers and integrators, such as Becton Dickinson and Jobin Yvon Horiba. Spectroscopy — including pump probe, fluorescence, Raman and anti-Stokes Raman techniques — remains the most prevalent application, but the hottest growth has been in multiphoton excitation, a field that one laser supplier described as halfway between a commercial and a scientific market. “The more exposure end users get to multiphoton techniques, the bigger the market will get,” said Paul Ginouves, business development manager for Coherent. “Multiphoton excitation is in the same kind of place that flow cytometry was 20 years ago.” Although still in an embryonic stage, multiphoton research is evolving into a hybrid field that encompasses physics, chemistry and biology. “So researchers, although they’re not laser jockeys, want to get the maximum functionality when they plunk down $250,000,” Ginouves said. “That drives them toward ultrafast technology because these lasers have the broadest tunability and functionality.” Typically, applications drive the research. Ultrafast Ti:sapphire lasers, however, continue to enable new and unanticipated experiments. Courtesy of Coherent Inc. But functionality does not equal complexity. Most multiphoton users are biologists and chemists who prefer to focus on the experiment rather than on its tools. “Physicists don’t mind as much because they have fun with lasers and they don’t mind tweaking them once and a while, but chemists and biologists expect more ease of use,” said Arndt Kruger, director of marketing for the industrial and scientific business unit of Spectra-Physics in Mountain View, Calif. Lasers on the brain When Marvin Minsky developed the first confocal microscope in the 1950s, he speculated on its potential for mapping interconnections of live brain cells. The application never coalesced, in part because the light sources available at the time were noncoherent and comparatively dim, which prolonged scanning times. Instead, confocal microscopy waited nearly 40 years for lasers that were compact and powerful enough to make the technique practical. In the relatively short time since then, multiphoton microscopy — evolved from confocal techniques — has realized Minsky’s dream. Both multiphoton and confocal microscopes rely on laser light to excite fluorescence in a sample, but multiphoton technology uses two or more infrared photons instead of confocal’s single visible or ultraviolet photon. Also, infrared radiation not only penetrates deeper into tissue, but also incurs less damage to cells and tissue outside of the focal plane, which enables extended three-dimensional imaging of living tissue. Multiphoton — specifically two-photon — techniques underlie neurobiologist Steve Potter’s attempts over the last three years to teach rat neurons a lesson. Potter affixes neurons taken from embryos into electrode-embedded petri dishes that maintain two-way communication between the cells and a computer. He hopes that if he can coax his simulated animals — called “animats” — to learn, he can make movies demonstrating how brain cells make connections that enable learning. The plot of his movies will unfold under a multiphoton microscope driven by a Ti:sapphire laser. A Ti:sapphire emitting 150-fs pulses at 850 nm enabled a multiphoton microscope to capture this image 100 to 150 μm inside a mouse’s lung and allowed biologist A.M. Gurney to examine samples. Confocal scanning microscopy, however, can reach depths of only about 40 μm. Courtesy of Strathclyde University. Within the context of his work, Potter regards Ti:sapphire lasers from Coherent and Spectra-Physics to be interchangeable, but his primary criterion for selecting the laser is how many milliwatts of power it can provide at various wavelengths. Hence, when he recently moved his research from the California Institute of Technology in Pasadena to the Georgia Institute of Technology in Atlanta, he obtained a 10-W Nd:YAG laser to pump his Ti:sapphire. The additional power will help the ultrafast laser tune from 700 to 1000 nm. The more wavelengths he can elicit from his system, the more fluorophores he can apply. In fact, laser selection for most fluorescence-based research stems from the fluorophores needed to “see the biology,” as one researcher put it. The fluorophore determines the wavelength of the excitation source and, to some extent, the power required. The biology determines the fluorophore, and the fluorophore determines the laser wavelength. Tunability, therefore, is crucial, particularly when imaging with two or more fluorophores, such as when Karen McCloskey captured this multiphoton image of an interstitial cell from a guinea-pig bladder labeled with actin (green) and DAPI (blue). Multiphoton excitation can also enable biologists to obtain two or more fluorescent signals with one measurementbecause it doesn’t bleach out a sample’s fluorophores with one shot. Courtesy of Strathclyde University. For instance, to image calcium within sample cells, you would probably look for a source emitting 800 to 820 nm in 100- to 200-fs pulses, said John Girkin, associate director of the Institute of Photonics at Strathclyde University in Glasgow, UK. However, he added, another researcher in the same lab might be capturing images with green fluorescent proteins, which would require the source to operate at around 920 nm as well. Their tunability and pulse widths, therefore, make Ti:sapphire lasers suitable for a broader range of applications. It also makes them more expensive. Girkin purchased four multiphoton microscopes from Bio-Rad Microscience in Hemel Hempstead, UK, in the last 18 months. The systems are based on Ti:sapphires that, by themselves, cost in the neighborhood of $100,000. Consequently, Strathclyde’s institute is working on a new generation of sources that are not tunable but that operate at one wavelength with a low component cost — about $5000. “This means that a personal multiphoton imaging system is possible,” Girkin said. “If one then wished to look at other samples later, one would just purchase a second laser source at 920 nm.” Such sources provide a tuning range of only about 30 nm. But by gradually adding systems, full spectral coverage would become available and cost-effective in comparison with a single Ti:sapphire. At the University of Virginia’s Keck Center for Cellular Imaging in Charlottesville, Ammasi Periasamy is writing grant proposals to obtain more multiphoton systems. In the last four months, he has added a Ti:sapphire laser to his lab’s arsenal of two. Integrated into multiphoton microscopes, the lasers enable his group to track the movement of cells in frog embryos. The researchers are also studying calcium emission in neurons, which could help them get a better handle on Alzheimer’s disease. Higher pump powers increase the tuning range of Ti:sapphires and the options for available fluorophores. In the case of this fluorescent resonance energy transfer image, a 10-W pump enabled a Ti:sapphire laser tuned to 820 nm to excite the donor molecules and, tuned to 950 nm, to excite the acceptor molecules. The images show a three-dimensional reconstruction of C/EBPα proteins expressed with CFP-dsRED in GHFT1-5 living cell nucleus. Courtesy of the University of Virginia’s Keck Center for Cellular Imaging. Parallel to this research, the group is monitoring several analytes together to compare their functional activity. Labeling and imaging three probes simultaneously is possible with confocal scanning microscopy, but it would require three measurements. Multiphoton excitation enables Periasamy to obtain all three signals in one measurement because it doesn’t bleach out a sample’s fluorophores with one shot. Unfortunately, there is no solution as simple as this for reducing the demand for more multiphoton instrumentation. “Once you set up a system for a particular experiment, you can’t use it for something else,” Periasamy explained. Ultrafast CARS Where multiphoton fluorescence studies tie up one laser, coherent anti-Stokes Raman scattering — better known as CARS — ties up two or more. And that’s just the Ti:sapphire instruments. The technique tunes the frequency difference of two or more laser beams to match a molecular resonance within the sample. When excited by laser pulses, targeted molecules emit intense Raman scattering toward the blue range of the spectrum (i.e., clear from the excitation signal). Coherent anti-Stokes Raman spectroscopy helped determine whether these spherical vesicles in mice hepatocytes were composed of water or lipids. The technique tunes two ultrafast lasers to correspond with a target molecule’s vibrational frequency. This eliminates the need for fluorophores but requires pulse widths on the order of 1 or 2 ps. Courtesy of Pacific Northwest National Laboratory. The technique provides an alternative for live-cell imaging without the use of fluorescent labels, but it exchanges the single laser and complex detection system of Raman and infrared-based microscopes for a complex laser system and a simple single-channel detector. “As you increase the number of lasers, the probability of getting everything working together at the same time becomes more and more remote,” said Gary Holtom, a chief scientist at Pacific Northwest National Laboratory in Richland, Wash. Holtom’s coherent Raman setup includes two Ti:sapphire lasers — an oscillator and an amplifier — an argon-ion laser pump and an optical parametric amplifier (OPA). Add an array of beamsplitters, neutral density wheels, bandpass filters and other components, and you begin to understand Holtom’s observation: “You have to have a commitment to these lasers to own one. Our total commercial investment is about $400,000. This is not an experiment you undertake lightly. To make a long story short, there’s way too much stuff on the table.” Argon-ion laser technology has continued to outlive its obituaries: Many scientists are loath to put their old workhorses to pasture. But 20-mW solid-state lasers emitting at 488 nm (shown) may be followed soon by 200-mW versions, which would deliver outputs comparable with water-cooled argon-ions while diminishing the draw on power consumption and bench space. Courtesy of Coherent Inc. Like fluorophore-based applications, the tunability of Ti:sapphire lasers is an advantage in anti-Stokes Raman techniques. But unlike straightforward fluorescence microscopy, in which the fluorophore determines laser wavelength, coherent anti-Stokes Raman scattering techniques tune the laser according to the target molecule’s vibrational frequency. Also, a laser’s peak power and spectral bandwidth play a critical role. Holtom aims for the highest peak power that he can get to elicit more intense signals. But that requires condensing the pulse widths to increase their power. The problem is that, when you shorten a pulse, you expand its bandwidth, which excites signals from additional molecules in the sample. As a rule of thumb, transfer-limited pulses from a Ti:sapphire are 10 to 100 fs in width. That translates into signals with 100 to more than 1000 wave numbers, which washes out all the useful molecular information that Holtom is after. Gires-Tournois interferometers integrated in the laser cavity enable pulses anywhere from 3 to 100 ps. “So I’m in kind of a no-man’s land,” Holtom said. “One picosecond just happens to correspond to the spectral width I’d like to get to. That’s where most molecules happen to have spectral widths of 10 to 30 wave numbers.” His former colleague Sunney Xie may have found a solution to this dilemma. Now at Harvard University in Cambridge, Mass., Xie has used two synchronized 5-ps pulse trains to yield better images. The difficulty is controlling jitter. The 5-ps pulse trains tolerate no more than 0.5 ps of jitter. A good compromise, as both Holtom and Xie said, is 2-ps pulse widths, which tolerate less than 200 fs of jitter. Recently, Xie’s group collaborated with Jun Ye’s group at JILA in Boulder, Colo., on an optimal laser source for coherent anti-Stokes Raman scattering microscopy that reduces jitter between two Ti:sapphire lasers to 20 fs using specialized feedback electronics. If commercialized, it would provide the higher resolution and sensitivity that Holtom seeks. Laser chemistry By definition, science explores the frontiers of knowledge, which limits the supply of tools designed for a specific experiment. Many researchers, such as the chemists at Stanford’s Zarelab, modify existing technology to extend the boundaries of their field. Others, such as Paul Corkum at the Steacie Institute for Molecular Sciences in Ottawa, study and hone the tools themselves. The array of lasers in the Zarelab encompasses old and new instruments that range from gas and solid-state lasers to OPOs to Ti:sapphires. Among other things, lasers allow chemists to: • Prepare molecules in known vibrational-rotational energy levels. • Detect reaction products and determine their internal state energy distribution as well as the correlation of one vector property, such as angular momentum, with another vector property, such as velocity. • Photodissociate molecules and look at the photofragments. • Photoionize molecules to learn about molecular structure and the photoionization dynamics. Representative of these applications is the research of Zee Hwan Kim and collaborators at Stanford. The chemists apply an Nd:YAG-pumped pulsed-dye laser to study chemical reactions in gas-phase to understand the reaction mechanism. The dye laser emits light between 600 and 800 nm, which is doubled to UV wavelengths. Its photons induce photolysis of a simple molecule, such as chlorine, and also help probe the reaction products. The most important attribute for the pump, Kim said, is shot-to-shot and long-term stability. The spatial mode quality, however, is critical for the dye laser because an optimal Gaussian beam shape facilitates frequency-doubling the beam. “Unfortunately, to our knowledge, none of the current commercial pulsed-dye lasers are designed for this requirement,” Kim said. However, he noted that helpful alternatives exist, such as Bethune cells. Under the auspices of Canada’s National Research Council, Corkum’s group is pushing laser instrumentation toward the attosecond mark and studying molecular dynamics under the intense pulses. At high intensities, researchers can share control of molecular processes and behaviors. In fact, Corkum collaborated with researchers at Technische Universität Wien in Vienna, Austria, and the Universität Bielefeld in Bielefeld, Germany, to develop a method to probe subatomic-scale reactions, which take place on the scale of attoseconds. Corkum believes it’s only a matter of years before it will be possible to achieve pulses of 100 as. “That will give us access to electron vibrational frequencies,” he said. His work relies largely on Ti:sapphires and OPOs for their tunability, although repetition rate is an important criterion. Higher repetition rates not only help diffuse some of the intensity of ultrashort pulses, but also enable Corkum to study smaller signals. He recently bought a high-power diode-pumped Q-switched Nd:YAG laser to amplify a Ti:sapphire and increase its repetition rate. In the future, he would like to see lasers with higher energy in the infrared, where wavelengths coincide with the vibrational frequency of molecules. One of the crucial points for lasers’ success in biophotonics, said Strathclyde’s Girkin, is that optical physicists try to solve the challenge posed by the biologist: “Any compromises have to be made for the biology rather than the physics. For example, in a given situation, one might trade off optimal resolution to ensure the viability of the sample, as this is the more important biological consideration.” Girkin also echoed other researchers by calling for cheaper lasers that are easier both to use and to maintain. The primary users in most of these labs, after all, are nonphysics students and researchers. “When students are first using the laser, they spend most of their time tweaking it,” Potter said. “Once they get good at it, they’re spending maybe 10 percent. The learning curve takes a couple of weeks.” But even seasoned users would rather spend their time on the science than on its tools. “Right now my [regenerator] is giving me ulcers,” said Holtom. “I typically go in and turn on the laser, go get my coffee and read the mail. If you’re lucky, after 15 minutes of real work, you’re ready to start your experiment.” Underlying appeal Cost, maintenance and ease of use all underlie the appeal of diode and diode-pumped lasers — not only for researchers, but for instrument manufacturers as well. John Gilchrist, director of Jobin Yvon’s optical spectroscopy division in Edison, N.J., is particularly interested in diode advances. “If their power levels could be made big enough, diode lasers could be the future,” he said. “Another problem is that, today, they’re not tunable.” Diode lasers have a particularly promising future as pumps for solid-state lasers. Although most researchers interviewed have used argon-ion lasers, they also tend to replace them whenever possible with diode-pumped solid-state lasers. Corkum’s lab, for example, is supplanting all of its argon-ion lasers with solid-state pumps because, he explained, “They have a little less noise and [cause] a little less headache if they don’t work.” In Georgia, Potter described his argon-ion as a “boat anchor,” but he would like to see more power from diode-pumped solid-state sources. Favor for these lasers increased with the appearance of 20-mW Nd:YAG lasers emitting at 488 nm, where they compete with air-cooled argon-ions. Although they cost more, solid-state sources eliminate the expense of water cooling and high power consumption. As the market activity shows, the capabilities and improvements that lasers lend scientific research outweigh the difficulties they introduce. “You have to find a technology that works, even if it’s expensive, but you also have to show that the information that comes from it is compelling,” Holtom said.