Multiphoton Microscopy Takes the Scatter Away

TWO ARE BETTER THAN ONE

MPM, also commonly known as two-photon microscopy, gets its strength from lasers that emit long-wavelength photons, which penetrate further into tissue than the shorter, visible-wavelength photons of confocal microscopy. The lasers excite fluorophores only at the focal point of the microscope, so no scatter occurs from surrounding tissue.

MPM may be a new technology, but it's based on a principle demonstrated decades ago by Nobel prize-winning physicist Maria Goeppert-Mayer: At high photon densities, a fluorophore can simultaneously absorb two photons, and the combined energy of the photons excites the fluorophore in the same way that a single, smaller-wavelength photon would. Thus, because a photon's energy is inversely proportional to its wavelength, two 720-nm photons excite a molecule just as a single 360-nm photon would.

In order to maximize the likelihood that a fluorophore will simultaneously absorb multiple photons, the photons must arrive at the same place at the same time. So high-power lasers are used to fire pulsed bundles of photons. The pulses, which range from about 100 femtoseconds (100 × 10^-15 sec.) to 1 picosecond (1 × 10^-12 sec.), give the cells respite from the heat so that they don't cook to death.

The most commonly used lasers are Ti:Sapphire mode-locked lasers (sapphire crystals embedded with titanium ions), which emit light in the near-infrared range (700–1100 nm). Other laser materials, most of which operate at even higher wavelengths, allow for even deeper penetration and three- and four-photon excitation.

BUILD, BUY, OR BORROW?

Two photons may be better than one, but they also cost more. Currently, there are two major multiphoton microscope manufacturers: Carl Zeiss (Advanced Imaging Microscopy Group) and Leica Microsystems, both in Germany. The former, as of June 1, 2004, holds the exclusive license to Webb's original, femtosecond-mode MPM technology. But even before they held the license, which was transferred from Bio-Rad Laboratories of Hercules, Calif., Zeiss was manufacturing MPM-adaptable microscopes: the LSM 510 NLO-META and LSM 510 NLO. Costing about $350,000 for a standard configuration, the microscopes are modified confocal systems able to accommodate Ti:Sapphire lasers.

Leica Microsystems manufactures three combined confocal-MPM systems, complete with lasers: the TCS SP2, TCS SP2 AOBS, and TCS SP2 RS. In contrast to Zeiss's products, the pulse durations of all three are on the order of picoseconds, not femtoseconds. But according to Scott Young, product and application specialist, the systems can readily accommodate femtosecond lasers as well.

Despite the flexibility and advantages afforded by combined confocal-MPM systems, some researchers hope the rapidly growing popularity of MPM will induce manufacturers to develop less-expensive MPM-dedicated products. As Zeiss product manager Sebastian Tille explains, when multiphoton systems are coupled with confocal systems, the user must bear the cost of the confocal technology. Confocal scopes rely on a specialized pinhole aperture through which emitted light must pass. But MPM-only scopes don't need that pinhole and could be manufactured and sold for considerably less money.

Another way to cut the cost: build your own. But, warns Tille, what looks easy on paper isn't always easy in the lab. You may be able to save yourself hundreds of thousands of dollars, but few neuroscientists have the background and knowledge to develop MPM systems or troubleshoot the technology when things go wrong.

At least the lasers are relatively simple to use. When MPM first emerged, Ti:sapphire lasers were difficult to operate. Over the last few years and partly driven by the MPM market, several companies have developed and improved easy-to-use, plug-and-play lasers. Instead of dealing with the actual laser, users interact via a more familiar computer interface. Still, with price tags as high as $200,000, cost is a problem.

Courtesy of Watt Webb

of an intact, axially oriented arteriole within a mouse lymph node. The characteristic invaginated elastin inner layer (red) lies directly under the endothelial cell lining. Collagen I and III fibrils (green) are dispersed among the smooth muscle cells and form a dense mesh surrounding the vessel, which is revealed by Second Harmonic Generation.

Coherent and Spectra-Physics, both in California, dominate the market, though there are a couple of small contenders. Coherent manufactures three MPM-suitable lasers: the turnkey, tunable Chameleon, which was designed specifically for MPM, the newer Chameleon XR, and the less expensive but more hands-on Mira. Customers who purchase a Mira usually also purchase the Verdi to serve as a pump laser (an energy source); both Chameleons have built-in pump lasers.

Spectra-Physics offers several options as well: the Mai Tai One Box Femtosecond Ti:Sapphire Laser (tunable range of 710 to 990 nm), a single box, plug-and-play product that was designed specifically for MPM users; the slightly less user-friendly Tsunami Ultrafast Ti:sapphire Laser, which requires the purchase of a second pump laser; and the OPA 800-C Ultrafast and Opal Femtosecond OPOs. The OPOs generate wavelengths in the 1100 to 1600 nm range and come coupled with Ti:Sapphire lasers, which product manager Ian Read says makes for a "really nice system." The longer wavelengths allow deeper imaging, but the lasers are more difficult to operate than the Mai Tai and Tsunami.

Switzerland-based Time-Bandwidth Products manufactures several tunable, MPM-suitable femtosecond lasers, including two Ti:Sapphire lasers (the Tiger and Pallas), two neodymium (Nd):glass lasers (GLX 200), and a new ytterbium (Yb):glass laser. Thomas Ruchti, CEO, calls the Tiger the "scientific version"; it requires more hands-on control than its turnkey counterpart, the Pallas. The GLX 200 and Yb:glass laser are high-wavelength alternatives (1-micron range) to the Ti:Sapphires.

In January 2004, Clark-MXR in Dexter, Mich., announced a Yb-doped product called the Magellan Palmtop Femtosecond Oscillator that, with its fixed 1300-nm wavelength pulses, doesn't provide the same flexibility as tunable systems but is nonetheless well-suited for long-wavelength MPM, according to CEO Bill Clark.

FLUOROPHORES DO THE FOOTWORK

Once your microscope is set up to do the work, how do you get started? Since changes in intracellular Ca²⁺ concentration reflect neuronal activity, Ca²⁺-dependent fluorophores are a popular choice for neuronal imaging. There are two types of calcium indicators: those that increase their fluorescence in the presence of calcium ion (e.g., Fluo-4, calcium green 1, calcium green 2), and those that have different excitation or emission wavelengths in the presence of Ca²⁺ (e.g., Fura-2 and Indo-1). The attached table includes a select list of Ca²⁺-indicator suppliers.

But the newest fluorophores on the block – so new that they're not commercially available – are genetically encoded Ca²⁺ indicators, also known as FCIPs (fluorescent calcium indicator proteins). As derivatives of the popular green fluorescent proteins, "many people place of lot of hope in this technology," says Rainer Freidrich, Max Planck Institute for Medical Research, Heidelberg. "Together with multiphoton microscopy, [FCIPs] will provide very new approaches."

So far, only a few groups have successfully used FCIPs to image vertebrate brain tissue; last year, Fetcho did so with his zebrafish. And in June 2004, an international team of researchers led by Winfried Denk, a codeveloper of MPM now at the Max Planck Institute, reported that it had produced the first FCIP-transgenic mice.³

FCIPs solve many problems associated with loading dyes into neural tissue, and they make it easier to target specific cell types (i.e., by using appropriate promoters). Perhaps most intriguing, Fetcho says, FCIPs pave the technological way for introducing molecules that silence or otherwise perturb molecular activity, thus providing experimenters with a powerful new way to test hypotheses about the neurology of behavior. Says Fetcho: "That's the future of systems neurobiology."

Leslie Pray lpray@the-scientist.com