A technique that can collect high-resolution fluorescence images 500 µm or more below the surface of living tissue without causing any sample damage would be an invaluable research tool for studying cellular and subcellular processes. It turns out that nonlinear fluorescence imaging is just such a technique, and these multiphoton imaging systems are fast becoming an essential instrument in today's biomedical research laboratories.
Do these imaging systems also have a future in the clinical laboratory?
Pioneered in 1990 at Cornell University by German physicist and neurobiologist, Winfried Denk (who, together with James Strickler and Watt Webb came up with the idea of combining a laser scanner with two-photon absorption), the multiphoton fluorescence microscopy enables in-vivo high-resolution, three-dimensional fluorescence imaging of living cells and tissues.
Among its advantages are deeper tissue penetration and less cell damage than alternatives like confocal microscopy, as well as inherent 3D optical sectioning. Since two-photon fluorescence excitation occurs only at the focal point of the microscope (a sub-femtoliter volume), by scanning the focus through a sample, 2D and 3D images can be gathered.
Multiphoton imaging has an ever-expanding range of applications in biological research, ranging from stem cells, to cancer and heart studies. Examples include characterizing many aspects of renal physiology and pathophysiology in living rats and mice. Microscopy of intact neural tissue has contributed to our understanding of a broad array of neurobiological phenomena, including the functional organization of cortical maps.
Despite its obvious appeal to biomedical researchers, the cost and complexity of the ultrafast lasers required for two-photon excitation microscopy slowed its adoption and limited its use. Such laser sources can drive system costs well into six figures, beyond the reach of many laboratories.
Labeled cells in the eye of a living zebrafish, taken with an Olympus 2P system. Courtesy Roy Quinlan.
More recently though, significant advances in sources for multiphoton microscopy and "single-box" turnkey femtosecond lasers have emerged from a number of laser manufacturers. These compact, fully automated, and widely wavelength-tunable femtosecond Ti:sapphire lasers can deliver pulses as short as 70 fs with peak powers up to 450 kW over a tuning range of more than 300 nm. Such "set and forget" systems enable researchers to focus on the biology without the need to become laser experts.
At the same time, commercial multiphoton microscopy systems are being introduced by microscope manufacturers. The result has been increasingly rapid global deployment of multiphoton systems for life science research.
Despite these advances, there are still significant barriers to overcome before multiphoton microscopy can find its way into a true clinical environment.
"With patients involved, the systems must work every day, be robust to the external environment, and be easy to use by several different staff members. This is an optomechanical challenge that should not be underestimated and even with today's 'one-package' femtosecond lasers, the technology is not ready," says John Girkin, director of the Biophysical Sciences Institute at Durham University (UK) in the October issue of Laser Focus World. "Cost also needs to be driven from the instrumentation if it is to have widespread acceptance because in the current financial climateeven in the clinicthe cost-benefit of new instruments is a growing factor in purchasing decisions," he says.
For makers of lasers, optics, and microscopy equipment, multiphoton microscopy represents a notable potential market opportunity if it can successfully address clinical diagnostics.
On 24 January at SPIE Photonics West 2012, an industry panel of representatives from laser makers and microscopy companies will explore the barriers and opportunities for this technique in a clinical environment and the ability of the key players to address the needs of this marketplace.
Discussing trends and opportunities as multiphoton microscopy is implemented will be:
Stephen G. Anderson, industry and market strategist for SPIE, panel moderator
Yiwei (Kevin) Jia, marketing manager, Olympus America, Inc.
Wilhelm Kaenders, president, Toptica Photonics AG (Germany)
Karsten König, CEO, JenLab GmbH (Germany)
Arnd Krueger, Sr., director of Strategic Marketing, Newport Spectra-Physics
Have a question or comment about this article? Write to us at email@example.com.