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  • Multiphoton Microscopy: Ready for Prime Time?

Jan 2010
Gary Boas, News Editor,

Having spent much of its admittedly short life in relative obscurity, multiphoton microscopy has, over the past decade, moved out of the shadows and into the mainstream of biological research. This is due in large part to the introduction of turnkey ultrafast laser systems that have put the technique in the hands of researchers in a variety of disciplines. Taking advantage of this newfound ease of use – not to mention the inherent advantages of the method – investigators are making great strides in neuroscience, immunology and cancer research, among other areas.

Researchers have developed low-cost, single-diode-pumped lasers – for example, the Cr:LiSAF shown here – for use with multiphoton microscopy. The total cost of materials for the lasers is less than $10,000, compared with $150,000 or more for the Ti:sapphire lasers conventionally used with the technique.

The final barrier to widespread implementation might just be cost: The lasers used in multiphoton microscopy systems cost $200,000 more than a standard confocal microscope, with the systems themselves costing significantly more. The prices could drop considerably, however, with a new generation of low-cost diode-pumped lasers developed for multiphoton microscopy, and this could encourage adoption on a much larger scale.

Turnkey systems lead to increased productivity

Laser systems such as Coherent Inc.’s Chameleon and Newport Corp. Spectra-Physics’ Mai Tai DeepSee have introduced a significant degree of automation to multiphoton microscopy, thus reducing considerably the hassle associated with performing multiphoton experiments.

To be sure, the publication rate has increased dramatically since these systems came onto the scene. This reflects two general trends, said David W. Piston, a professor of molecular physiology and biophysics at Vanderbilt University in Nashville, Tenn. First, researchers who came onboard with earlier generations of multiphoton microscopy systems have doubled and tripled their productivity with the latest generation. At the same time, other investigators in multi-user facilities, for example, are getting into the game – having witnessed just how accessible the systems have become.

Accessible in certain ways, at least. Although multiphoton microscopy systems have become much more user-friendly in recent years, the cost has barely budged. “The performance has gone up like crazy,” Piston said, “but the price has not changed in twenty years.” This is in large part because they now offer additional features. He suggests that there is a certain psychology behind the inclusion of such features. “People know that multiphoton microscopy systems cost $200,000 more than a standard confocal microscope, and that’s what they’re willing to pay, and the companies give them as much as they can for that price.”

Now, however, researchers are designing low-cost lasers for use with multiphoton microscopy. At MIT in Cambridge, a group has built Cr3+:colquiriite lasers pumped by single-mode laser diodes that cost only $150 each. When these diodes are used as a pump source, the total cost of materials for the lasers amounts to less than $10,000.

Franz X. Kaertner, a senior investigator with the MIT team, said, “Ti:sapphire lasers are very wide, and broadband-tunable and powerful, but they need expensive pump sources. So we have always been interested in looking for alternative laser materials that can be pumped directly with diodes. Chromium lithium fluoride is one such material.”

The researchers have described the lasers in a series of Optics Express and Optics Letters papers, noting that the low cost and high efficiency of the technology recommend it for a variety of applications in nonlinear optics, pump probe spectroscopy and amplifier seeding, and multiphoton microscopy. Also, because neither the diodes nor the laser crystals requires water cooling and the diodes can run off batteries, the lasers are compact and offer low power consumption. Finally, the semiconductor saturable absorber structure used facilitates turnkey mode-locked operation.

The lasers do not offer all the advantages of the Ti:sapphire types typically used in multiphoton microscopy. They do not have the same wavelength range, for instance. Whereas Ti:sapphire lasers exhibit a range of approximately 680 to 1030 nm, the diode-pumped Cr3+:colquiriite lasers can go down to only about 800 nm. “This is important for biophotonics applications,” Kaertner said, “because some chromophores are only accessible in that lower range.”

Also, the lasers are not as tunable as Ti:sapphire ones. The investigators have thus far demonstrated tunability over 30 nm, compared with more than 200 nm for Ti:sapphire lasers. Kaertner noted that they are seeking to achieve tunability over more than 100 nm, adding that the femtosecond pulse generation requires further development. “We haven’t yet pushed for the shortest pulses.”

Even with the current specs, however, the lasers could offer a less expensive alternative to Ti:sapphire lasers for a range of multiphoton microscopy applications, with the lower costs helping to expand the user base for the technique. Piston believes that eventually we will see commercial systems incorporating a series of $10,000 diode-pumped lasers rather than the single, $150,000 lasers currently in use. “There will be a day, and that day might be very soon.”

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