Multiphoton Microscopy Comes Alive

In 1989, multiphoton microscopy (MPM) was developed as an offshoot of confocal microscopy to provide deeper imaging capabilities while causing less damage to samples. The multiphoton imaging market is expected to grow by 12% in 2009 to over $27 million. As the market matures, the largest providers of multiphoton products are addressing many of the obstacles to further adoption, such as the expense and complexity of the equipment. At the same time, multiphoton technology is advancing, allowing for the researchers to develop new applications within fields such as neuroscience, immunology and tumor research.

Although a fairly new technology, the advantages of MPM are already clear. Both confocal microscopy and MPM are based on exposing a sample to a light source, and using computerized imaging technology to detect the path and movement of the light in order to provide a detailed image of the sample’s interior. MPM is based on a targeted fluorophore-molecule simultaneously absorbing two or more long-wavelength, low-energy photons generated by a pulsed laser. In comparison to confocal microscopy, MPM imaging allows researchers to more precisely choose the sites within a sample to excite and causes less photoxicity or photobleaching above and below a targeted site. As a result, researchers are able to see deeper within a live sample while causing the sample less harm, a desirable trait when working with live or intact samples. One drawback is that, because the technique is relatively new, some researchers are still learning about MPM systems. Another drawback is that, historically, MPM systems have been packaged with confocal microscopes, making systems rather expensive.

The growing acceptance of MPM technology has paved the way for new research techniques, which have also informed how companies approach the development of new systems. These techniques include photoactivation and uncaging, which are based on the controlled stimulation of a site within a sample, causing the site to release compounds. By using photoactivation techniques, for example, researchers have mapped glutamate receptors within a cell and have naturally delivered calcium to a location within a sample through the uncaging of calcium.

The number of applications that can be done by MPM are wide ranging. “Some main applications [for MPM] are brain imaging and deep tissue imaging. Another application, within developmental biology, is the imaging of transgenic embryos,” said Dr. Bernd Saegmueller, team leader of Product Management for Leica’s confocal business. The nondestructive nature of MPM techniques also allows for improved in vivo applications. For example, immunological studies previously conducted in vitro, such as the tracking of lymphocyte behaviors, can now be conducted in vivo with multiphoton imaging. Dennis Donley, group manager of Laser Scanning Confocal Microscopes at Olympus, provided details concerning one of the larger application sectors. “Neuroscience is one of the big ones where everybody is doing research as to neuronal changes and electrophysiology changes within the brain. [Researchers] do a lot of neurology where [they] look at dendrites and research possibilities of photoactivating the soma to see what happens later on in the neuron,” he said.

The MPM market has advanced with the release of a dedicated MPM system by Carl Zeiss. The company entered the MPM market in the late 1990s, and introduced one of its major MPM products, the LSM 510, in 2001. In 2004, the company acquired Bio-Rad Laboratories’ confocal business (see IBO 5/31/04), which held the patent on a femtosecond laser developed by Cornell University. Femtosecond lasers allow for a shorter pulse-width and a higher peak-power, allowing for imaging deeper than 500 µm. After the acquisition, Leica remained Zeiss’s only competition within the market among major confocal microscopy companies. In 2005, Zeiss and Cornell licensed the femtosecond laser patent to Olympus.

Carl Zeiss’s introduction of a dedicated system is an example of an effort to increase the use of MPM systems. The LSM 7 MP, introduced in November, is a straightforward MPM system. “The way that MPM developed is that confocal microscopes were converted into multiphoton through the addition of a femtosecond-pulsed IR laser. Some researchers found it easier to transition into MPM with their confocal ‘safety wheels’ in place,” said Duncan McMillan, product marketing manager of Laser Scanning Microscopy at Carl Zeiss. “However, the technique has now matured to the extent that there are a significant number of researchers who have become very familiar with MPM and they don’t want the added expense of the components that make up the confocal part of that system.” With a lower price than combination systems, Mr. McMillan explained, the LSM 7 MP addresses one of the barriers preventing MPM from wider usage. Another advantage of providing a dedicated MPM system is that the instrument’s components have been configured specifically for MPM applications. Equipped for photoactivation or uncaging a target site, the LSM 7 MP has two scanners to allow for stimulation of a sample with two lasers of individual wavelengths while imaging.

Zeiss’s multipurpose offerings also feature advances in MPM technology. The company’s LSM 710 NLO, with MPM and confocal capabilities, which was released in February, can handle up to five non de-scanned detectors (devices that capture fluorescent light before it is picked up by the scanner). The LSM 710 NLO also includes a new optional detector. “As an alternative to the photomultiplier tube (PMT) detector, we offer a detector which is built into the body of the microscope. It’s a gallium arsenide phosphide detector (GaAsP), which is positioned very close to the back of the objective lens. It has very high quantum efficiency-somewhere between two and three times more efficient than a PMT.” Both the LSM 7 MP and the LSM 710 NLO can use the Axio Examiner microscope as a base, which is equipped with a stage designed to mount live animals.

Like Zeiss, Leica has long been a major MPM provider, but the company’s approach to the market is founded on its distinct technology. Leica’s first system in its multipurpose confocal and MPM TCS line, the TCS MP, was released in 1998. Unlike the MPM systems offered by Carl Zeiss and Olympus, Leica’s MPM systems are based on picosecond-pulsed lasers. According to Leica, the lower peak-power of picosecond lasers is more applicable to research with a higher threshold for cell damage. Leica also states that a picosecond laser is more suitable than a femtosecond laser when undertaking three-photon absorption experiments. According to Dr. Saegmueller, the ranges of applications covered by MPM instruments and the rapid pace at which new multiphoton techniques are being developed has led the company to take a broad approach to the market. “The field of MPM expands all the time because new fields of investigation are always opening up. The present fields are now going from simple imaging to the discovery of the dynamics of life.” He explained that researchers are continually discovering new applications for MPM as they experiment with the system. Dr. Saegmueller noted that the company’s scanners are what separates its MPM systems from the competition. “We have a tandem scanner. The system provides a very high speed and is able to image at video rates, say 25 frames per second at 512 x 512.” In 2005, the company introduced the TCS SP5 system, for MPM and confocal imaging. This year, the company released the TCS STED, a system based on the TCS SP5, with super-resolution capabilities for the confocal microscope.

Olympus’s increased presence within the MPM market is an indication of the field’s growth. When compared to Carl Zeiss and Leica, Olympus is a newcomer to the MPM market. The company introduced its first MPM system, the FluoView MPE-1000, a confocal and MPM system with a femtosecond pulsed laser licensed from Zeiss and Cornell University, in 2006. In November, Olympus considerably expanded its MPM product line with products based on the FluoView MPE-1000.

The new MPM systems cater to both basic MPM applications as well as more advanced research. The FluoView-1000 MPE line contains four basic systems that can accommodate up to 14 different configurations. The most basic system is the FV-1000 MPE Multiphoton Exclusive, which comes only with essential MPM capabilities. Along with MPM capabilities, the FV-1000 MPE BASIC comes with a visible laser for confocal imaging. Other systems come with two independent scanners, allowing for further IR and visible light stimulation options. With the FV-1000 MPE SIM, researchers can do visual imaging while also stimulating samples with an IR laser. While the SIM’s capabilities allow for some IR imaging, the FV-1000 MPE TWIN is the system within the line designed to IR image, while also allowing for IR stimulation. “On the TWIN, I can do the imagining with the IR and do multiple points of stimulation while I am imaging simultaneously. The TWIN goes deeper. The differences are not market specific, but application specific,” said Mr. Donley.

The direction MPM is currently headed in is quite clear: live animal imaging. “There are groups who we are familiar with who are working on using MPM to image the inherent fluorescence in human tissue. They’re trying to do that to develop real-time diagnostics for cancer. That’s the whole way biomedical research is going: it’s looking at organisms and physiology in as natural a state as possible,” said Mr. McMillan. Dr. Saegmueller’s stated “you’re going from a very precise view to an overview. And that means deep penetration, which means having the sample in contact with surrounding tissue types, and getting the dynamics of life.” Mr. Donley touched on how the derivation of MPM from confocal microscopy does not mean that they will directly compete. “I think what you’re going to see is that the technology will become more prevalent, and become as standardized a research tool as any other standardized research tool. It’s hard to judge where we are going to go from here. Every researcher we talk to has a different idea of where this concept will go.”

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