Multiphoton microscopy techniques – including two-photon fluorescence and single-harmonic generation (SHG) – have blossomed over the more than 20 years since they first became widespread, but they continue to evolve, becoming more useful than ever to biologists. Much of the new work being performed with multiphoton techniques is additive: using adaptive optics, for example, or combining it with an adjunct methodology such as coherent anti-Stokes Raman scattering (CARS)-based spectroscopy. The following are a few examples of exciting work centered on these multimodal methods. Sorting signals Optical coherence microscopy (OCM), which offers high-lateral-resolution structural information, and multiphoton microscopy (MPM), which provides high contrast and submicron-resolution functional data, are a natural fit. The two techniques highly complement each other, but the combined systems tend to be quite large, primarily because it has been difficult to switch MPM to work with fiber lasers. Pulse broadening, weak signals and nonlinearity have plagued such attempts. Now, however, a team of researchers at the University of California, Irvine, has developed a fiber-based Fourier-domain OCM/MPM system that operates off a single laser source. The result is a compact multimodal system that could lead to clinical use of endoscopic imaging at the bedside. Figure 1. This schematic shows how researchers at the University of California, Irvine, combined optical coherence microscopy (OCM) with multiphoton microscopy (MPM) techniques. FBFL = fiber-based femtosecond laser; AMP = preamplifier; CL = collimator; G = grating; GS = galvanometer mirror; M = mirror; O = objective; PMT = photomultiplier tube; PP = prism pair; SM = single mode. Courtesy of Gangjun Liu. Led by Gangjun Liu, the Irvine team based its system on a lab-built fiber-based femtosecond laser emitting 80-fs pulses at a wavelength of 1.04 µm and a repetition rate of 76 MHz (Figure 1). Crucial to the ability to adopt a fiber laser, however, is that the system incorporates a (2+1):1 pump/signal combiner. The device features a pair of multimode fibers fused to a double-clad fiber, which effectively permits the proper light paths for the signals necessary for both OCM and MPM operation (Figure 2). Double-clad fibers typically are used in multiphoton applications because of their collection efficiency. Figure 2. This schematic illustrates the light paths through a (2+1):1 pump/signal combiner, which makes it possible for OCM and MPM techniques to use the same light source. Reused with permission of the Journal of Biomedical Optics. The collection efficiency for the MPM signal is 20 percent using this combiner, but the group reports that other combiners – with configurations up to (19+1):1 – could collect as much as half the MPM signal. The OCM and MPM paths do not at all affect one another, despite the use of a single-fiber light source. Liu and his colleagues successfully used the system to obtain OCM, MPM and second-harmonic generation images from a variety of samples, with the multimodal system providing both structural and functional information. They now are working to further reduce the size of the probe to less than 2 mm and are testing fiber lasers of various wavelengths. Figure 3. Shown are OCM images (a, c) of fixed and stained rabbit heart tissue along with single-harmonic generation (SHG) images (b, d) of a dense rat tail tendon. Image reused with permission of the Journal of Biomedical Optics. In your eye Two-photon excitation fluorescence is an excellent method for studying tissues, especially in the eye, where there is higher transparency. The technique provides the ability to see samples in sections along the Z-axis, offering insight into both healthy and diseased components at various levels within the depths of a tissue section. “Very different cells and structures appear at different depth positions,” said Juan M. Bueno of Universidad de Murcia in Spain. “It is especially interesting for basic research to analyze the ocular structures under intact conditions and visualize them with optimized contrast and resolution.” The technique, however, is particularly affected by the intrinsic wavefront aberrations in the microscope’s optics. Aberrations such as defocus and astigmatism are the most common causes of poor image quality, although defocus does not play a role in microscopy. Researchers at various institutions have tried to correct astigmatism and other aberrations using software to alter the shape of a deformable mirror or liquid crystal modulator in the optical path and have found some success with fixed and stained samples ex vivo. Until recently, however, none used a wavefront sensor to close the loop between aberrations in the emission wavelength and the corrective mirror or modulator. To bring two-photon fluorescence techniques to the realm of in vivo ocular research, Bueno and his colleagues needed to obtain the best performance from the corrective tool – in their case, a 140-element deformable mirror coated with gold for protection from the high-power laser they used. Other researchers have focused on correcting aberrations generated within samples without knowing the amount of aberration at each plane they looked at, Bueno said. Instead, they used basic algorithms to change the shape of the mirror or modulator in their systems. “In our work, we measured and compensated for the exact aberrations of the laser beam,” he said. The rest of the optics and the tissue itself cause wavelength aberrations, but the group focused on the illuminating beam. “Although this is only a portion of the total aberration of the entire system, we were able to significantly improve the quality of the images (of ocular tissues).” Bueno and his collaborators, Emilio J. Gualda and Pablo Artal, based their imaging system on a Nikon TE-2000U inverted microscope, a 760-nm Ti:sapphire laser, a Hartmann-Shack wavefront sensor made by Thorlabs Inc. of Newton, N.J., and a deformable mirror from Boston Micromachines Corp. of Cambridge, Mass. (Figure 4). Figure 4. Introducing a Hartmann-Shack wavefront sensor (HS) and deformable mirror (DM) into a multiphoton microscope system enables sharper imaging of ocular tissues. AO = adaptive optics; M1/M2 = galvanometric mirrors; PMT = photomultiplier tube. As described in the November/December 2010 issue of the Journal of Biomedical Optics, the researchers tested their system on intact human and porcine eyes as well as on fixed and nonfixed ocular tissues. They found that the corrected beam provided a better point spread function for image generation and permitted the use of lower laser power, which will be essential for future in vivo ocular studies in patients (Figure 5). “In a few years … adaptive optics multiphoton microscopy might be used in living eyes, if not for retinal imaging, at least for corneal tissue analysis,” Bueno said. Figure 5. Two-photon excitation fluorescence images of a sample of fluorescent paper are shown before (a) and after (b) adaptive optics equipment corrected wavefront aberrations in the laser beam. Scale bar = 70 μm. Reused with permission of Journal of Biomedical Optics. Next, the investigators intend to increase the depth of focus of the system by manipulating the beam aberrations. They expect that doing so will reduce the signal at the best-imaged plane but will provide higher contrast at other levels along the Z-axis, Bueno said. Under your skin Digging through layers is not just the concern of ophthalmologists. Skin also is of great concern, and research into better ways to examine skin layers in situ – no tagging, no biopsies – is supported by the pharmaceutical and cosmetic industries alike. MPM, and the closely related multiphoton tomography, are useful for imaging everything from single cells to whole animals, said Karsten König, the head of JenLab GmbH in Jena, Germany, and a researcher affiliated with both the University of California, Irvine, and Saarland University in Saarbrücken, Germany. Neither technique, however, can find nonfluorescing biomolecules such as lipids, water and hemoglobin. To get that information, König and his colleagues add CARSto the mix. “The combination of fluorescence/SHG (for collagen)/CARS is the most interesting approach to image the most important biomolecules as well as exogenous pharmaceutical substances and cosmetics,” he said. Multiphoton/CARS tomography provides information on the diffusion of materials through the stratum corneum (the outermost layer of skin), in situ pharmacokinetics and the stimulated synthesis of collagen, among other interactions between drugs and cosmetics with skin at contact and beyond, König said. In an article published March 1, 2011, in the online edition of Laser Physics Letters, König and his colleagues at JenLab and at the Berlin-based APE GmbH and Charité (the medical school for both Humboldt University and the Free University of Berlin) described the first use of a clinical CARS/MPM/SHG skin biopsy system on human patients. The group used a commercial multiphoton tomography instrument made by JenLab. The overall system is based on a near-IR femtosecond laser operating at 80 MHz combined with an optical parametric oscillator (OPO) emitting in the 1000- to 1300-nm range. CARS stimulation came from femtosecond pulses from both the laser and the OPO. The researchers kept the power below 50 mW to avoid tissue damage. They examined the skin of healthy volunteers, patients with psoriasis and people who had omega-3 oil applied to their otherwise healthy skin. Figure 6. Researchers recently reported the first CARS/MPM imaging performed on human volunteers. Here, two-photon fluorescence imaging (left), CARS (center) and an overlay of the two techniques (right) are shown. Scale bar = 40 μm. Reused with permission of Laser Physics Letters. Overall, the researchers reported that the resulting images provided detailed information on several skin factors, including tissue structure and cell morphology (Figure 6). They obtained good discrimination in psoriasis patients between nonfluorescent lipids and water, and fluorescent molecules such as coenzyme NAD(P)H, melanin, elastin and keratin. JenLab already has sold several of its multiphoton tomography systems but is working to enhance the system to detect more chemicals in the skin as well as to further shrink it for clinical use.