Multiphoton excitation microscopy (MPM) was first reported in 1990 (Denk, Strickler and Webb). Since then, it has grown to become a ubiquitous imaging technique thanks to its in-vivo, in-depth optical sectioning capability. In multiphoton microscopy, a fluorescent molecule – attached to the specimen, genetically expressed or naturally present – is excited by two or more photons of infrared (IR) light. This is in contrast with confocal microscopy where the same types of molecules are excited by a single photon of visible or UV light. Multiphoton excitation offers several advantages over confocal microscopy: IR light penetrates more deeply in the tissue because of lower absorption and scattering, its longer wavelength is less damaging and the non-linear process excites fluorescence only on the focal plane so that the confocal aperture required with single-photon excitation is no more necessary.
MPM has many applications, primarily in neuroscience, diseases studies and immunology. The development of fluorescent proteins like GFP (Green Fluorescent Protein) and genetically encoded Calcium indicators (GECIs) like GCaMP enables to image structure and brain activity in model animals like mice, zebra fish and fruit flies. In mice, observation of this activity is possible across several cortical layers down to about 500 m, once the skull is replaced with an optical window. Structural imaging down to 1 mm or even beyond has also been demonstrated.
The development of newer fluorescent structural and functional probes – either synthetic or genetic expression – at different, usually longer wavelength - goes hand in hand with an expansion of the tuning range of femtosecond lasers specifically designed for biologist and neuroscientist, like Coherent Chameleon and Discovery lasers. Discovery’s octave tuning range (660 nm-1320 nm) is the broadest in the industry and addresses every existing and foreseeable fluorescent probe. Conversely, cost-effective lasers addressing specific wavelengths of broad interest (920 nm to excite GFP or GCaMP, as an example) are now available as add-on to an existing laser system or as a convenient dedicated system (Coherent Axon).
To further address the specific needs of the non-linear imaging community, some of these lasers are now equipped with application-specific features like pulse pre-chirping and – more recently – integrated, fast modulation of the output power (Coherent Discovery TPC and Axon).
Recent trends in MPM include the two-photon optogenetics studies and deeper imaging with three-photon microscopy. Two-photon optogenetic stimulation (or silencing) is used to activate or silence many arbitrarily selected neurons to simulate the effect of external stimuli (i.e. olfactive or visual). Since stimulation of a ~ 10 µm neuron does not necessarily require a diffraction-limited laser spot, new optical techniques have been developed to address many arbitrarily selected neurons at the same time. This parallel approach is made possible by spatial light modulators and requires higher laser power, to be spread on the targeted neuron population. Mode-locked lasers able to generate 1-3 watt at 80 MHz are perfectly suitable for point scanning but not ideal for this parallel approach. Fortunately, Yb fiber amplifiers (Coherent Monaco) producing tens of watts average power, tens of µJ energies and very flexible repetition rates are a perfect match for this type of optogenetic experiments.
Three-photon excitation has been shown to increase by a factor two or more the depth of functional imaging in the mouse brain and other model animals. Three-photon excitation enables to image the function in a volume that extends to the depth of the entire cortex but requires longer wavelengths (1.3 µm and 1.7 µm) and higher peak power. High peak power is required by the highly non-linear 3-photon process and is achieved increasing the energy per pulse and decreasing the pulse duration. To maintain a heat load compatible with in-vivo subjects, the average power is reduced by lowering the repetition rate. Here too, Yb amplifiers like Monaco provide the right energy and repetition rate (1-4 MHz) to pump OPAs (Opera-F) producing 50-80 fs pulses at these wavelengths.
While all these techniques can be used for neuroscience or disease studies in model animals or in any type of ex-vivo sample or culture, applications to in-vivo imaging of human subjects is not compatible with the use of markers- with a few exceptions. To acquire “information-rich” images in absence of exogenous markers, it is often necessary to use multiple non-linear techniques. In the most general cases these include two-photon excitation of endogenous fluorescence, SHG and THG imaging, and CARS/SRS imaging. This “multimodal” approach requires the availability of several wavelengths from the same laser or multiple (single-wavelength) lasers.
Coherent recognizes that microscopists, neurologists and biologists are not expected to be laser experts to do their work. For this reason, we design and manufacture an expansive line of lasers designed specifically for non-linear imaging. MPE microscopy users can therefore focus on their sample, not on the laser equipment.
Learn more about Multiphoton Excitation Microscopy by visiting our OASIS™ page.
