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.
Laser scanning microscopy has become a ubiquitous tool in biology with many thousands of confocal and other types of laser-based microscopes installed in research, diagnostics and industrial laboratories. In all laser scanning microscopes, a continuous wave (CW) laser with a power of a few milliwatts is used to excite the fluorescence of markers that are either attached to specific biological structures of the sample or naturally occurring in the specimen. In addition, the diffraction-limited focusability of a TEMoo laser beam provides the ideal tool to achieve maximum resolution when combined with the confocal excitation scheme or with one of several sub-diffraction imaging methods like STED, PALM/STORM or structured illumination, collectively named also as super-resolution techniques.
Many fluorescent probes are excited by blue or green laser light and exhibits relatively wide (~ 50 nm) excitation and emission spectra. Most of these probes were developed when the only visible laser sources were ion gas lasers emitting in the blue-green region of the spectrum. The availability of solid-state or semiconductor laser emitting also at longer visible wavelengths and the wish to image different cellular structures with different colors in the same sample led to the development of many newer dyes and genetic expression that fluoresce in every part of the visible spectrum and even in the near infrared region.
While the excitation spectra of organic dyes are broad, imaging samples with many different stains requires the availability of spectral filtering, multi-channel detection and deconvolution techniques on the detection side, and of laser wavelengths matching the excitation peak of a stain, or able to excite multiple stains at the same time.
For this reason, lasers for laser scanning microscopy are available in many wavelengths with the same form/fit/function to simplify control and integration with the microscope. They provide powers of a few milliwatts up to a few hundreds of milliwatts to satisfy also the requirement of power-hungry super-resolution techniques like STED or PALM. Moreover, these lasers should be capable of direct modulation– to adjust power to a level that does not damage the sample and provide enough excitation, and rapidly – to blank the beam or for time-resolved imaging methods, like FLIM (Fluorescence Lifetime Imaging). The optical noise of these lasers should also be very low under any output power level to guarantee uniform image illumination, especially when used for rapid sample scanning (resonant galvos).
Coherent offers many family of CW lasers especially designed for confocal laser microscopy – OBIS, OBIS Core and their integration module OBIS Galaxy and Cell-X, Genesis and Sapphire. These families of lasers are based either on laser diodes or on Coherent patented and exclusive Optically Pumped Semiconductor Lasers (OPSLs). All these families are designed to satisfy each of the requirements previously described. OPSLs offers also the exclusive advantages of invariant beam parameters as the output power is changed from minimum to maximum, power scalability up to watt level and above, and availability of a virtually unlimited selection of wavelengths by tailoring the design of the active medium semiconductor chip.
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