two-photon microscopy

two-photon microscopy
Alberto Diaspro, Paolo Bianchini, Giuseppe Vicidomini, Mario Faretta, Paola Ramo · CC BY 2.0 · source
Name	Two-photon microscopy
Caption	Schematic representation of a two-photon microscope
Inventors	Maria Goeppert Mayer; Winfried Denk; Watt W. Webb; James H. Strickler
Introduced	1990s
Field	Optical microscopy; Neurobiology; Cell biology

two-photon microscopy is a fluorescence imaging technique that uses simultaneous absorption of two photons to excite fluorophores, enabling deep tissue imaging with intrinsic optical sectioning. It is widely used in neuroscience, developmental biology, and biomedical research for live imaging of intact specimens and scattering tissues. The method combines principles from nonlinear optics, laser physics, and microscopy instrumentation to produce high-resolution, three-dimensional images.

Principles

Two-photon excitation relies on quantum-mechanical two-photon absorption originally predicted by Maria Goeppert Mayer and later applied experimentally in microscopy by teams including Winfried Denk, Watt W. Webb, and collaborators. The technique exploits simultaneous absorption of two lower-energy photons to reach an excited electronic state typically achieved by one higher-energy photon; this nonlinear process scales with the square of photon flux, enabling spatial confinement of excitation to the focal volume as in confocal methods used by researchers at Nikon Corporation and Olympus Corporation. Two-photon excitation reduces out-of-focus excitation and photobleaching compared with single-photon widefield excitation approaches developed earlier by inventors associated with Ernst Abbe and companies like Carl Zeiss AG.

The excitation uses ultrafast pulsed lasers such as mode-locked titanium-sapphire lasers pioneered by groups at Bell Labs and later commercialized by firms related to Coherent, Inc. and Spectra-Physics. The temporal confinement of femtosecond pulses produced by laser designers like Theodore Maiman and laboratories including Stanford University allows high peak intensities without excessive average power, a strategy influenced by work at Massachusetts Institute of Technology and Harvard University on laser applications in biology.

Instrumentation and Techniques

Two-photon microscopes integrate pulsed laser sources, beam delivery optomechanics, scanning systems, and sensitive detectors. Laser sources include titanium-sapphire lasers, optical parametric oscillators developed at institutions such as Bell Labs and Lawrence Berkeley National Laboratory, and compact fiber lasers engineered by companies like Yb-doped fiber manufacturers. Beam scanning is often achieved with galvanometric scanners conceptualized by industrial groups including Cambridge Technology or resonant scanners used in imaging centers at Max Planck Society sites.

Objective lenses with high numerical aperture from manufacturers such as Olympus Corporation, Carl Zeiss AG, and Nikon Corporation are essential; immersion media and specialized objectives developed in collaboration with groups at University of Oxford and Harvard Medical School extend imaging depth. Non-descanned detection schemes adopted by laboratories at Cold Spring Harbor Laboratory and University of California, Berkeley use large-area photomultiplier tubes from producers like Hamamatsu Photonics or GaAsP detectors influenced by work at Princeton University to collect scattered fluorescence efficiently.

Advanced modalities—such as three-photon excitation, fluorescence lifetime imaging pioneered at University of Illinois Urbana–Champaign, and adaptive optics developed with input from European Southern Observatory and Max Planck Institute teams—enhance penetration, contrast, and aberration correction. Multiplexed imaging strategies used in facilities at Howard Hughes Medical Institute combine spatial light modulators produced by companies like Meadowlark Optics and computational methods influenced by groups at California Institute of Technology and ETH Zurich.

Fluorophores and Contrast Mechanisms

Fluorophore selection is critical: genetically encoded indicators such as enhanced green fluorescent protein from Martin Chalfie’s legacy and red-shifted proteins engineered by labs at Howard Hughes Medical Institute and Max Planck Institute enable targeted imaging. Synthetic dyes developed in collaborations with chemists at University of Cambridge and ETH Zurich and small-molecule probes from groups at Salk Institute expand spectral options. Near-infrared probes optimized for multiphoton cross-sections were advanced by researchers at Riken and Janelia Research Campus.

Contrast mechanisms include intrinsic signals such as second-harmonic generation used in studies at Columbia University and third-harmonic generation explored at University of Munich; autofluorescence imaging derives from metabolic cofactors characterized by teams at University of Pennsylvania and Yale University. Fluorescence lifetime contrast and Förster resonance energy transfer applied by researchers at University of California, San Diego provide functional readouts of calcium, voltage, and signaling dynamics adopted in projects at Scripps Research and Massachusetts General Hospital.

Applications

Two-photon microscopy is extensively applied in neuroscience by groups at Allen Institute for Brain Science, Cold Spring Harbor Laboratory, and Max Planck Institute for Brain Research for imaging neuronal activity, dendritic spines, and synaptic plasticity. Developmental biology labs at European Molecular Biology Laboratory and University of Cambridge use it for live embryo imaging; immunology research at Dana–Farber Cancer Institute and National Institutes of Health employs it for intravital imaging of immune cell dynamics. Cancer biology centers at Memorial Sloan Kettering Cancer Center and MD Anderson Cancer Center use multiphoton approaches to study tumor microenvironments, while ophthalmology researchers at Bascom Palmer Eye Institute investigate retinal structure.

Clinical translations and in vivo imaging studies occur in collaborations involving Mayo Clinic, Johns Hopkins University School of Medicine, and biotechnology firms such as ZEISS Meditec and Leica Microsystems.

Advantages and Limitations

Advantages include deep tissue penetration and reduced photodamage emphasized in reviews by teams at National Institutes of Health and Howard Hughes Medical Institute; intrinsic optical sectioning avoids confocal pinhole losses as highlighted by engineers at Carl Zeiss AG. Limitations include high equipment cost and complexity noted by core facilities at Wellcome Trust-funded centers and restricted imaging speed compared to widefield methods developed at MIT Media Lab; scattering and absorption in highly pigmented tissues limit depth despite innovations from groups at University of California, San Francisco and University College London.

Historical Development and Key Contributors

Foundational theory was provided by Maria Goeppert Mayer, while experimental adaptation to microscopy was driven by Watt W. Webb and Winfried Denk with early implementations at Cornell University and Bell Labs. Subsequent technological advances were propelled by researchers at Harvard University, Stanford University, Max Planck Society, and commercial firms such as Coherent, Inc. and Spectra-Physics. Major contributors include optical engineers and neuroscientists from Cold Spring Harbor Laboratory, Janelia Research Campus, and Howard Hughes Medical Institute who expanded applications and instrumentation.

Safety and Practical Considerations

Safe operation requires laser safety protocols established by organizations like American National Standards Institute and institutional guidelines at National Institutes of Health and European Commission-funded facilities. Biological safety practices from Centers for Disease Control and Prevention and animal-care standards from Institutional Animal Care and Use Committee influence in vivo imaging workflows. Maintenance considerations involve service relationships with manufacturers such as Hamamatsu Photonics, Coherent, Inc., and Leica Microsystems and training programs run by core facilities at Wellcome Trust and Howard Hughes Medical Institute.

Category:Microscopy