Technique - (12) Two-photon fluorescence microscopy

Type: Experimental

Description: Non-linear laser excitation providing intrinsic optical sectioning for deep-tissue and in vivo imaging of labeled structures.

Department(s)/lab(s): Physics & Astronomy – AMOPP | Bain Lab (Femtosecond Laser Spectroscopy and Super-Resolution Biosensing) @ UCL
Summary:

Bain develops advanced laser spectroscopy and super-resolution microscopy techniques for biological applications. Research directions: (1) Femtosecond time-resolved STED (stimulated emission depletion) β€” combining sub-diffraction spatial resolution with picosecond time resolution to study FRET dynamics in live cells with both spatial and lifetime precision; (2) Time-resolved polarized fluorescence β€” probing orientation distributions and rotational dynamics of fluorophores; (3) CW STED fluorescence lifetime reconstruction β€” lower-photodose STED for longer live-cell imaging; (4) Single-molecule FRET to study protein-protein interactions; (5) Single-particle tracking of membrane receptors relevant to viral entry and cancer signaling. Former PhD students include SiΓ’n Culley (now King's College, SMLM).

Department(s)/lab(s): Biological Engineering | Synthetic Neurobiology Group @ MIT
Summary:

PREFERRED. Boyden co-invented optogenetics and expansion microscopy, the latter physically swelling fixed tissue in a hydrogel to achieve nanoscale-resolution imaging on conventional diffraction-limited microscopes; his Synthetic Neurobiology Group continues to push these techniques (expansion revealing, thousandfold expansion microscopy) alongside genetically encoded voltage/activity indicators and brain-wide circuit mapping. The group's Media Lab page notes it is currently accepting new students.

Department(s)/lab(s): Imaging Physics (ImPhys) | Brinks Lab @ TU Delft
Summary:

Daan Brinks develops all-optical electrophysiology tools for neuroscience. His lab engineers genetically-encoded voltage indicators (GEVIs) and combines them with optogenetics to read out and control neural circuit activity. Key directions: (1) engineering bright, fast GEVIs with improved photostability and voltage sensitivity; (2) multiplexed all-optical neural circuit mapping; (3) identifying rare aggressive cancer cells using voltage-sensitive dyes. His voltage imaging approach represents cutting-edge biosensing at the intersection of photonics and neuroscience.

Department(s)/lab(s): Physics & Astronomy – Photon Science Institute | Bioimaging and Microscopy Group (Dickinson Group) @ Manchester
Summary:

Dickinson's group develops advanced optical microscopy methods for biological and biomedical imaging. Research directions: (1) STORM super-resolution microscopy β€” stochastic optical reconstruction for nanoscale imaging of biological structures at ~20 nm lateral resolution; imaging cytoskeletal dynamics, cellular organelles, and pathological structures; (2) Optical coherence tomography (OCT) β€” depth-resolved, label-free imaging for biomedical diagnostics (retinal, cardiovascular tissues); (3) Laser speckle imaging β€” blood flow and perfusion measurements in tissues; (4) Multiphoton microscopy β€” second harmonic generation (SHG) and two-photon for collagen structure imaging in connective tissues and cancer. Part of the Manchester Photon Science Institute biophotonics theme.

Department(s)/lab(s): Physics – Laboratoire Kastler Brossel, Sorbonne UniversitΓ© | Optical Imaging in Complex Media Group (Gigan Group / LKB) @ Sorbonne
Summary:

Gigan leads the Optical Imaging group at LKB, pioneering wavefront shaping and computational imaging through scattering media. Research directions: (1) Wavefront shaping / transmission matrix β€” measuring the ~10^5 optical modes of a scattering sample's transmission matrix to focus and image through highly scattering biological tissues; roadmap on deep tissue imaging (J. Phys. Photonics 2022, lead author); (2) Multimode quantum optics through complex media β€” spatially multimode squeezed states transmitted through scattering media for quantum-enhanced imaging; (3) Optical computing / AI β€” using multiple scattering as a physical neural network for reservoir computing and nonlinear machine learning (LightOn spin-off, 2016); (4) Neurophotonics applications β€” focusing through the skull for deep brain imaging. Two ERC grants (2011, 2017). Optica Fellow. IUF member (2016–2021).

Department(s)/lab(s): Physics / LKB | PICO Group (Gigan Lab) @ ENS Paris
Summary:

Sylvain Gigan's PICO (Photonics, Information, and Complexity) group focuses on imaging through and with complex and scattering media. Research: (1) wavefront shaping through scattering media β€” adaptive optics and transmission matrix approaches for deep-tissue fluorescence imaging; (2) multimode quantum optics through complex media β€” pushing quantum light through scattering and multi-mode fibres; (3) analogue computing with random optical scattering media. Key for biosensing: deep tissue imaging at high spatial resolution and quantum-enhanced light manipulation.

Department(s)/lab(s): Imaging Physics (ImPhys) | Hoogenboom Lab (Integrated Microscopy) @ TU Delft
Summary:

Jacob Hoogenboom develops integrated correlative light and electron microscopy (CLEM) and molecular nanophotonic imaging. Research: (1) 3-in-1 microscopy combining light, electron beam, and ion beam for precise biological sample sectioning and protein localisation; (2) integrated CLEM for mapping proteins in cellular context; (3) single-molecule nanophotonic sensing using fluorescence. Relevant to advanced single-molecule biosensing approaches.

Department(s)/lab(s): Neurobiology | Kozorovitskiy Laboratory @ Northwestern
Summary:

Prof. Kozorovitskiy (Neurobiology) studies neuromodulation and plasticity in the striatum and basal ganglia, with a distinctive emphasis on developing and applying advanced optical imaging methods. Imaging technique innovations: (1) Oblique plane illumination (OPI / scanned oblique plane illumination, SOPi) microscopy β€” a single-objective light-sheet technique achieving tilt-invariant volumetric imaging for rapid 3D capture of fluorescently labeled neural structures without mechanical tilting; (2) Two-photon fluorescence imaging and two-photon glutamate/neuromodulator photorelease for single-synapse resolution in live tissue; (3) Near-infrared genetically-encoded calcium indicators (with Verkhusha group) for in vivo multi-color neural recording with reduced photobleaching. The lab's technical contributions are centered on extending the spatial and volumetric resolution of live-tissue fluorescence imaging. Irving M. Klotz Research Professor of Neurobiology; Beckman Young Investigator 2015.

Department(s)/lab(s): Physics & Astronomy – Photon Science Institute | Parkinson Group (Ultrafast Spectroscopy of Photonic Materials) @ Manchester
Summary:

Parkinson's group uses ultrafast optical spectroscopy to study carrier dynamics in photonic materials with quantum device applications. Research directions: (1) Time-resolved photoluminescence β€” TRPL with single-photon counting to map exciton lifetimes, diffusion, and defect trapping in GaN, perovskite, and 2D semiconductor quantum wells; (2) Optical single-particle spectroscopy β€” isolating single nanowires or nanocrystals for defect-free measurements of intrinsic optical properties; (3) Photon-number statistics β€” Hanbury Brown–Twiss measurements of single-photon purity from quantum dots and localized excitons; (4) Semiconductor quantum sensing interfaces β€” studying how carrier dynamics affect the fidelity of semiconductor-based quantum sensors and emitters.

Department(s)/lab(s): Biology | Schnitzer Lab @ Stanford
Summary:

Schnitzer's lab invents miniaturized and fiber-based two-photon microscopes and voltage/calcium imaging methods that allow single-cell-resolution recording of neural activity in freely behaving animals, including recent wide-field fluorescence-lifetime voltage imaging developed with the Kasevich group for high-throughput readout of neuronal spiking.