Two-photon excitation microscopy

Are you an EPFL student looking for a semester project?

Work with us on data science and visualisation projects, and deploy your project as an app on top of GraphSearch.

Two-photon excitation microscopy (TPEF or 2PEF) is a fluorescence imaging technique that is particularly well-suited to image scattering living tissue of up to about one millimeter in thickness. Unlike traditional fluorescence microscopy, where the excitation wavelength is shorter than the emission wavelength, two-photon excitation requires simultaneous excitation by two photons with longer wavelength than the emitted light. The laser is focused onto a specific location in the tissue and scanned across the sample to sequentially produce the image. Due to the non-linearity of two-photon excitation, mainly fluorophores in the micrometer-sized focus of the laser beam are excited, which results in the spatial resolution of the image. This contrasts with confocal microscopy, where the spatial resolution is produced by the interaction of excitation focus and the confined detection with a pinhole. Two-photon excitation microscopy typically uses near-infrared (NIR) excitation light which can also excite fluorescent dyes. Using infrared light minimizes scattering in the tissue because infrared light is scattered less in typical biological tissues. Due to the multiphoton absorption, the background signal is strongly suppressed. Both effects lead to an increased penetration depth for this technique. Two-photon excitation can be a superior alternative to confocal microscopy due to its deeper tissue penetration, efficient light detection, and reduced photobleaching. Two-photon excitation employs two-photon absorption, a concept first described by Maria Goeppert Mayer (1906–1972) in her doctoral dissertation in 1931, and first observed in 1961 in a CaF2:Eu2+ crystal using laser excitation by Wolfgang Kaiser. Isaac Abella showed in 1962 in caesium vapor that two-photon excitation of single atoms is possible. Two-photon excitation fluorescence microscopy has similarities to other confocal laser microscopy techniques such as laser scanning confocal microscopy and Raman microscopy.

About this result

This page is automatically generated and may contain information that is not correct, complete, up-to-date, or relevant to your search query. The same applies to every other page on this website. Please make sure to verify the information with EPFL's official sources.

Related publications (32)

Despite a long history of research in motor control, the exact mechanism of how the brain communicates with the the invertebrate ventral nerve cord (VNC) and the vertebrate spinal cord on a single neu

EPFL2022

Diamond magnetometry makes use of fluorescent defects in diamonds to convert magnetic resonance signals into fluorescence. Because optical photons can be detected much more sensitively, this technique

WILEY-V C H VERLAG GMBH2022

High sensitivity, incoherent differential imaging

Chiara Bonati

Microscopic visualisation of optically transparent samples has been a topic of interest for several decades. Features such as density or chemical composition can influence the optical phase of transmi

EPFL2022

Related people (40)

Related units (1)

Related concepts (8)

Related courses (6)

Related lectures (43)

Related MOOCs (2)

Second-harmonic generation

Second-harmonic generation (SHG, also called frequency doubling) is a nonlinear optical process in which two photons with the same frequency interact with a nonlinear material, are "combined", and generate a new photon with twice the energy of the initial photons (equivalently, twice the frequency and half the wavelength), that conserves the coherence of the excitation. It is a special case of sum-frequency generation (2 photons), and more generally of harmonic generation.

Confocal microscopy

Confocal microscopy, most frequently confocal laser scanning microscopy (CLSM) or laser scanning confocal microscopy (LSCM), is an optical imaging technique for increasing optical resolution and contrast of a micrograph by means of using a spatial pinhole to block out-of-focus light in image formation. Capturing multiple two-dimensional images at different depths in a sample enables the reconstruction of three-dimensional structures (a process known as optical sectioning) within an object.

Two-photon excitation microscopy

Cellular Mechanisms of Brain Function

This course aims for a mechanistic description of mammalian brain function at the level of individual nerve cells and their synaptic interactions.

Cellular Mechanisms of Brain Function

This course aims for a mechanistic description of mammalian brain function at the level of individual nerve cells and their synaptic interactions.

The course starts from general discussion of the microscopy spatial resolution problem and different proposals to beat classical criteria in the field. Afterwards, modern scanning probe microscopy met

This module serves as an introduction to the area of biophotonics. The approach is multidisciplinary .The course is mainly knowledge-based but students will benefit from the skills learned by carrying

For further information, please get in contact with the instructor or have a look on the following web-site: http://biop.epfl.ch/

Explores the principles and applications of two-photon microscopy, emphasizing its advantages in deep tissue imaging and reduced phototoxicity.

Covers the concept of a photon, its emission, coherence length, and the electromagnetic spectrum.

Explores photon detection using avalanche photodiodes and position-sensitive detectors.