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Life Science

Two-Photon Excitation (TPE)

Deep-tissue imaging with Two-Photon Excitation

In Two-Photon Excitation (TPE), a high power pulsed laser with very short pulse width is focused into the sample. The high photon density in the focus leads to a certain probability that a fluorophore absorbs two photons quasi simultaneously. The wavelength of the excitation laser is chosen such that the combined energy of two photons spans the gap between the ground state and first excited electronic state of the molecule. Lasers that are usually used for this application are Ti:Sa lasers with femtosecond pulse width and a tunable wavelength in the range between 690 nm and 1040 nm. At these infrared wavelengths, the excitation light is scattered only little inside the sample, and a good focal spot can be created even millimeters deep into the sample. Since the probability of excitation depends quadratically on the photon density, z-sectioning is provided by the excitation alone, and the pinhole in the detection path can be omitted.

Generalized set-up of a fluorescence lifetime imaging microscope with TPE excitationA Two-Photon Excitation (TPE) set-up is, in principle, not very different from a confocal fluorescence set-up. A collimated beam is focused by an objective, and the fluorescence is collected by the same objective and and passed on to the detection unit. Depending on the realization, scanning can be performed by sample- or objective scanning (MicroTime 200), or laser scanning (LSM Upgrade Kit). For laser scanning, descannend as well as non-descanned detection are possible. In the latter case, no pinhole is used.

Consequently the essential components of a time-resolved TPE set-up are:

  • Femtosecond pulsed laser source
  • Single photon sensitive detector
  • Dichroic mirror (to separate fluorescence signal from excitation light)
  • Objective (to focus the excitation light into the sample and collect fluorescence signal)
  • Scanning device
  • TCSPC unit to measure the time between excitation and fluorescence emission

Related technical and application notes:

PicoQuant offers several systems that can be used to perform Two-Photon Excitation:

MicroTime 200 - inverse time-resolved confocal microscopeMicroTime 200

Inverse time-resolved confocal microscope

The MicroTime 200 time-resolved fluorescence microscope system is a powerful instrument capable of Fluorecence Corelation Spectroscopy and its daughter techniques as well as Fluorescence Lifetime Imaging (FLIM) with single molecule detection sensitivity. It contains the complete optics and electronics for recording virtually all aspects of the fluorescence dynamics of microscopic samples or femtoliter volumes. The instrument gains its exceptional sensitivity and flexibility in combination with unprecedented ease-of-use from a unique fusion of miniaturized and highly sophisticated state-of-the-art technologies. Although, these technologies enable to run an instrument of comparable complexity and power without having to spend more time on instrument maintenance than on original scientific content, the MicroTime200 remains an open platform that allows the advanced scientist to easily built upon the open character of the instrument in order to realize highly customized applications

LSM Upgrade kit - add time-resolution to your laser scanning microscopeLSM Upgrade Kit

Add FLIM and FCS to a laser scanning microscope

Confocal Laser Scanning Microscopes (LSMs) are widely used tools in cell and molecular biology, biochemistry and other related sciences. PicoQuant's LSM Upgrade Kits greatly enhance the capabilities of these microscopes by extension to time-resolved techniques, and thereby providing not only Fluorescence Lifetime Imaging (FLIM) but also Fluorescence Correlation Spectroscopy (FCS) and a wealth of other time resolved techniques. The LSM Upgrade Kit combines PicoQuant products to a ready-to-use kit that fits your specific application on a state of the art Laser Scanning Microscope of your choice from Leica, Nikon, Olympus or Zeiss.

The following core components are needed to build a system capable of FLIM measurements, which are (partly) available from PicoQuant: