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This paper provides an overview of recent advances in neural circuit imaging technologies, including the resulting technical challenges of signal processing and data analysis tools. You can read more about the 3D Acousto-Optic Scanning.  

Advances in Two-Photon Scanning and Scanless Microscopy Technologies for Functional Neural Circuit Imaging IEEE 2016 Simon R. Schultz ;  Caroline S. Copeland ;  Amanda J. Foust ;  Peter Quicke ;  Renaud Schuck

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Introduction to two-photon microscopy

Two-photon laser scanning microscopy is a fluorescence imaging technique that allows the imaging of living tissues such as brain slices, embryos, whole organs, and even entire animals up to a depth of more than one millimeter. Two-photon microscopes acquire the images by scanning the sample with a special mode-locked laser beam which is capable to produce a special kind of non-linear fluorescent excitation, the so-called two-photon effect. Two-photon microscopes are sometimes also called scanning, non-linear, or multiphoton microscopes and are often abbreviated as 2PLSM. Two-photon microscopy is in many cases a viable alternative to confocal microscopy due to its deeper tissue penetration and reduced photo toxicity. Two-photon technology was pioneered by Winfried Denk in the Watt W. Webb Laboratory of the Cornell University in 1990.

The concept of two-photon excitation is based on the idea that two photons of low energy can excite a fluorophore in the same quantum event, resulting in the emission of a fluorescence photon at a higher energy than either one of the two excitatory photons. The two photons need to be absorbed by the dye molecule at nearly the same time (within about 10 fs).

The probability of such a near-simultaneous two-photon absorption is extremely low. However, as the absorption cross section is proportional to the square of the intensity of the exciting laser, it is possible to balance the low absorption probability by using extremely high photon fluxes. Such high fluxes are only present in the focus of a high numerical aperture lens illuminated by a strong, pulsed near-infrared laser.

The optical sectioning capability of a two-photon microscope is based on the fact that excitation takes place only in the focal spot of the objective. As a result, a two-photon microscope does not need a pinhole and it is possible to redesign its optical pathway and detect an outstanding high percentage of the excited photons. This makes it possible to measure signals from small cellular compartments with a high signal-to-noise ratio over a long time.

The most commonly used fluorophores have excitation spectra in the 400–500 nm range, whereas the laser wavelength used to excite the fluorophores lies within the ~700–1000 nm (near infrared, nIR) range. If the fluorophore absorbs two infrared photons simultaneously, it will absorb enough energy to be raised into its excited state. The fluorophore will then emit a single photon with a wavelength that depends on the type of fluorophore used (typically in the visible spectrum). Because two photons need to be absorbed to excite a fluorophore, the probability for fluorescent emission from the fluorophores increases quadratically with the excitation intensity. Therefore, much more two-photon fluorescence is generated where the laser beam is tightly focused than where it is more diffuse. Effectively, fluorescence is observed in any appreciable amount in the focal volume only, resulting in a high degree of rejection of out-of-focus objects. The fluorescence from the sample is then collected by a high sensitivity detectors, such as a photomultiplier tubes. The observed light intensity gives one three-dimensional pixel in the final image. Usually, the focal point is scanned through the desired region of the sample to map all pixels of the image.

2-photon microscopy has the following main characteristics:

  • Deep penetration in the tissue because of infrared frequencies
  • Outstanding fluorescent light detection efficiency
  • Tunability for different kinds of fluorescent dyes
  • Minimal photo damage
  • Low dye concentrations enabled by the excellent signal-to-noise ratio
  • Long measurement times due to low dye concentrations
  • Enables thick slice techniques for in vivo applications
  • Costs slightly more than confocal microscopes due to the lasers used

2PLSM has several advantages over commonly used fluorescence microscopy techniques. If the average excitation power is moderate, two-photon absorption takes place only in the focus of the laser beam, providing three-dimensional resolution. Radial and axial resolutions of well below 0.5 μm and 1 μm, respectively, are achieved for typical excitation wavelengths of 800 nm by using microscope objectives with a high numerical aperture. The high axial resolution of 2PLSM is a great advantage compared to classical one-photon fluorescence microscopy, such as using a confocal set-up, where the three-dimensional spatial resolution is compromised by the lower signal intensity compared to 2PLSM. Therefore, confocal microscopy requires higher excitation intensities. This reduces the observation time of photo-vulnerable objects – like cells or biological tissues.

Using infrared light to excite fluorophores in light-scattering tissues has additional benefits. Longer wavelength light is scattered to a smaller degree than shorter wavelength light bringing a benefit to high-resolution imaging. In addition, lower energy photons are less likely to cause damage outside of the focal volume. On the other hand, there are several caveats to using two-photon microscopy: pulsed lasers are quite expensive, the microscope requires special optics to withstand the high-intensity light pulses, the two-photon absorption spectrum of a molecule may vary significantly from its one-photon counterpart, and wavelengths above 1400 nm may be significantly absorbed by the water in the living tissue.