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What is two-photon microscopy used for?

Two-photon microscopy (also called multiphoton microscopy) can be used for live cell imaging of thick biological specimens, as it has several advantages over confocal microscopy. Molecules can be visualized deeply within the specimen with a maximal penetration depth of about 1 mm.

How much does a 2 photon microscope cost?

A base model, turnkey two-photon microscope will cost an additional $125,000 on top of the laser. “By the time you add components, you’re starting at around $225,000 [for the microscope],” says Rafter. A complete setup could cost “probably close to a half-million dollars,” Robey estimates.

What is two-photon fluorescence microscopy?

Two-photon fluorescence microscopy allows three-dimensional imaging of biological specimens in vivo. Two-photon excitation is a fluorescence process in which a fluorophore (a molecule that fluoresces) is excited by the simultaneous absorption of two photons (Figure 1).

Who invented two-photon microscopy?

Denk et al
Two-photon fluorescence microscopy (TPM), invented by Denk et al in 1990 (1), is a three-dimensional (3D) imaging technology based on the nonlinear excitation of fluorophores. TPM is considered a revolutionary development in biological imaging because of its four unique capabilities.

How does multi photon microscopy work?

In practice, two-photon excitation is generated by focusing a single pulsed laser through the microscope optics. As the laser beam is focused, the photons become more crowded (their spatial density increases), and the probability of two of them interacting simultaneously with a single fluorophore increases.

What is two-photon calcium imaging?

Two-photon calcium imaging is a powerful means for monitoring the activity of distinct neurons in brain tissue in vivo. In vivo two-photon Ca2+ recordings, obtained by imaging through the intact skull, indicated that whisker deflection-evoked Ca2+ transients occur in a subset of layer 2/3 neurons of the barrel cortex.

What is serial two-photon tomography?

Here we describe an automated method, named serial two-photon (STP) tomography, that achieves high-throughput fluorescence imaging of mouse brains by integrating two-photon microscopy and tissue sectioning.

Why does two-photon microscopy not require a pinhole?

The localization of two-photon excitation to solely the focal plane provides most of the advantages over confocal microscopy. In contrast, two-photon excitation only generates fluorescence at the focal plane, so there is no background; thus, no pinhole is required.

What is the resolution of two-photon microscopy?

STED microscopy has been done with two-photon excitation and has achieved a spatial resolution of ~60 nm in optical systems with two lasers [13–15] and a single laser [16,17]. However, the imaging depth of two-photon STED microscopy is still limited to being less than ~100 µm.

How does 2 photon calcium imaging work?

In a technique called two-photon microscopy, light-sensitive proteins are introduced into the brain cells. A laser then shines light of a specific wavelength into the brain. Whenever one of the proteins in an active brain cell absorbs some light from the laser, it gives off light that a sensor can detect.

What is calcium imaging used for?

Calcium imaging can be used to track the activity of neurons over time and investigate how networks grow or change during learning. This is especially important for the longitudinal study of animal models.

Why do we use two photon microscopy for live cells?

Why use 2-photon microscopy? For questions that require imaging live cells for extended time periods deep within tissues, two-photon microscopy is the current method of choice. Like confocal microscopy, two-photon microscopy uses a laser to excite a fluorescent tag within a sample and detectors to measure the emitted light.

How many channels can a two photon microscope image?

In addition, there is no out-of-focus light that could make the image blurry and difficult to interpret. Two photon microscopes can look deep into fluorescent samples, typically 5-20 times deeper than other types of fluorescent microscopes. Our 2-P microscope can image four simultaneous color channels at 30 frames/second.

How is two photon excitation used in laser scanning microscopy?

The considerable advantages of using two-photon excitation in laser-scanning microscopy arise from the basic physical principle that the absorption depends on the square of the excitation intensity. In practice, two-photon excitation is generated by focusing a single pulsed laser through the microscope optics.

Why are two photons required for two photon excitation?

The phenomenon of two-photon excitation arises from the simultaneous absorption of two photons in a single quantitized event. Since the energy of a photon is inversely proportional to its wavelength, the two absorbed photons must have a wavelength about twice that required for one-photon excitation.