What is two-photon fluorescence microscopy?
Two-photon microscopy is a fluorescence imaging technique that allows the visualisation of living tissue at depths unachievable with conventional (one-photon) fluorescence or confocal microscopy.
How does two-photon excitation microscopy work?
The concept of two-photon excitation is based on the idea that two photons, of comparably lower photon energy than needed for one photon excitation, can also excite a fluorophore in one quantum event. Each photon carries approximately half the energy necessary to excite the molecule.
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?
Two-photon calcium imaging has been widely used to image the activity of neurons in awake behaving animals. Neurons are loaded with a calcium-sensitive dye or, more commonly, made to express a genetically encoded calcium indicator, such that their fluorescence signal reflects spiking activity of the neurons.
What is a 2 photon laser?
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.
What is two-photon polymerization?
Two-photon polymerization is a non-linear optical process based on the simultaneous absorption of two photons in a photosensitive material (photoresist). This process changes the photosensitive material, i.e. it leads to a polymerization by activating so-called photo-initiators in the resist.
What is the purpose of calcium imaging?
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 is calcium imaging important?
In neurons, electrical activity is always accompanied by an influx of Ca2+ ions. Thus, calcium imaging can be used to monitor the electrical activity in hundreds of neurons in cell culture or in living animals, which has made it possible to dissect the function of neuronal circuits.
How much is a two-photon microscope?
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.
What is two-photon resonance?
Two-photon absorption is one of a variety of two-photon processes. In this specific process, two photons are absorbed by a sample simultaneously. Neither photon is at resonance with the available energy states of the system, however, the combined frequency of the photons is at resonance with an energy state.
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 the mammalian brain, such imaging studies have been restricted largely to calcium recordings from neurons that were individually dye-loaded through microelectrodes.
What is two-photon absorption coefficient?
The two‐photon absorption (TPA) coefficient has been measured for a single‐mode GaAs/AlGaAs quantum well laser at 0.86 μm, near the lasing wavelength of 0.83 μm. Picosecond laser pulses were employed to resolve the ultrafast TPA from long‐lived carrier‐dependent effects.
Which enzyme is activated by calcium ions?
Though extremely short-lived, these calcium spikes lead to the activation of the enzyme calcium/calmodulin-dependent kinase II (CaMKII).
Why is ca2+ an important second messenger?
Calcium ion (Ca(2+)) plays an important role in stimulus-response reactions of cells as a second messenger. This is done by keeping cytoplasmic Ca(2+) concentration low at rest and by mobilizing Ca(2+) in response to stimulus, which in turn activates the cellular reaction.