What is the benefit of two-photon microscopy?

What is the benefit of two-photon microscopy?

The principal advantages of two-photon microscopy are reduced phototoxicity, increased imaging depth, and the ability to initiate highly localized photochemistry in thick samples.

How does a two-photon microscopy work?

When using two-photon microscopy, two or three photons of a higher wavelength do the work of one: When they hit the fluorophore at the very same time (typically within several femtoseconds), they are absorbed, resulting in fluorophore excitation and emission of light.

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 serial two-photon tomography?

STP tomography generates high-resolution datasets that are free of distortions and can be readily warped in three dimensions, for example, for comparing multiple anatomical tracings. This method opens the door to routine systematic studies of neuroanatomy in mouse models of human brain disorders.

What is Vivo 2 photon imaging?

Abstract. 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 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 in vivo calcium imaging work?

Calcium imaging measures changes in intracellular calcium concentrations, providing an indirect indicator of neural activity. Compared to changes in voltage, fluctuations in calcium levels are much slower and may reflect a summation of signals rather than individual spikes (Wei et al. 2019).

What is two-photon luminescence?

Two-photon fluorescence microscopy allows three-dimensional imaging of biological specimens in vivo. Compared with confocal microscopy, it offers the advantages of deeper tissue penetration and less photodamage but has the disadvantage of slightly lower resolution.

How do you measure calcium levels in cells?

Changes in intracellular calcium concentration can be measured using the calcium sensitive fluorescent ratiometric dye fura-2 AM. This method is a high throughput way to measure agonist mediated calcium responses.

How does Fura 2 work?

Fura-2 has the advantage that its excitation spectrum shifts upon the binding of calcium. This property enables measurement of [Ca 2+]i independent of the intracellular concentration of the dye by using the ratio of fluorescence intensities measured at two wavelengths (“ratio method”) (Grynkiewicz et al., 1985).

What is a calcium flux assay?

A calcium flux assay is a fluorescence-based assay used to detect intracellular calcium mobilization in cells and follow the release of Ca2+ into the cytoplasm. It involves preloading cells with a calciumsensitive fluorescent dye that is capable of crossing the cell membrane.

What is a calcium assay?

Calcium Assay Kit (Colorimetric) ab102505 provides a simple assay to determine calcium concentration within the physiological range of 0.4 – 100 mg/dL (0.1 – 25 mM). In the calcium assay protocol, a chromogenic complex is formed between calcium ions and 0-cresolphthalein. The complex is measured at OD = 575 nm.

What Fura-2 ratio?

Fura-2 is a ratiometric and sensitive indicator dye for measuring intracellular calcium. Since its introduction in 1985, fura-2 has been cited in thousands of papers that describe its applications in a wide variety of cells.

What is fluorescence ratio?

Fluorescence ratio imaging is a spectroscopic technique and is usable for any technique that can be expressed by the relationship of signals at two or more wavelengths.

What are ratiometric probes?

Ratiometric fluorescence is the method where intensities at two or more wavelengths of an excitation or emission spectrum are measured to detect changes to local environment. Typically, a probe is used that is specifically sensitive to an environmental parameter such as ion concentration, pH, viscosity, or polarity.