Fluorescence microscope

Typical components of a fluorescence microscope are a light source (xenon arc lamp or mercury-vapor lamp are common; more advanced forms are high-power LEDs and lasers), the excitation filter, the dichroic mirror (or dichroic beamsplitter), and the emission filter (see figure below).

The filters and the dichroic beamsplitter are chosen to match the spectral excitation and emission characteristics of the fluorophore used to label the specimen.

[citation needed] Fluorescence microscopy requires intense, near-monochromatic, illumination which some widespread light sources, like halogen lamps cannot provide.

[1] In the life sciences fluorescence microscopy is a powerful tool which allows the specific and sensitive staining of a specimen in order to detect the distribution of proteins or other molecules of interest.

Others are drugs, toxins, or peptides which bind specific cellular structures and have been derivatised with a fluorescent reporter.

Several techniques exist to reduce photobleaching such as the use of more robust fluorophores, by minimizing illumination, or by using photoprotective scavenger chemicals.

Furthermore, fluorescent molecules have a tendency to generate reactive chemical species when under illumination which enhances the phototoxic effect.

Computational techniques that propose to estimate the fluorescent signal from non-fluorescent images (such as brightfield) may reduce these concerns.

[8] In general, these approaches involve training a deep convolutional neural network on stained cells and then estimating the fluorescence on unstained samples.

Fluorescence microscopy is central to many techniques which aim to reach past this limit by specialized optical configurations.

[citation needed] Several improvements in microscopy techniques have been invented in the 20th century and have resulted in increased resolution and contrast to some extent.

This method and all techniques following the RESOLFT concept rely on a strong non-linear interaction between light and fluorescing molecules.

The molecules are driven strongly between distinguishable molecular states at each specific location, so that finally light can be emitted at only a small fraction of space, hence an increased resolution.

This stochastic response of molecules on the applied light corresponds also to a highly nonlinear interaction, leading to subdiffraction resolution.