Status Updates From Laser Spectroscopy and Lase...

The concept of localizing individual molecules with high precision is closely linked to Heisenberg's work on uncertainty in the 1930s, but its mathematical (and computational) treatment was put into a formalistic framework only more than 50 years later.

.., superresolution fluorescence techniques are classified according to the approach by which they introduce or achieve the subdiffraction limit. .., they can be grouped into two main categories: techniques that introduce the subdiffraction limit by spatially patterned excitation and techniques that go beyond the diffraction limit implementing single-molecule imaging.

The shortest visible light wavelength is at about λ = 400 nm (blue-violet light); thus, according to the Abbe limit, the smallest lateral resolution that can be achieved in a conventional microscope is d≈ 200 nm.

.., the PSF size determines the resolu-tion of the microscope, and it is well known that the FWHM of the PSF in the lateral directions x and y, i.e., perpendicular to the optical axis,..

Recently the discovery of the green fluorescent protein (GFP) and its different color protein variants ... has significantly expanded the fluorescence imaging field by the development of so-called indirect fluorescence imaging methods that are based on genetic manipulation to induce fluorescence contrast, i.e., the introduction of a gene into a cell that encodes it for a fluorescent protein (FP).

There are two main criteria for classifying fluorescence imaging methods: the first one is the size of the object to be imaged and the second one is based on the contrast mechanism employed to obtain the image.

Fluorescence imaging is the visualization of fluorescence radiation emitted at different wavelengths by molecules present in a given sample that has been illuminated by incident photons. Thus, the color image of the fluorescence radiation obtained with a sufficient resolution provides a map of fluorophore distribution within the investigated object.