Multi-photon excitation microscopy for advanced biomedical imaging

Abstract

Fluorescence microscopy (FM) is a technique traditionally used for determining biological structures [33]; its basic concept is summarised in Figure 1a. The biological specimen under examination is labelled with one or more fluorescent probes before being placed in the microscope. A single photon from the light source (usually a Hg lamp) has sufficient energy to excite an electron in the fluorescence moiety of the specimen-bound probe, taking it from an un-excited 'ground' state to an excited state. The excited electron subsequently decays back to its ground state and in so doing loses energy in the form of an emitted photon that has a longer wavelength than the photon from the light source. This phenomenon is known as the Stokes shift. The objective of FM is to detect only probe-emitted photons, leaving wavelengths originating from the light source invisible. An excitation filter is placed in the light's pathway to ensure the specimen is illuminated only by light with a wavelength-band that will optimally excite the specific specimen-bound probe. The microscope also contains an emission filter and a dichroic mirror that reflects the excitation light away from the filter but allows emitted photons of a specific wavelength to pass through to the detector. Specific filter settings are used when specimens have been stained with multiple probes, for instance for co-localisation of molecules of interest. To establish the precise location of a bound probe in a specimen, fluorescent images can be recorded in combination with (contrast) bright field images (Figure 2). To image live cells an inverted fluorescence microscope is used with the sample placed in a cell chamber, and cells are adhered to a glass rather than a plastic support because of the superior optical properties of glass and its lack of autofluorescence.