Multi-Photon Microscope

RECOMMENDED SAMPLE SIZE : Cellular tissue blocks < 5 mm³. MAGNIFICATION: up to 180X.

The multi-photon consists of Bio-Rad Radiance2000MP visualising system, Nikon Eclipse TE300 inverted microscope and Coherent Mira900-F titanium:sapphire ultrafast laser. The available excitation spectrum is 700 – 980 nm.

In multi-photon microscope, the fluorescence excitation is produced by 2 (or more) photons of light, which individually have insufficient energy to excite the fluorescence molecule, but under certain conditions may interact co-operatively to produce enough energy to achieve fluorescent excitation. The excitation process depends on the 2 (or more) photons arriving at the point of focus within 10-16 seconds of each other. As in 1-photon fluorescence, the excited molecule relaxes to a state from which it decays back to ground state with the emission of fluorescence.

The laser is the all important feature of the multi-photon fluorescence microscope. The Ti-Sapphire laser which operates in the BioRad Radiance2000 MP system is tunable within the range 700-980 nm, producing pico-second pulses of light to achieve 2 photon excitation effect. This means that red- infra-red light can be used to excite dyes normally only excited by short wavelength uv – blue light.

For example, if a fluorochrome requires an excitation wavelength of about 360 nm (eg DAPI) in continuous light (as in conventional epi-fluorescence or confocal microscopy), it will be excited by 2-photons of approximately 720 nm wavelength infra-red (IR) light.

Under the multi-photon imaging system, the spectral distribution of excitation of fluorescent stains exhibits a plateau rather than a peak as in single photon fluorescence. This means that several stains can be visualised simultaneously. The spectral distinction between these stains is possible because of the differences in their emission peaks, which behave as in conventional fluorescence.

Multi-photon excitation has several advantages over confocal (single photon excitation) microscopy

Photobleaching is reduced because the fluorescence excitation is localised to the small focal region only, so that photobleaching does not occur in regions of the sample that have are not being imaged

Cytotoxic effects are reduced because only red or IR light is used to excite fluorescent probes which conventionally are only excited by uv or green/blue light

Deeper optical sectioning can be achieved because of the reduced scattering effects of IR light often resulting in improved sensitivity and optical sectioning.