Pharmacy and Biomedical Sciences

Total Internal Reflected Fluorescence (TIRF) Microscope

Total Internal Reflectance Fluorescence (TIRF) microscopy is similar to standard fluorescence microscopy (inverted microscope), but the sample (mounted between a microscope slide and coverslip using a particular fixative) is subjected to a laser (488 nm only) at the critical angle of reflection. This produces an evanescent wave of electromagnetic radiation at a depth of about 20 nm from the coverslip surface (nearest the objective) and so only fluorophores at the surface are activated. This allows features close to the surface to be analysed without fluorescing the whole sample. It has been particularly useful for investigating positions of receptors on cancer cell surfaces.

Our instrument consists of a Carl Zeiss Axiovert 200M inverted microscope fitted with a laser (488 nm). TIRF measurements have to be carried out using the 100x objective . The microscope can be run without TIRF, where brightfield, phase, DIC and fluorescence imaging is possible, as well as a wider range of objectives 5x - 100x. The microscope is also fitted with a microinjection facility. Images are acquired and processed using AxioVision 4.7.1 software (Carl Zeiss).

Contacts: Dr James Smith, Professor Geoff Pilkington