We have two custom-built TIRF microscopes. One microscope, assembled in 1997, is based on a Zeiss 135 Axiovert microscope and is used primarily in the prism mode with a 1.2 N.A. 60x water-immersion objective lens. The other is an objective-based TIRF microscope and is currently under construction by Andrew Hudson and Clive Bagshaw. The microscope is based on a 1.45 NA 100x Zeiss objective lens with plane mirrors for the introduction of the excitation light. The design features a single light path with minimal components between the objective and the emCCD camera (emission filter and imaging lens), as well as options for two channel detection via two cameras and split view mode on a single camera.

Objective-based TIRF microscope under construction

Zeiss Microscope with Xe flash lamp, adapted for TIRF illumination

Laser optics for TIRF excitation

Image beam splitter for Dual View detection

Removable mirrors on magnetic mounts allow interchange between different detectors (ICCD and CCD cameras and Avalanche Photodiode photon counters) as well as a split view on a single camera for simultaneous recording of two colour or polarisation signals. The optics and electronics were assembled using commercial and custon-built components made in the Biomedical Joint Workshops.

Single molecule fluorescence of the splicing factor eGFP-U1-A (panel A) and Cy5-oligonulceotide annealed to mRNA (panel C) and FRET signal (panel B) between the labels as measured using dual view optics (collaboration with Dmitry Cherny & Ian Eperon)

Single molecule FRET between Cy3 (green) and Cy5 (red) in an RNA-DNA duplex showng Cy5 blinking (see Bagshaw & Cherny, 2006)

GFP-myosin in Hela cells observed by epifluorescence and TIRF microscopy (collaboration with Dr Marina Kriajevska)