Multidimensional single molecule TIRF microscopy
29 Jan 2010







For more information please contact: Dr. Chris Tynan.

​Instrument and technique summary

​​Technique and applications
Total Internal Reflection (SM-TIRF) Microscopy Widefield multidimensional fluorescence imaging where signal is limited to 100 nm layer adjacent to coverslip.

Biological and medical research, material science, chemistry

Lasers: 405, 488, 532, 561, 642 nm or white-light excitation. Three-colour simultaneous imaging.

Detectors: Andor iXon EMCCDs.

Objectives: 100x TIRF oil immersion.

Extra information: Excitation and emission polarisation resolvable.

Single molecule fluorescence sensitivity is achieved by using Total Internal Reflection Fluorescence (TIRF) or High Inclination illumination to restrict background fluorescence, using extremely sensitive cooled EMCCDs and judicious sample preparation to minimise non-specific probe binding and molecular crowding in the sample.

Single molecule fluorescence imaging allows a target to be studied without ensemble averaging of its properties. It can be applied to a range of commercially available fluorescent dyes, commonly used fluorescent proteins and quantum dots and applied to a wide range of sample types. It is particularly useful for investigating biological systems and OCTOPUS single molecule microscopes have been heavily utilised for studying the behaviour of membrane proteins in cultured cells.

OCTOPUS single molecule microscopes are flexible, multidimensional instruments. This means that they can be adapted for use with any technique that requires multiple image channels resolved by fluorescence wavelength or polarisation.


Multi-channel TIRF image of HeLa cells

Technical specification

Imaging modes
Epi-fluorescence, Total Internal Reflection Fluorescence (TIRF)

Illumination/fluorescence excitation sources
405nm, 488nm, 561nm, 640nm continuous wave excitation

Available channels and detectors
1 - 6 channels split by wavelength. Andor cooled EMCCDs. Maximum frame rate 50 Hz (faster frame rates are possible by reducing the effective chip size).

Techniques available

Multicolour TIRF microscopy, Single Particle Tracking (SPT), Fluorescence Localisation Imaging with Photobleaching (FLImP), Single Molecule Förster Resonance Energy Transfer (SM-FRET), combined wavelength and polarisation resolved single molecule imaging.

Single molecule tracks reveal co-localisation of fluorescently labelled features

Sample environment

Samples can created in glass bottom dishes or mounted on coverslips. Biological samples can be live or chemically fixed. Temperature control is available in the range 18-40°C.

Case studies

Contact: Tynan, Christopher (STFC,RAL,CLF)