Total internal reflectance fluorescence (TIRF) microscopy across multiple wavelengths simultaneously

摘要

A multiple wavelength total internal reflection fluoresence 'TIRF' microscopy system has an objective (304). A dispersion unit (350,650) of the system comprises a high-dispersion optical element (354,654). The dispersion unit receives illumination light having at least a first wavelength » 1 and a second wavelength » 2 , where » 1 ‰  » 2 , and splits the illumination light into a first monochromatic beam having the first wavelength » 1 and a second monochromatic beam having the second wavelength » 2 . The monochromatic beams are focused onto a back focal plane (306) of the objective, near an outer edge of the objective, at different radial distances from an optical axis (346) of the objective. The dispersion unit is rotatable in order to adjust angles of incidence of the monochromatic beams onto an interface (316) between a substrate (312) and a sample (314) to be imaged, wherein the angles of incidence are greater than the critical angle of the interface.