A method for a multiple wavelength total internal reflection fluorescence 'TIRF' microscopy system comprising an objective, the method comprising: receiving, at a dispersion unit of the microscopy system, illumination light having at least a first wavelength λ 1 and a second wavelength λ 2, where λ 1 ≠ λ 2, wherein the dispersion unit comprises a first optical flat and a second optical flat mounted on a rotatable plate such that there is a non-zero angle between a normal of the first optical flat and a normal of the second optical flat, and wherein one optical flat is made of a high-dispersion optical material and the other optical flat is made of a low-dispersion optical material; splitting, at the dispersion unit, the illumination light into a first monochromatic beam having the first wavelength λ 1 and a second monochromatic beam having the second wavelength λ 2, the illumination light passing through the first optical flat before passing through the second optical flat; focusing the first monochromatic beam and the second monochromatic beam onto a back focal plane of the objective, near an outer edge of the objective, such that the first monochromatic beam is focused onto the back focal plane at a first focal point located at a first radial distance R 1 from an optical axis of the objective and the second monochromatic beam is focused onto the back focal plane at a second focal point located at a second radial distance R 2 from the optical axis of the objective, where R 1 ≠ R 2; and while maintaining a radial separation between the first focal point and the second focal point at the back focal plane, rotating the dispersion unit in order to adjust angles of incidence of the monochromatic beams onto an interface between a substrate and a sample to be imaged, thereby adjusting depths of evanescent waves within the sample to reduce a difference between the depths, wherein the angles of incidence differ from each other and are greater than the critical angle and wherein the evanescent waves are generated by the monochromatic beams, the non-zero angle, the optical materials, and a thickness of each of the first and second optical flats having been selected to minimize a shift in axial focus of the first focal point and the second focal point at the back focal plane while the dispersion unit is rotated between two angular extremes. receiving, at a dispersion unit of the microscopy system, illumination light having at least a first wavelength λ 1 and a second wavelength λ 2, where λ 1 ≠ λ 2, wherein the dispersion unit comprises a first optical flat and a second optical flat mounted on a rotatable plate such that there is a non-zero angle between a normal of the first optical flat and a normal of the second optical flat, and wherein one optical flat is made of a high-dispersion optical material and the other optical flat is made of a low-dispersion optical material; splitting, at the dispersion unit, the illumination light into a first monochromatic beam having the first wavelength λ 1 and a second monochromatic beam having the second wavelength λ 2, the illumination light passing through the first optical flat before passing through the second optical flat; focusing the first monochromatic beam and the second monochromatic beam onto a back focal plane of the objective, near an outer edge of the objective, such that the first monochromatic beam is focused onto the back focal plane at a first focal point located at a first radial distance R 1 from an optical axis of the objective and the second monochromatic beam is focused onto the back focal plane at a second focal point located at a second radial distance R 2 from the optical axis of the objective, where R 1 ≠ R 2; and while maintaining a radial separation between the first focal point and the second focal point at the back focal plane, rotating the dispersion unit in order to adjust angles of incidence of the monochromatic beams onto an interface between a substrate and a sample to be imaged, thereby adjusting depths of evanescent waves within the sample to reduce a difference between the depths, wherein the angles of incidence differ from each other and are greater than the critical angle and wherein the evanescent waves are generated by the monochromatic beams, the non-zero angle, the optical materials, and a thickness of each of the first and second optical flats having been selected to minimize a shift in axial focus of the first focal point and the second focal point at the back focal plane while the dispersion unit is rotated between two angular extremes.