We show that the position of single molecules in all three spatial dimensions can be estimated alongside its emission color by diffractive optics based design of the Point Spread Function (PSF). The phase in a plane conjugate to the aperture stop of the objective lens is modified by a diffractive structure that splits the spot on the camera into closely spaced diffraction orders. The distance between and the size of these sub-spots are a measure of the emission color. Estimation of the axial position is enabled by imprinting aberrations such as astigmatism and defocus onto the orders. The overall spot shape is fitted with a fully vectorial PSF model. Proof-of-principle experiments on quantum dots indicate that a spectral precision of 10 to 20 nm, an axial localization precision of 25 to 50 nm, and a lateral localization precision of 10 to 30 nm can be achieved over a 1 μm range of axial positions for on average 800 signal photons and 17 background photons/pixel. The method appears to be rather sensitive to PSF model errors such as aberrations, giving in particular rise to biases in the fitted wavelength of up to 15 nm.
- Fluorescence microscopy
- Image formation theory