Abstract
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.
Highlights
The resolution in single molecule localization microscopy based on widefield fluorescence imaging [1,2,3,4] is limited by the localization precision and the density of fluorescent labels [5] and can be on the order of several tens of nanometers in practice
The study of molecular interaction and function necessitates the detection of multiple molecular species, which is conventionally done by labeling the target molecules with fluorophores that differ in their emission color
We have proposed a new method for the simultaneous measurement of the 3Dposition and the emission wavelength of single emitters
Summary
The resolution in single molecule localization microscopy based on widefield fluorescence imaging [1,2,3,4] is limited by the localization precision and the density of fluorescent labels [5] and can be on the order of several tens of nanometers in practice. These are variants of the astigmatic method that use higher order astigmatism for splitting the astigmatic focal lines into halter shaped spot pairs, which improves precision and axial range Based on these developments we set out to explore if 3D spatial and color information can be encoded simultaneously on a single camera. The shape of the diffractive zones is no longer straight as in a conventional grating but instead curved so as to generate a desired aberration (astigmatism, defocus) in the different diffraction orders This enables the detection of the emission color from the distance and relative magnitude of the sub-spots and of the axial position from the shape of and the shape differences between the sub-spots.
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