Microscopy apparatus and method for three-dimensional positioning of point-like targets in a sample

Abstract

A microscopic device for three-dimensional positioning of a point target in a sample, including a detection optical system, which images the point target arranged in the object space into the image space in the form of a three-dimensional focused light distribution; a color separation device divides the light into at least two independent beams, the wavelengths of which fall into different wavelength regions; at least two detection units, arranged in the image space, one of which receives one of the two beams and the other receives the other of the two beams, Each detection unit comprises a detection surface arranged perpendicular to the direction of incidence of the respective light beam for sensing the light spot; an evaluation unit, by evaluating the sensed light spot on the detection surface, determines the transverse direction X-Y of the point-like object in question position, and the axial Z position of the associated point-like target relative to the sharpness plane in the direction of the optical axis perpendicular to the sharpness plane; at least one Z position correction value for at least one of the two wavelength regions, stored in In the evaluation unit, the correction value represents the longitudinal chromatic aberration of the detection optical system in the wavelength region; and the evaluation unit uses the relevant Z-position correction value to correct the Z-position determined in each wavelength region.

Description

Background technique [0001] In recent years, light microscopy methods have been developed based on the continuous, random positioning of independent labels of, inter alia, fluorescent molecules, capable of revealing sample structures with dimensions smaller than the diffraction-limited resolution limit of conventional light microscopy. 这种方法描述在下述文献中,例如WO2006 / 127692A2;DE102006021317B3;WO2007 / 128434A1,US2009 / 0134342A1;DE102008024568A1;WO2008 / 091296A2;"Sub-diffraction-limitimaging bystochastic optical reconstruction microscopy(STORM)",Nature Methods3,793- 796 (2006), M.J. Rust, M. Bates, X. Zhuang; "Resolution of Lambda / 10 illumination microscopy using fast single molecule photo-switching", Geisler C. et al., Appl. Phys. A, 88, 223-226 (2007). This new branch of microscopy is also known as "positioning microscopy". Applied methods are known from the existing literature and are, for example, named (F)PALM ((fluorescence) photosensitive localization microscopy), PALMIRA (PALM acquir...

AI Technical Summary

Problems solved by technology

[0010] However, in 3D positioning, the fundamental problem is that the PSF obtained from a point-like target is symmetric about the detection plane

This means that although the PSF changes when a point-like object moves away from the intelligibility plane and thus the distance of the object from the intelligibility plane can be determined, the change in PSF is symmetrical on both sides of the intelligibility plane, so Difficulty determining which side of the sharpness plane a point target is on

[0011] Three-dimensional localization of point objects becomes more difficult when so-called multicolor measurements need to be performed, where the sample is marked with different dyes and the probe signals for sensing these different dyes must be separated

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