Method and microscope for three-dimensional resolution-enhanced microscopy

摘要

The invention relates to a high resolution microscope for three-dimensionally determining the position of objects, in particular individual fluorophores, and preferably for the high spatial resolution luminescence microscopy of a sample, which is marked with marker molecules that can be activated or switched using a signal such that they can be induced to emit certain luminescent radiation only in the activated state. The object is represented by means of an imaging system, preferably the microscope lens, on a surface detector consisting of individual detector elements. At least one microlens array is located in front of the detector elements, and different, preferably adjacent, detector elements receive light from microlenses having different focal lengths and from different object planes, or wherein by means of at least once microlens array, located in part in front of the detector elements, a different object plane is represented on the detector elements in the direction of the light behind the microlenses than on detector elements having no microlenses in front of the latter.

主权项

1. A method for high-resolution microscopy for three-dimensional determination of the position of objects, in particular individual fluorophores, for spatial high-resolution luminescence microscopy of a sample labeled with marker molecules which can be activated or switched by means of a signal in such a way that they can be excited to emit certain luminescent radiation only in the activated state, wherein the method comprises the following steps:
a) introduction of a signal onto the sample in such a way that only a subquantity of the marker molecules present in the sample are activated, wherein the sample contains subquantities in which the distance between the closest neighboring activated marker molecules is at least greater than or equal to a length resulting from a predefined optical resolution, b) excitation of the activated molecules to emit luminescent radiation, c) detection of the luminescent radiation with the predefined optical resolution, and d) generation of an individual picture from the luminescent radiation captured in step c), wherein the geometrical locations of the marker molecules emitting luminescent radiation are identified by means of a spatial resolution increased above the predetermined optical resolution, wherein the steps are repeated several times and the plurality of individual pictures obtained in this fashion are combined into a complete image, and wherein an object is imaged onto a surface detector consisting of individual detector elements by means of an imaging system, preferably the microscope lens, wherein a different object plane is imaged onto the detector element in the direction of light behind the microlenses than onto the detector element in front of which no microlenses are located through at least one microlens array which is partly located in front of the detector elements.