Method for determining a height position of an object

Abstract

A method for determining a height position of an object at of using a microscope which images using a point-spread function along a z-direction (height direction), comprising the steps of imaging the object in the far field and determining a far-field intensity, calculating a maximum intensity expected by multiplying the far-field intensity by a scaling factor, partially confocally imaging the object with the focus in the z-direction within the depth-of-field range, and determining a partially-confocal intensity of the imaging, calculating the intensity of the point-spread function (at the first location) by forming a difference between the partially-confocal intensity and a product of the far-field intensity and a predefined combination factor, calculating the z-coordinate of the focus at a point-spread function maximum, using a previously-known form of the point spread function, its calculated intensity, and the calculated expected maximum intensity, and using the z-coordinate as the height position of the object.

1 . A method for determining a height position of an object at a lateral first location of the object by using a microscope, which images the object with a point spread function along a z-direction that coincides with the height direction, the method comprising the following steps:
 imaging the object with the microscope in wide field and determining a wide field intensity,   calculating a maximum intensity expected at the first location by multiplying the wide field intensity with a predetermined scaling factor,   partially confocally imaging the object at the first location with the focus located at a measurement position in the z-direction, wherein a pinhole device having an opening which is greater than a diffraction limit is used for the partially confocal imaging, and wherein the partially confocal imaging defines a depth of field range and the focus is within the depth of field range, and determining a partially confocal intensity of the partially confocal image at the first location,   calculating an intensity corresponding to the point spread function at the first location by way of subtraction between the partially confocal intensity and the product of the wide field intensity and a predetermined linking factor,   computing a z-coordinate of the focus at which the point spread function is maximum using a predetermined form of the point spread function, the calculated intensity corresponding to the point spread function and the calculated expected maximum intensity, and   using the z-coordinate as the height position of the object at the first location.   
     
     
         2 . The method as claimed in  claim 1 , wherein the scaling factor is determined by performing the following steps:
 producing a z-stack by repeatedly shifting the focus position in the z-direction and partially confocally imaging a lateral first calibration location of a first calibration object or of the object   at each focus position, and determining the partially confocal intensity for each of the partially confocal images thus obtained,   calculating a z-direction intensity profile at the first calibration location, and determining a maximum calibration intensity of the intensity profile at the first calibration location,   imaging the first calibration location with the microscope in wide field, and determining a first calibration wide field intensity at the first calibration location, and   calculating the scaling factor as a ratio between the maximum calibration intensity and the first calibration wide field intensity.   
     
     
         3 . The method as claimed in  claim 1 , wherein the linking factor is determined by performing the following steps:
 partially confocal imaging a lateral second calibration location of a second calibration object or of the object with the focus located at a calibration measurement position in the z-direction which position is within the depth of field range, and determining a calibration intensity of the partially confocal image at the second calibration location,   confocally imaging the second calibration location with the focus located at the calibration measurement position, and determining a confocal calibration intensity of the confocal image at the second calibration location,   imaging the second calibration location with the microscope in wide field, and determining a second calibration wide field intensity at the second calibration location, and   calculating the linking factor as a ratio between the difference between the partially confocal calibration intensity and the confocal calibration intensity to the second second calibration wide field intensity.   
     
     
         4 . The method as claimed in  claim 1 , wherein the first location is imaged with the microscope in wide field and in that the wide field intensity is determined at the first location. 
     
     
         5 . The method as claimed in  claim 1 , wherein the following steps:
 partially confocally imaging the first location with the focus located at least at two measurement positions which are spaced apart in the z-direction and are situated both in the depth of field range, and determining partially confocal intensities for each partially confocal image at the first location,   calculating the intensities corresponding to the point spread function at the first location as a difference between the partially confocal intensity and a product of wide field intensity and predetermined linking factor,   computing the z-coordinate using the form of the point spread function, the calculated intensities corresponding to the point spread function and the calculated expected maximum intensity.   
     
     
         6 . The method as claimed in  claim 1 , wherein for producing a live determination of a height position in the object at a laterally second location of the object, which differs from the first one, the second location is imaged in partially confocal fashion and in that, for calculating a z-coordinate at the second location, the z-coordinate of the focus at the first location is taken into consideration. 
     
     
         7 . The method as claimed in  claim 1 , further comprising the following steps:
 partially confocally imaging the first location with at least two different pinholes and with the focus at a measurement position in the z-direction which is situated within the depth of field range, and determining a further partially confocal intensity for the partially confocal image with the further pinhole,   calculating the z-coordinate using the form of the point spread function, the calculated intensity corresponding to the point spread function, the further partially confocal intensity and the calculated expected maximum intensity.   
     
     
         8 . The method as claimed in  claim 1 , wherein a laser scanning microscope is used as the microscope, and wherein the size of the pinhole is changed for partially confocal imaging and for imaging in wide field. 
     
     
         9 . The method as claimed in  claim 1 , wherein a laser scanning microscope is used as the microscope, wherein, for partially confocal imaging and for imaging in wide field, two detectors are provided behind pinholes of different size or wherein a wide field camera is used for imaging in wide field. 
     
     
         10 . The method as claimed in  claim 1 , wherein a confocal topography microscope is used as the microscope, in which a grating is used as a pinhole, wherein a first camera with the grating is used for partially confocal imaging and a second camera is used for imaging in wide field. 
     
     
         11 . The method as claimed in  claim 1 , wherein a confocal Airy microscope is used as the microscope, in which the object is imaged onto a detector device which comprises a plurality of pixels and resolves a diffraction structure of the partially confocal image, wherein the wide field intensity and the partially confocal intensity are determined from one recording or wherein a wide field camera is used for imaging in wide field. 
     
     
         12 . A microscope for producing a partially confocal image of an object and an image of the object in wide field, comprising:
 a detector device,   a pinhole device for partially confocal imaging and recording in wide field,   a focusing device, which is embodied for setting a z-position of a focus of the partially confocal image, and   a control device for controlling the pinhole device, which is connected to the detector device,   wherein the microscope images the object in a z-direction, which coincides with the height direction of the object, with a point spread function,   wherein the control device is configured to carry out the method as claimed in  claim 1 .   
     
     
         13 . The microscope as claimed in  claim 12 , wherein a size of a pinhole of the pinhole device is adjustable. 
     
     
         14 . The microscope as claimed in  claim 12 , wherein the detector device comprises a first detector for partially confocal imaging and a second detector for imaging in wide field.