3D Biplane Microscopy

Abstract

A microscopy system is configured for creating 3D images from individually localized probe molecules. The microscopy system includes a sample stage, an activation light source, a readout light source, a beam splitting device, at least one camera, and a controller. The activation light source activates probes of at least one probe subset of photo-sensitive luminescent probes, and the readout light source causes luminescence light from the activated probes. The beam splitting device splits the luminescence light into at least two paths to create at least two detection planes that correspond to the same or different number of object planes of the sample. The camera detects simultaneously the at least two detection planes, the number of object planes being represented in the camera by the same number of recorded regions of interest. The controller is programmable to combine a signal from the regions of interest into a 3D data.

1 - 40 . (canceled) 
     
     
         41 . A microscopy system configured for creating 3D images from individually localized probe molecules, the system comprising:
 an activation light source configured to illuminate a sample with an activation light, the activation light source being configured to activate probes of at least one probe subset of the plurality of photo-sensitive luminescent probes;   a readout light source configured to illuminate the sample with a readout light, the readout light source being configured to cause luminescence light from the activated probes;   a beam splitting device located in a detection light path that splits the luminescence light into at least two paths, the beam splitting device creating at least two detection planes that correspond to the same or different number of object planes of the sample;   at least one camera positioned to detect simultaneously the at least two detection planes, the number of object planes being represented in the camera by the same number of recorded regions of interest; and   a controller programmable to
 combine a signal from the regions of interest into a 3D data stack, 
 calculate a figure of merit as a measure of the difference between imaged pixel data and a reference data set modifiable by at least one parameter, and 
 optimize the figure of merit by adjusting the at least one parameter. 
   
     
     
         42 . The microscopy system of  claim 41 , wherein the controller is further programmable to perform a least-squares fit for finding a best fit by successively contracting an n-dimensional polytope around a minimum of a figure-of-merit function in m-dimensional parameter space. 
     
     
         43 . The microscopy system of  claim 41 , wherein the controller is further programmable to calculate the figure-of-merit function as a square of error-weighted differences between observed number of photons and a model function that depends on a set of fit parameter values summed over all pixels, 
     
     
         44 . The microscopy system of  claim 41 , wherein the figure-of-merit function χ 2  is expressed as 
       
         
           
             
               
                 
                   
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       wherein x j  describes 3D coordinates in the sample, n j , is the observed number of photons, j is the number of all pixels, F v,b,a (x)=vh a (x)+b, a is the particle 3D position in a specific dimension, v is the number of photons at the intensity maximum detected over the area of one pixel, b is the number of background photons per pixel, and h a (x) a describes the normalized instrument response at point x for a particle located at position a. 
     
     
         45 . The microscopy system of  claim 41 , wherein fitting particle data is based on 5 parameters, including (i) particle 3D position in x direction a x , (ii) particle 3D position in y direction a y , (iii) particle 3D position in x direction a z , (iv) number of photons v at the intensity maximum detected over the area of one pixel, and (v) the number of background photons b per pixel. 
     
     
         46 . The microscopy system of  claim 41 , wherein the reference data set is defined for a lattice of voxel coordinates ξ l , the controller being further programmable to determine values h a (x j ) from the reference data set by interpolation. 
     
     
         47 . The microscopy system of  claim 46 , wherein interpolation is based on Fourier transforms. 
     
     
         48 . The microscopy system of  claim 46 , wherein the controller is further programmable to convolve the reference data set with a shifted Dirac delta distribution. 
     
     
         49 . The microscopy system of  claim 41 , wherein the at least one parameter includes a particle position, an amplitude, and a background signal. 
     
     
         50 . The microscopy system of  claim 41 , wherein the at least one parameter includes an interference phase parameter, a polarization parameter, and a wavelength parameter.