Sub-diffraction limit image resolution in three dimensions

Abstract

The present invention generally relates to sub-diffraction limit image resolution and other imaging techniques, including imaging in three dimensions. In one aspect, the invention is directed to determining and/or imaging light from two or more entities separated by a distance less than the diffraction limit of the incident light. For example, the entities may be separated by a distance of less than about 1000 nm, or less than about 300 nm for visible light. In some cases, the position of the entities can be determined in all three spatial dimensions (i.e., in the x, y, and z directions), and in certain cases, the positions in all three dimensions can be determined to an accuracy of less than about 1000 nm. In one set of embodiments, the entities may be selectively activatable, i.e., one entity can be activated to produce light, without activating other entities. A first entity may be activated and determined (e.g., by determining light emitted by the entity), then a second entity may be activated and determined. The emitted light may be used to determine the x and y positions of the first and second entities, for example, by determining the positions of the images of these entities, and in some cases, with sub-diffraction limit resolution. In some cases, the z positions may be determined using one of a variety of techniques that uses intensity information or focal information (e.g., a lack of focus) to determine the z position. Non-limiting examples of such techniques include astigmatism imaging, off-focus imaging, or multi-focal-plane imaging. Other aspects of the invention relate to systems for sub-diffraction limit image resolution, computer programs and techniques for sub-diffraction limit image resolution, methods for promoting sub-diffraction limit image resolution, and the like.

What is claimed is: 
     
         1 - 77 . (canceled) 
     
     
         78 . A microscope apparatus comprising:
 an illumination system that irradiates light to activate and excite a fraction of a plurality of entities in a sample;   an imaging system comprising a non-circularly symmetric lens,   wherein the imaging system forms a plurality of images of the fraction of the plurality of entities by fluorescence light emitted from the fraction of the plurality of entities on a detector; and   a processor that determines x, y, and z positions of at least some of the plurality of entities in the sample based on the plurality of images, wherein the x, y, and z positions of the plurality of entities are 3-D spatial coordinates.   
     
     
         79 . The microscope apparatus according to  claim 78 , wherein the processor determines the z positions of the plurality of entities based on shapes of the plurality of images. 
     
     
         80 . The microscope apparatus according to  claim 79 , wherein the processor determines the z positions of the plurality of entities based on shapes of the plurality of ellipsoidal images. 
     
     
         81 . The microscope apparatus according to  claim 78 , wherein the lens is a cylindrical lens. 
     
     
         82 . The microscope apparatus according to  claim 78 , wherein the processor uses a Gaussian function to determine the x, y, and z positions. 
     
     
         83 . The microscope apparatus according to  claim 78 , wherein the processor uses an elliptical Gaussian function to determine the x, y, and z positions. 
     
     
         84 . The microscope apparatus according to  claim 78 , wherein the processor calculates the z position from the shape of images within the plurality of images. 
     
     
         85 . The microscope apparatus according to  claim 78 , wherein the processor calculates the z position from the intensity of images within the plurality of images. 
     
     
         86 . The microscope apparatus according to  claim 78 , wherein the processor applies drift correction when determining the x, y, and z positions. 
     
     
         87 . The microscope apparatus according to  claim 86 , wherein applying drift correction comprises using fiducial markers. 
     
     
         88 . The microscope apparatus according to  claim 86 , wherein applying drift correction comprises using fluorescent beads. 
     
     
         89 . The microscope apparatus according to  claim 86 , wherein applying drift correction comprises identifying a fixed point, determining apparent movement of the fixed point, and correcting the x, y, and z positions based on the apparent movement of the fixed point. 
     
     
         90 . The microscope apparatus according to  claim 86 , wherein applying the drift correction comprises using a correlation function. 
     
     
         91 . The microscope apparatus according to  claim 78 , wherein the processor determines x, y, and z positions as a function of time. 
     
     
         92 . The microscope apparatus according to  claim 78 , wherein the illumination system irradiates activation light to activate the fraction of a plurality of entities and excitation light to excite the fraction of a plurality of entities, wherein the activation light and the excitation light have substantially the same wavelengths. 
     
     
         93 . The microscope apparatus according to  claim 78 , wherein the illumination system irradiates activation light to activate the fraction of a plurality of entities and excitation light to excite the fraction of a plurality of entities, wherein the activation light and the excitation light have substantially different wavelengths. 
     
     
         94 . The microscope apparatus according to  claim 78 , wherein at least some of the plurality of entities are separated by a distance of separation less than a wavelength of the fluorescence light emitted from the fraction of the plurality of entities. 
     
     
         95 . The microscope apparatus according to  claim 78 , wherein the x, y, and z positions are calculated at a precision better than the diffraction limit of the fluorescence light emitted from the fraction of the plurality of entities. 
     
     
         96 . The microscope apparatus according to  claim 78 , wherein at least some of the plurality of entities comprise cyanine dyes. 
     
     
         97 . The microscope apparatus according to  claim 78 , wherein at least some of the plurality of entities comprises fluorescent protein.