US 9,075,013 B2 · Apparatus and methods for microscopy having resolution beyond the Abbe limit

Abstract

Microscope apparatus and methods for imaging an object with a resolution beyond the Abbe limit are disclosed. The apparatus employs an object selectively patterned with a fluorescing material that is induced to fluoresce with one wavelength and inhibited from fluorescing with a second wavelength. Two orthogonal interference-fringe patterns are generated from four diffracted light beams of an inhibiting wavelength and superimposed on the object along with light that induces fluorescence. The interference-pattern image allows only sub-resolution-sized emission areas of the object to fluoresce. Multiple images of the fluorescing object are obtained, each corresponding to a slightly different position of the fringe patterns on the substrate. Each image is processed to yield a sparsely sampled super-resolution image. Multiple sparse images are interwoven to form a complete super-resolution image of the object.

Claims20

Dependent claims

A method according to forming a super-resolution image of an object that fluoresces at an activation wavelength and is inhibited from fluorescing at an inhibition wavelength, comprising:

a) using light having the inhibition wavelength, sequentially forming n2 interference-pattern images on the object to define fluorescence emission areas of the object, where n is an integer equal to 2 or greater;
b) illuminating the object with light of the activation wavelength for each of the n2 interference-pattern images to cause the fluorescence emission areas to emit fluorescent light at a fluorescent wavelength;
c) capturing n2 sparse object images that include images of the emission areas convolved with respective point spread functions (PSF), wherein the emission areas are smaller than the PSFs;
d) for each of the sparse object images, de-convolving each of the emission-area images to obtain emission-area image intensities;
e) combining the emission-area image intensities from the n2 sparse object images to form the super-resolution image of the object;
and
further comprising performing a calibration that comprises at least one of:
i) measuring and compensating for a distortion in the interference-pattern image;
and ii) measuring and compensating for a non-uniformity of the activation-wavelength illumination.

6 more elements

A microscope for forming a super-resolution image of an object that fluoresces at an activation wavelength and is inhibited from fluorescing at an inhibition wavelength, comprising:

an objective lens arranged adjacent the object, the objective lens having a resolution and at least one point spread function (PSF);
an interference pattern generator that operates cooperatively with the objective lens to generate an interference-pattern image on the object with first light of the inhibition wavelength, wherein the interference-pattern image comprises at least first and second sets of interference fringes;
a light-source system that operates cooperatively with the objective lens to illuminate the object with second light having the activation wavelength, thereby defining on the object, in combination with the interference-pattern image, a plurality of sub-resolution fluorescence emission areas that emit fluorescent light, and wherein the image-pattern generator is adapted to shift at least one of the first and second sets of interference fringes relative to the object to shift a location of the fluorescence emission areas;
a fluorescence camera and filter that operate cooperatively with the objective lens to capture an image of the fluorescence emission areas and in response generate a photodetector signal;
and
a controller that receives the photodetector signal, the controller having instructions embodied in a computer-readable medium that cause the controller to perform a de-convolution of the captured image based on the at least one PSF to determine emission-area image intensities that define the super-resolution image.

3 more elements

Dependencies are inferred from the claim text.

Apparatus and methods for microscopy having resolution beyond the Abbe limit

Life of this patent

Apparatus and methods for microscopy having resolution beyond the Abbe limit

Life of this patent

Abstract

Claims20