Method and apparatus for three-dimensional microscopy with enhanced resolution

A method for three-dimensional optical microscopy, comprising the steps of:

• (a) placing a sample between first and second opposing objective lenses;
• (b) focusing said objective lenses on a section of said sample;
• (c) directing observed light from said section of said sample along first and second paths to imaging means for detecting and recording images, said first and second paths leading from said section of said sample to said imaging means through said first and second objective lenses respectively, and causing said observed light from said first and second paths to coincide on said imaging means;
• (d) adjusting optical lengths of at least one of said first and second paths so as to make said first and second optical path lengths be closely equal, thereby causing said observed light from said first and second objective lenses to interfere on said imaging means;
• (e) recording said interfering observed light on said imaging means;
• (f) focusing said objective lenses on another section of said sample;
• and
• (g) repeating steps (c), (d), (e) and (f) until a plurality of sections of said sample have been observed and recorded, forming a data set of recorded images.

A method for three-dimensional optical microscopy, comprising the steps of:

• (a) placing a sample between first and second opposing objective lenses;
• (b) focusing said first and second objective lenses onto a section of said sample;
• (c) directing illuminating light from an extended, spatially incoherent light source along first and second illumination paths to said section of said sample, said first illumination path extending from said light source to said section of said sample via said first objective lens, said second illumination path extending from said light source to said section of said sample via said second objective lens;
• (d) directing observed light from said sample along first and second observation paths to imaging means for detecting and recording images, said first and second observation paths extending from said section of said sample to said imaging means via said first and second objective lenses respectively, and causing said observed light from said first and second observation paths to coincide on said imaging means;
• (e) adjusting optical lengths of at least one of said first and second illumination paths, so as to make said optical lengths of said first and second illumination paths be closely equal thereby causing said illuminating light from said first and second illumination paths to interfere in said section of said sample;
• (f) recording said observed light on said imaging means;
• (g) refocusing said first and second objective lenses onto another section of said sample;
• and
• (h) repeating steps (c), (d), (e) (f) and (g) until a plurality of sections of said sample have been observed and recorded, forming a data set of recorded images.

A method for three-dimensional optical microscopy, comprising the steps of:

• (a) placing a sample between first and second opposing objective lenses;
• (b) focusing said first and second objective lenses onto a section of said sample;
• (c) directing illuminating light from an extended, spatially incoherent light source along first and second illumination paths to said section of said sample, said first illumination path extending from said light source to said section of said sample via said first objective lens, said second illumination path extending from said light source to said section of said sample via said second objective lens;
• (d) directing observed light from at least one of said first and second objective lenses to imaging means for detecting and recording images;
• (e) adjusting optical lengths of at least one of said first and second illumination paths, so as to make said optical lengths of said first and second illumination paths be closely equal, thereby causing said illuminating light from said first and second illumination paths to interfere in said section of said sample;
• (f) recording said observed light on said imaging means;
• (g) refocusing said first and second objective lenses onto another section of said sample;
• and
• (h) repeating steps (c), (d), (e), (f) and (g) until a plurality of sections of said sample have been observed and recorded, forming a data set of recorded images.

A three dimensional optical microscopy apparatus, comprising:

• (a) a first objective lens and a second objective lens, said objective lenses mounted opposite to each other;
• (b) means for supporting a microscopy sample between said objective lenses;
• (c) means for beam splitting light;
• (d) first and second optical paths, said first optical path extending from said beam splitting means to said microscopy sample via said first objective lens, said second optical path extending from said beam splitting means to said microscopy sample via said second objective lens;
• (e) illuminating means for producing extended, spatially incoherent light, said illuminating means positioned to provide illuminating light to said beam splitting means;
• (f) a plurality of means for directing light, at least one of said light directing means positioned along each of said first and second optical paths to direct illuminating light from said beam splitting means along said first and second optical paths to said sample;
• (g) optical path length balancing means for adjusting optical path lengths of at least one of said first and second optical paths, so as to make said optical path lengths of said first and second optical paths be closely equal;
• and
• (h) imaging means for detecting and recording images, said imaging means positioned to record observed light from at least one of said objective lenses.

An apparatus for optical microscopy, comprising:

• (a) means for supporting a sample;
• (b) means for providing spatially structured illuminating light to said sample, said spatially structured illuminating light containing lateral structure, said means for providing spatially structured illuminating light comprising light source means for producing light, an illuminating path from said light source means to said sample, and at least one mask located along said illuminating path;
• (c) optical magnification means for producing magnified images of said sample illuminated by said spatially structured illumination light;
• (d) imaging means for detecting and recording said magnified images of said sample;
• and
• (e) processing means for processing said recorded images from said imaging means to obtain a reconstruction of said sample with improved resolution including improved lateral resolution, said processing means arranged to cause information components from said recorded images to assume new positions in Fourier space.

An apparatus for optical microscopy, comprising:

• (a) means for supporting a sample;
• (b) means for providing spatially structured illuminating light to said sample, said spatially structured illuminating light containing lateral structure, said means for providing spatially structured illuminating light comprising means for providing at least two mutually coherent beams of light to said sample, said at least two mutually coherent beams of light arranged so as to interfere with each other at said sample;
• (c) optical magnification means for producing magnified images of said sample illuminated by said spatially structured illumination light;
• (d) imaging means for detecting and recording said magnified images of said sample;
• and
• (e) processing means for processing said recorded images from said imaging means to obtain a reconstruction of said sample with improved resolution, including improved lateral resolution.

A method of optical microscopy comprising the steps of:

• (a) placing a luminescent sample in a microscope containing image detecting and recording means;
• (b) illuminating said sample with an illumination pattern that contains lateral structure;
• (c) recording at least one image of said sample using said image detecting and recording means;
• (d) altering said illumination pattern at least one time, each time recording at least one image of said sample illuminated with said altered illumination pattern;
• (e) collecting said images into a data set;
• and
• (f) computationally processing said data set to obtain a reconstruction of said sample with improved resolution, including improved lateral resolution, said step of computationally processing said data comprising the steps of separating a plurality of information components, causing said information components to assume new positions in Fourier space, and recombining said information components.