Bessel beam plane illumination microscope
AssigneeHoward Hughes Medical Institute
InventorRobert E. Betzig
Life of this patent
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Abstract
A microscope has a light source for generating a light beam having a wavelength, λ, and beam-forming optics configured for receiving the light beam and generating a Bessel-like beam that is directed into a sample. The beam-forming optics include an excitation objective having an axis oriented in a first direction. Imaging optics are configured for receiving light from a position within the sample that is illuminated by the Bessel-like beam and for imaging the received light on a detector. The imaging optics include a detection objective having an axis oriented in a second direction that is non-parallel to the first direction. A detector is configured for detecting signal light received by the imaging optics, and an aperture mask is positioned.
Claims27
A microscope comprising:
- a light source for generating a light beam having a wavelength, λ;
- beam-forming optics configured for receiving the light beam and generating a Bessel-like beam that is directed into a sample, the beam-forming optics including an excitation objective having a numerical aperture (NA) and an axis oriented in a first direction;
- imaging optics configured for receiving signal light from a position within the sample that is illuminated by the Bessel-like beam and for imaging the received light on a detector, the imaging optics including a detection objective having an axis oriented in a second direction that is non-parallel to the first direction;
- beam-translation optics configured for translating the position of the Bessel-like beam within the sample in discrete steps of more than or equal to λ/2NA to create a first excitation pattern of multiple Bessel-like beams having a spatial period, Λ, equal to the distance between beam positions of neighboring steps and configured to create N−1 additional excitation patterns that are spatially phase shifted from the first excitation pattern by (N−1)Λ/N;
- a detector configured for detecting signal light received by the imaging optics, the detector having individual detection units;
- and
- a processor configured to generate N images from the received signal light, each n image, for n=1 to N, being based on detected light due to excitation of the sample by the nth excitation pattern and further configured generate a final image of the sample through a linear combination of the N individual images.
A method comprising:
- generating a Bessel-like beam having a wavelength, λ;
- directing the Bessel-like beam through an excitation objective having a numerical aperture (NA) and an axis oriented in a first direction and into a sample;
- receiving signal light through a detection objective having an axis oriented in a second direction that is non-parallel to the first direction from a position within the sample that is illuminated by the Bessel-like beam and imaging the received light onto a detector, wherein the detector includes individual detection units;
- translating the position of the Bessel-like beam within the sample in discrete steps of more than or equal to λ/2NA to create a first excitation pattern of multiple Bessel-like beams having a spatial period, Λ, equal to the distance between beam positions of neighboring steps and configured to create N−1 additional excitation patterns that are spatially phase shifted from the first excitation pattern by (N−1)Λ/N;
- generating N images from the received signal light, each n image, for n=1 to N, being based on detected light due to excitation of the sample by the nth excitation pattern;
- and
- generating a final image of the sample through a linear combination of the N individual images.
Dependencies are inferred from the claim text.