STRUCTURED PLANE ILLUMINATION MICROSCOPY
First Claim
1. An apparatus comprising:
- a light source configured for generating a coherent light beam having a wavelength, λ
;
beam-forming optics configured for receiving the generated light beam and for generating a plurality of substantially parallel Bessel-like beams directed into a sample in a first direction, wherein each of the Bessel-like beams has a fixed phase relative to the other Bessel-like beams;
a light detector;
imaging optics configured for receiving light from a position within the sample that is illuminated by the Bessel-like beams and for imaging the received light onto the detector, the imaging optics including a detection objective having an axis oriented in a second direction that is non-parallel to the first direction, wherein the detector is configured for detecting light received by the imaging optics; and
a processor configured to generate an image of the sample based on the detected light.
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Abstract
An apparatus includes a light source configured for generating a coherent light beam having a wavelength, λ, a light detector, and beam-forming optics configured for receiving the generated light beam and for generating a plurality of substantially parallel Bessel-like beams directed into a sample in a first direction. Each of the Bessel-like beams has a fixed phase relative to the other Bessel-like beams. Imaging optics are configured for receiving light from a position within the sample that is illuminated by the Bessel-like beams and for imaging the received light onto the detector. The imaging optics include a detection objective having an axis oriented in a second direction that is non-parallel to the first direction, where the detector is configured for detecting light received by the imaging optics. A processor configured to generate an image of the sample based on the detected light.
124 Citations
115 Claims
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1. An apparatus comprising:
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a light source configured for generating a coherent light beam having a wavelength, λ
;beam-forming optics configured for receiving the generated light beam and for generating a plurality of substantially parallel Bessel-like beams directed into a sample in a first direction, wherein each of the Bessel-like beams has a fixed phase relative to the other Bessel-like beams; a light detector; imaging optics configured for receiving light from a position within the sample that is illuminated by the Bessel-like beams and for imaging the received light onto the detector, the imaging optics including a detection objective having an axis oriented in a second direction that is non-parallel to the first direction, wherein the detector is configured for detecting light received by the imaging optics; and a processor configured to generate an image of the sample based on the detected light. - View Dependent Claims (2, 3, 3, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29)
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30. A method comprising:
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generating a coherent light beam having a wavelength, λ
;generating from the coherent beam a plurality of substantially parallel Bessel-like beams directed into a sample in a first direction, wherein the each Bessel-like beam has a fixed phase relative to the other Bessel-like beams; imaging, with imaging optics, light received from a position within the sample that is illuminated by the Bessel-like beams onto detector, the imaging optics including a detection objective having an axis oriented in a second direction that is non-parallel to the first direction; and generating an image of the sample based on the detected light. - View Dependent Claims (31, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52)
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53. A method comprising:
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(a) providing activation radiation to a sample that includes phototransformable optical labels (“
PTOLs”
) to activate a statistically sampled subset of the PTOLs in the sample, wherein the PTOLs are distributed in at least a portion of the sample with a density greater than an inverse of the diffraction-limited resolution volume (“
DLRV”
) of imaging optics;(b) providing a sheet of excitation radiation to the sample to excite at least some of the activated PTOLs; (c) detecting with the imaging optics radiation emitted from activated and excited PTOLs within the first subset of PTOLs, wherein the imaging optics include an objective lens having an axis along a second direction that is substantially perpendicular to the plane; (d) determining locations within the sample of the PTOLs from which radiation is detected with superresolution accuracy; (e) controlling the activation radiation such that the density of activated PTOLs in the subset is less than the inverse of the (“
DLRV”
) of the imaging optics;(f) repeating (a)-(e) to determine location of more PTOLs within the sample; and (g) generating a sub-diffraction-limited image of the sample based on the determined locations of the PTOLs. - View Dependent Claims (53, 54, 55, 55, 56, 57, 57, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85)
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87. An apparatus comprising:
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a first light source for generating a first light beam having a wavelength, λ
1;first beam-forming optics configured for receiving the generated light beam and for generating activation radiation to a sample that includes phototransformable optical labels (“
PTOLs”
) to activate a statistically sampled subset of the PTOLs in the sample, wherein the PTOLs are distributed in at least a portion of the sample with a density greater than an inverse of the diffraction-limited resolution volume (“
DLRV”
) of imaging optics;a second light source for generating a second light beam having a wavelength, λ
2;second beam-forming optics configured for receiving the second generated beam and providing a second sheet of excitation radiation to the sample to excite at least some of the activated PTOLs; a detector configured for detecting radiation emitted from activated and excited PTOLs within the first subset of PTOLs; imaging optics configured for receiving radiation emitted from activated and excited PTOLs and for imaging the received light onto the detector, the imaging optics including a detection objective having an axis oriented in a second direction that is substantially perpendicular to the plane; a controller configured for controlling the activation radiation such that the density of activated PTOLs in the subset is less than the inverse of the (“
DLRV”
) of the imaging optics;one or more processors of a computing system configured for determining, with superresolution accuracy, locations within the sample of the PTOLs from which radiation is detected; one or more processors of the computing system configured for generating a sub-diffraction-limited image of the sample based on the determined locations of the PTOLs. - View Dependent Claims (86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115)
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Specification