Abstract: A microscope directs light through an excitation objective to generate a lattice light sheet (LLS) within a sample. A detection objective collects signal light from the sample in response to the LLS and images the collected light onto a detector. Second and third light beams are imaged onto focal planes of the excitation objective and detection objective, respectively. One or more wavefront detectors determine wavefronts of light emitted from the sample and through the excitation objective in response to the imaged second light beam and emitted from the sample through the detection objective in response to the imaged third light beam. A wavefront of the first light beam is modified to reduce a sample-induced aberration of the LLS within the sample, and a wavefront of the signal light emitted from the sample is modified to reduce a sample-induced aberration of the signal light at the detector.

Abstract: Imaging a sample that includes phototransformable optical labels (“PTOLs”) with an optical system having a diffraction-limited resolution volume (DLRV), includes providing activation radiation to the PTOLs to activate a statistical subset of the PTOLs. A density of the PTOLs of the activated subset is less than an inverse of the DLRV. Excitation radiation is provided to the activated subset to excite activated PTOLs. Radiation emitted from the activated and excited PTOLs located at different focal planes of the optical system within the sample is detected with the optical system. The preceding steps are repeated one or more times, each time activating a different statistical subset of the plurality of PTOLs. Three-dimensional locations within the sample are determined, with a sub-diffraction-limited accuracy, of the activated and excited PTOLs based on the radiation emitted from the activated and excited PTOLs that is detected from the different focal planes of the optical system.

Abstract: A microscope directs light through an excitation objective to generate a lattice light sheet (LLS) within a sample. A detection objective collects signal light from the sample in response to the LLS and images the collected light onto a detector. Second and third light beams are imaged onto focal planes of the excitation objective and detection objective, respectively. One or more wavefront detectors determine wavefronts of light emitted from the sample and through the excitation objective in response to the imaged second light beam and emitted from the sample through the detection objective in response to the imaged third light beam. A wavefront of the first light beam is modified to reduce a sample-induced aberration of the LLS within the sample, and a wavefront of the signal light emitted from the sample is modified to reduce a sample-induced aberration of the signal light at the detector.

Abstract: Imaging a sample that includes phototransformable optical labels (“PTOLs”) with an optical system having a diffraction-limited resolution volume (DLRV), includes providing activation radiation to the PTOLs to activate a statistical subset of the PTOLs. A density of the PTOLs of the activated subset is less than an inverse of the DLRV. Excitation radiation is provided to the activated subset to excite activated PTOLs. Radiation emitted from the activated and excited PTOLs located at different focal planes of the optical system within the sample is detected with the optical system. The preceding steps are repeated one or more times, each time activating a different statistical subset of the plurality of PTOLs. Three-dimensional locations within the sample are determined, with a sub-diffraction-limited accuracy, of the activated and excited PTOLs based on the radiation emitted from the activated and excited PTOLs that is detected from the different focal planes of the optical system.

Abstract: A sample is bathed in a solution that includes labels having a binding affinity for a structure on the sample and that emit light in response to excitation light. A sheet of excitation light having a FWHM thickness is provided to the sample. Light emitted from labels in response to the excitation light is imaged onto a detector, where the light is imaged with a detection objective having a depth of focus comparable to or greater than the FWHM thickness. The bathing of the sample and the imaging of the light emitted from the bound labels is controlled, such that the imaged light from different individual labels bound to the structure is resolved on the detector. The providing, imaging, and controlling are repeated to image, at different times, light from labels bound to the structure at different locations on or within the sample.

Abstract: Imaging a sample that includes phototransformable optical labels (“PTOLs”) with an optical system having a diffraction-limited resolution volume (DLRV), includes providing activation radiation to the PTOLs to activate a statistical subset of the PTOLs. A density of the PTOLs of the activated subset is less than an inverse of the DLRV. Excitation radiation is provided to the activated subset to excite activated PTOLs. Radiation emitted from the activated and excited PTOLs located at different focal planes of the optical system within the sample is detected with the optical system. The preceding steps are repeated one or more times, each time activating a different statistical subset of the plurality of PTOLs. Three-dimensional locations within the sample are determined, with a sub-diffraction-limited accuracy, of the activated and excited PTOLs based on the radiation emitted from the activated and excited PTOLs that is detected from the different focal planes of the optical system.

Abstract: A microscope directs light through an excitation objective to generate a lattice light sheet (LLS) within a sample. A detection objective collects signal light from the sample in response to the LLS and images the collected light onto a detector. Second and third light beams are imaged onto focal planes of the excitation objective and detection objective, respectively. One or more wavefront detectors determine wavefronts of light emitted from the sample and through the excitation objective in response to the imaged second light beam and emitted from the sample through the detection objective in response to the imaged third light beam. A wavefront of the first light beam is modified to reduce a sample-induced aberration of the LLS within the sample, and a wavefront of the signal light emitted from the sample is modified to reduce a sample-induced aberration of the signal light at the detector.

Abstract: Imaging a sample that includes phototransformable optical labels (“PTOLs”) with an optical system having a diffraction-limited resolution volume (DLRV), includes providing activation radiation to the PTOLs to activate a statistical subset of the PTOLs. A density of the PTOLs of the activated subset is less than an inverse of the DLRV. Excitation radiation is provided to the activated subset to excite activated PTOLs. Radiation emitted from the activated and excited PTOLs located at different focal planes of the optical system within the sample is detected with the optical system. The preceding steps are repeated one or more times, each time activating a different statistical subset of the plurality of PTOLs. Three-dimensional locations within the sample are determined, with a sub-diffraction-limited accuracy, of the activated and excited PTOLs based on the radiation emitted from the activated and excited PTOLs that is detected from the different focal planes of the optical system.

Abstract: Imaging a sample that includes phototransformable optical labels (“PTOLs”) with an optical system having a diffraction-limited resolution volume (DLRV), includes providing activation radiation to the PTOLs to activate a statistical subset of the PTOLs. A density of the PTOLs of the activated subset is less than an inverse of the DLRV. Excitation radiation is provided to the activated subset to excite activated PTOLs. Radiation emitted from the activated and excited PTOLs located at different focal planes of the optical system within the sample is detected with the optical system. The preceding steps are repeated one or more times, each time activating a different statistical subset of the plurality of PTOLs. Three-dimensional locations within the sample are determined, with a sub-diffraction-limited accuracy, of the activated and excited PTOLs based on the radiation emitted from the activated and excited PTOLs that is detected from the different focal planes of the optical system.

Abstract: A sample is bathed in a solution that includes labels having a binding affinity for a structure on the sample and that emit light in response to excitation light. A sheet of excitation light having a FWHM thickness is provided to the sample. Light emitted from labels in response to the excitation light is imaged onto a detector, where the light is imaged with a detection objective having a depth of focus comparable to or greater than the FWHM thickness. The bathing of the sample and the imaging of the light emitted from the bound labels is controlled, such that the imaged light from different individual labels bound to the structure is resolved on the detector. The providing, imaging, and controlling are repeated to image, at different times, light from labels bound to the structure at different locations on or within the sample.

Abstract: Imaging a sample that includes phototransformable optical labels (“PTOLs”) with an optical system having a diffraction-limited resolution volume (DLRV), includes providing activation radiation to the PTOLs to activate a statistical subset of the PTOLs. A density of the PTOLs of the activated subset is less than an inverse of the DLRV. Excitation radiation is provided to the activated subset to excite activated PTOLs. Radiation emitted from the activated and excited PTOLs located at different focal planes of the optical system within the sample is detected with the optical system. The preceding steps are repeated one or more times, each time activating a different statistical subset of the plurality of PTOLs. Three-dimensional locations within the sample are determined, with a sub-diffraction-limited accuracy, of the activated and excited PTOLs based on the radiation emitted from the activated and excited PTOLs that is detected from the different focal planes of the optical system.

Abstract: Imaging a sample that includes phototransformable optical labels (“PTOLs”) with an optical system having a diffraction-limited resolution volume (DLRV), includes providing activation radiation to the PTOLs to activate a statistical subset of the PTOLs. A density of the PTOLs of the activated subset is less than an inverse of the DLRV. Excitation radiation is provided to the activated subset to excite activated PTOLs. Radiation emitted from the activated and excited PTOLs located at different focal planes of the optical system within the sample is detected with the optical system. The preceding steps are repeated one or more times, each time activating a different statistical subset of the plurality of PTOLs. Three-dimensional locations within the sample are determined, with a sub-diffraction-limited accuracy, of the activated and excited PTOLs based on the radiation emitted from the activated and excited PTOLs that is detected from the different focal planes of the optical system.

Abstract: Microscopy techniques in which a rear pupil of an optical system is segmented and the segments are individually controlled with a wavefront modulating device to control the direction and phase of individual beamlets of an excitation or emission beam in the optical system, thereby providing an adaptive optics correction to sample and system induced aberrations.

Abstract: An apparatus includes a pulsed laser source that produces a pulsed laser beam at an input repetition rate and an input pulse power; a passive pulse splitter that receives the pulsed laser beam and outputs a signal including a plurality of sub-pulses for each input pulse of the pulsed laser beam, where the sub-pulses have a repetition rate that is greater than the input repetition rate and at least two of the sub-pulses have power less than the input pulse power; a sample accommodating structure configured to accommodate a sample placed in the path of a sample beam that is formed from the beam that exits the pulse splitter; and a detector that receives a signal of interest emitted from a sample accommodated by the sample accommodating structure based on the incident sample beam.

Abstract: A method of manipulating a focused light beam includes focusing a beam of excitation light with a lens to a focal spot within a sample, where a cross-section of the beam includes individual beamlets. Directions and/or relative phases of the individual beamlets of the excitation beam at a rear pupil of the lens are individually varied with a wavefront modulating element, and emission light emitted from the focal spot is detected while the directions or relative phases of individual beamlets are varied. The directions of individual beamlets are controlled to either maximize or minimize the emission light from the focal spot, and the relative phases of individual beamlets are controlled to increase the emission light from the focal spot.

Abstract: An apparatus includes a position-sensitive detector to detect intensities of radiation as a function of position on the detector, and an optical system, characterized by a diffraction-limited resolution volume, adapted for imaging light emitted from activated and excited phototransformable optical labels (“PTOLs”) in a sample onto the position sensitive-detector. A first light source provides activation radiation to the sample to activate a subset of the PTOLs that are distributed in the sample with a density greater than an inverse of the diffraction-limited resolution volume of the optical system. A second light source provides excitation radiation to the sample to excite a portion of the PTOLs in the subset of the PTOLs. A controller controls one both of the activation radiation and the excitation radiation provided to the sample such that a density of PTOLs in the portion of the PTOLs is less than the inverse of the diffraction-limited resolution volume.

Abstract: A method of manipulating a focused light beam includes focusing a beam of excitation light with a lens to a focal spot within a sample, where a cross-section of the beam includes individual beamlets. Directions and/or relative phases of the individual beamlets of the excitation beam at a rear pupil of the lens are individually varied with a wavefront modulating element, and emission light emitted from the focal spot is detected while the directions or relative phases of individual beamlets are varied. The directions of individual beamlets are controlled to either maximize or minimize the emission light from the focal spot, and the relative phases of individual beamlets are controlled to increase the emission light from the focal spot.

Abstract: An apparatus includes a position-sensitive detector to detect intensities of radiation as a function of position on the detector, and an optical system, characterized by a diffraction-limited resolution volume, adapted for imaging light emitted from activated and excited phototransformable optical labels (“PTOLs”) in a sample onto the position sensitive-detector. A first light source provides activation radiation to the sample to activate a subset of the PTOLs that are distributed in the sample with a density greater than an inverse of the diffraction-limited resolution volume of the optical system. A second light source provides excitation radiation to the sample to excite a portion of the PTOLs in the subset of the PTOLs. A controller controls one both of the activation radiation and the excitation radiation provided to the sample such that a density of PTOLs in the portion of the PTOLs is less than the inverse of the diffraction-limited resolution volume.

Abstract: An apparatus includes a pulsed laser source that produces a pulsed laser beam at an input repetition rate and an input pulse power; a passive pulse splitter that receives the pulsed laser beam and outputs a signal including a plurality of sub-pulses for each input pulse of the pulsed laser beam, where the sub-pulses have a repetition rate that is greater than the input repetition rate and at least two of the sub-pulses have power less than the input pulse power; a sample accommodating structure configured to accommodate a sample placed in the path of a sample beam that is formed from the beam that exits the pulse splitter; and a detector that receives a signal of interest emitted from a sample accommodated by the sample accommodating structure based on the incident sample beam.

Abstract: A microscope includes a source of electromagnetic radiation, having a wavelength, ?1 and dividing optical elements configured for dividing the radiation from the source into multiple excitation beams. The microscope also includes a detector and directing optical elements, which are configured for directing each excitation beam in unique directions, such that the beams intersect in an excitation region within a sample to create a two-dimensional or three-dimensional interference pattern of multiple excitation maxima within the sample. The detector has individual detector elements, where the detector elements are configured for detecting light resulting from an interaction of an individual excitation maximum and the sample.