Abstract: Multiple fluorophores within a sample can be imaged by merging a plurality of beams from different wavelength light sources of excitation light into a single path, directing the excitation light into the sample, and detecting light emitted by the fluorophores within the sample on two different arrays of pixels (e.g., two regions within a single camera sensor chip). The light sources are activated during respective timeslots, and captured image data is processed. For at least one of the timeslots, the processing of the image data comprises using the image data captured using the first array of pixels to detect a presence of a given fluorophore, and using the image data captured using the second array of pixels to detect a presence of a different fluorophore. This arrangement enables a system that includes only N light sources to image more than N fluorophores.

Abstract: This application describes Swept, Confocally-Aligned Planar Excitation (SCAPE) microscopy systems that incorporate a gradient index (GRIN) lens to relay images from their distal tip to their proximal tip so that 3D images of deep tissue can be captured, without undue loss of light. A zero working distance feature can be designed into the third objective to ensure that the light that exits the second objective in the SCAPE system is not lost. Alternatively, a tapered fiber bundle may be positioned between the second objective and the third objective in the SCAPE system to ensure that the light that exits the second objective is not lost. As yet another alternative, direct detection at the intermediate image plane without using a third objective can ensure that the light that exits the second objective in the SCAPE system is not lost.

Abstract: A spacer for an immersion objective lens can be fabricated by pressing a set of sidewalls onto a mirror to form a liquid-tight cavity, filling the liquid-tight cavity with a first quantity of a UV-curable polymer, and curing the first quantity of the UV-curable polymer into a first solid mass that will be adhered to the mirror. The upper surface of the first solid mass is then positioned near the objective lens, with a second quantity of a UV curable polymer occupying the space between the first solid mass and the objective lens. Next, the position of the first solid mass is adjusted until it reaches a final position with respect to the objective lens. This adjustment may be assisted by checking the collimation of light reflected back through the mirror. The second quantity of the UV curable polymer is then cured.

Abstract: A microscope routes excitation light through a first set of optical components so the excitation light is projected into a sample and forms a sheet of excitation light at an oblique angle. The position of the sheet varies depending on an orientation of the scanning element. The first set of optical components routes detection light back to the scanning element, which routes the detection light into a second set of optical components. The second set of optical components forms an intermediate image plane that is imaged onto a detector. In some embodiments, a folding mirror is disposed between the first and second sets of optical components. In some embodiments, an optically transparent spacer covers the first objective and is configured to press against tissue being imaged. This spacer sets the working distance for the first objective to capture a particular range of depths within the tissue.

Abstract: Optical imaging or spectroscopy described can use laminar optical tomography (LOT), diffuse correlation spectroscopy (DCS), or the like. An incident beam is scanned across a target. An orthogonal or oblique optical response can be obtained, such as concurrently at different distances from the incident beam. The optical response from multiple incident wavelengths can be concurrently obtained by dispersing the response wavelengths in a direction orthogonal to the response distances from the incident beam. Temporal correlation can be measured, from which flow and other parameters can be computed. An optical conduit can enable endoscopic or laparoscopic imaging or spectroscopy of internal target locations. An articulating arm can communicate the light for performing the LOT, DCS, or the like. The imaging can find use for skin cancer diagnosis, such as distinguishing lentigo maligna (LM) from lentigo maligna melanoma (LMM).

Abstract: A pulsed beam of NIR excitation light is projected into a sample at an oblique angle and scanned by a scanning element through a volume in the sample. 2-photon excitation excites fluorescence within the sample. The fluorescence is imaged onto an intermediate image plane that remains stationary regardless of the orientation of the scanning element. The image is captured by a linear array of light detecting elements or a linear portion of a rectangular array. At any given position of the scanning element, the linear array (or portion) images all depths simultaneously. A plurality of images are captured for each of a plurality of different orientations of the scanning element. The orientation of the scanning element is controlled to move in a two dimensional pattern, which causes the beam of excitation light to sweep out a three dimensional volume within the sample.

Abstract: Methods, devices and systems for up to three-dimensional scanning of target regions at high magnification are disclosed.

Abstract: In some embodiments of SCAPE imaging systems, a Powell lens is used to expand light from a light source into a sheet of illumination light. An optical system sweeps the sheet of illumination light through a sample, and forms an image at an intermediate image plane from detected return light. A camera captures images of the intermediate image plane. In some embodiments of SCAPE imaging systems, an optical system sweeps the sheet of illumination light through a sample, and forms an image at an intermediate image plane from detected return light. A camera captures images of the intermediate image plane. In the latter embodiments, the optical system is deliberately misaligned with respect to a true alignment position so that a significant portion of light that would be lost at the true alignment position will arrive at the camera.

Abstract: In some embodiments, a SCAPE system routes light from a tilted intermediate image plane to an infinity space disposed behind a third objective. A first beam splitter positioned in the infinity space routes light from the intermediate image plane with different wavelengths in different directions. First and second light detector arrays capture first and second wavelength images, respectively, and optical components route light having the first and second wavelength towards the first and second light detectors, respectively. In some embodiments, a SCAPE system is used to capture a plurality of images while a sample is perturbed (e.g., vibrated, deformed, pushed, pulled, stretched, or squeezed) in order to visualize the impact of the perturbation on the sample.

Abstract: Implementing swept, confocally aligned planar excitation (SCAPE) imaging with asymmetric magnification in the detection arm provides a number of significant advantages. In some preferred embodiments, the asymmetric magnification is achieved using cylindrical lenses in the detection arm that are oriented to increase the magnification of the intermediate image in the width direction but not in the depth direction. SCAPE imaging may also be improved by using an SLM to modify a characteristic of the sheet of excitation light that is projected into the sample. Additional embodiments include a customized version of SCAPE that is optimized for imaging the retina at the back of an eyeball in living subjects.

Abstract: A pulsed beam of NIR excitation light is projected into a sample at an oblique angle and scanned by a scanning element through a volume in the sample. 2-photon excitation excites fluorescence within the sample. The fluorescence is imaged onto an intermediate image plane that remains stationary regardless of the orientation of the scanning element. The image is captured by a linear array of light detecting elements or a linear portion of a rectangular array. At any given position of the scanning element, the linear array (or portion) images all depths simultaneously. A plurality of images are captured for each of a plurality of different orientations of the scanning element. The orientation of the scanning element is controlled to move in a two dimensional pattern, which causes the beam of excitation light to sweep out a three dimensional volume within the sample.

Abstract: In some embodiments of SCAPE imaging systems, a Powell lens is used to expand light from a light source into a sheet of illumination light. An optical system sweeps the sheet of illumination light through a sample, and forms an image at an intermediate image plane from detected return light. A camera captures images of the intermediate image plane. In some embodiments of SCAPE imaging systems, an optical system sweeps the sheet of illumination light through a sample, and forms an image at an intermediate image plane from detected return light. A camera captures images of the intermediate image plane. In the latter embodiments, the optical system is deliberately misaligned with respect to a true alignment position so that a significant portion of light that would be lost at the true alignment position will arrive at the camera.

Abstract: A pulsed beam of NIR excitation light is projected into a sample (345) at an oblique angle and scanned by a scanning element through a volume in the sample. 2-photon excitation excites fluorescence within the sample. The fluorescence is imaged onto an intermediate image plane that remains stationary regardless of the orientation of the scanning element. The image is captured by a linear array of light detecting elements (392) or a linear portion of a rectangular array. At any given position of the scanning element, the linear array (or portion) images all depths simultaneously. A plurality of images are captured for each of a plurality of different orientations of the scanning element. The orientation of the scanning element is controlled to move in a two dimensional pattern, which causes the beam of excitation light to sweep out a three dimensional volume within the sample.

Abstract: Methods, devices and systems for up to three-dimensional scanning of target regions at high magnification are disclosed.

Abstract: In a first SCAPE imaging system, a Powell lens (105) is used to expand light from a light source (100) into a sheet of illumination light. An optical system (125,131,132,140) sweeps the sheet of illumination light through a sample (145), and forms an image at a tilted intermediate image plane (170) from detected return light. A camera (190) captures images of the intermediate image plane. In a second SCAPE imaging system, an optical system (125,131,132,140) sweeps the sheet of illumination light through a sample (145), and forms an image at a tilted intermediate image plane (170) from detected return light. A camera (190) captures images of the intermediate image plane. The detection optics are deliberately misaligned with respect to a true alignment position so that a significant portion of light that would be lost at the true alignment position will arrive at the camera.

Abstract: A scanning element routes a sheet or beam of excitation light through a first set of optical components and into a sample at an oblique angle. The position of the excitation light within the sample varies depending on the orientation of the scanning element. Fluorophores within the sample emit fluorescent detection light. The first set of optical components route the detection light back to the scanning element, and the scanning element routes the detection light through a second set of optical components and into a camera. The second set of optical components includes a phase modulating element that induces a controlled aberration so as to homogenize point spread functions of points at, above, and below a focal plane when measured at the camera. In some embodiments, the images captured by the camera are processed to correct for aberration by performing deconvolution of a point spread function.

Abstract: In a first invention, a SCAPE system routes light from a tilted intermediate image plane (170) to an infinity space disposed behind a third objective (180). A first dichroic beam splitter (52) positioned in the infinity space routes light from the intermediate image plane with different wavelengths in different directions. First and second light detector arrays (90) capture first and second wavelength images, respectively, and optical components (54,56,58,82) route light having the first and second wavelengths towards the first and second light detectors, respectively. In a second invention, a SCAPE system is used to capture a plurality of images while a sample is perturbed (e.g., vibrated, deformed, pushed, pulled, stretched, or squeezed) in order to visualize the impact of the perturbation on the sample.

Abstract: Methods, devices and systems for up to three-dimensional scanning of target regions at high magnification are disclosed.

Abstract: A scanning element routes a sheet or beam of excitation light through a first set of optical components and into a sample at an oblique angle. The position of the excitation light within the sample varies depending on the orientation of the scanning element. Fluorophores within the sample emit fluorescent detection light. The first set of optical components route the detection light back to the scanning element, and the scanning element routes the detection light through a second set of optical components and into a camera. The second set of optical components includes a phase modulating element that induces a controlled aberration so as to homogenize point spread functions of points at, above, and below a focal plane when measured at the camera. In some embodiments, the images captured by the camera are processed to correct for aberration by performing deconvolution of a point spread function.

Abstract: In some embodiments, a SCAPE system routes light from a tilted intermediate image plane to an infinity space disposed behind a third objective. A first beam splitter positioned in the infinity space routes light from the intermediate image plane with different wavelengths in different directions. First and second light detector arrays capture first and second wavelength images, respectively, and optical components route light having the first and second wavelength towards the first and second light detectors, respectively. In some embodiments, a SCAPE system is used to capture a plurality of images while a sample is perturbed (e.g., vibrated, deformed, pushed, pulled, stretched, or squeezed) in order to visualize the impact of the perturbation on the sample.