Highly inclined swept tile (HIST) imaging apparatus, methods, and applications
AssigneeUniversity of Central Florida Research Foundation, Inc.
InventorsKyu Young Han, Jialei Tang
Life of this patent
Estimating the term…
Abstract
A fluorescent emission HIST microscope utilizes a highly inclined tile beam that is scanned over a biological sample object. Fluorescence emission from the sample is propagated through a confocal slit into an sCMOS camera supporting a rolling shutter mode. The tile beam is synchronously swept with the readout of the camera to facilitate the rejection of background. The system provides for decoupling of the total imaging area from the beam thickness, which now solely depends on the width of the tile beam, enabling a thinner illumination and larger FOV imaging. Clear visualization of single molecules across a 130 μm×130 μm FOV provided a greater than 40× FOV than conventional HILO imaging. An associated imaging method is disclosed.
Claims15
A highly inclined swept tile (HIST) microscope, comprising:
- an optical train having a Cartesian z-direction optical axis, said optical train including a first optical component or assembly disposable to receive a sample object fluorescence excitation input light, wherein said first optical assembly has optical characteristics suitable to transform the input light into a collimated output tile beam in the shape of a line oriented in an x-y-plane and characterized by a length, Ly, along the y-axis, a width, wx, along the x-axis, and a thickness, dz, along the z-axis, further wherein Ly>>wx>>dz;
- a second optical component or assembly disposable to receive the collimated tile-beam and having optical characteristics suitable to orient the tile beam at a highly inclined angle, θ, about the y-axis in the x-z plane;
- a third optical component or assembly having optical characteristics suitable to focus the highly inclined tile beam at an image plane;
- a fourth optical component or assembly disposed in a conjugate back focal plane of the image plane having optical characteristics suitable to sweep the highly inclined tile beam along a horizontal x-axis in the image plane;
- and
- a detector/camera assembly disposable in a conjugate image plane including either a confocal slit or a functionally equivalent electro-optical confocal slit to receive a fluorescence emission from the sample object,
- wherein said HIST microscope is to be configured as a confocal, fluorescence emission microscope,
- further wherein the tile beam thickness, dz, and the tile beam length, Ly, are decoupled, further wherein the HIST microscope has a total imaging area that depends solely on the tile beam width, wx.
A method for imaging a sample object, comprising:
- generating a collimated tile beam having a wavelength suitable for generating a fluorescent emission from a region of the sample object, wherein the tile beam has a line shape of length, Ly, thickness, dz, and width, wx, wherein Ly and wx are measured in an x-y Cartesian plane and dz is measured in an orthogonal z-axis beam propagation direction, further wherein Ly>>wx>>dz;
- disposing the tile beam at a highly inclined angle, θ, in the x-z Cartesian plane;
- sweeping the highly inclined tile beam along the x-axis across a region of the sample object and generating a fluorescent emission from the sample object;
- propagating the fluorescent emission from the sample object to a detector comprising a confocal slit, wherein the tile beam is synchronously swept with an output of the detector.
Dependencies are inferred from the claim text.