Yale University

University of Maine System

The Jackson Laboratory

University of Utah Research Foundation

Isis Innovation Limited

Joerg Bewersdorf

Sam Hess

Travis J Gould

Fang Huang

Brian Thomas Bennett

Manuel F Juette

Martin James Booth

Erik Jorgensen

Michael Darin Mason

Yongdeng Zhang

Daniel Burke

Mudalige Siyath Gunewardene

Andrew Barentine

Sam T Hess

One aspect of the invention provides a method of continuously scanning with a localization microscope. The method includes: modifying a position of a sample relative to a field of view (FOV) of the localization microscope to capture a plurality of image frames of the sample, each captured image frame having a limited FOV; acquiring image frames with the localization microscope during at least one position modification; determining a set of localization position coordinates for at least one localizable object in the sample within at least one image frame of the plurality of image frames; determining one or more field of view (FOV) position coordinates for the at least one image frame; and modifying the set of localization position coordinates based on the one or more FOV position coordinates to produce a collection of coordinates covering a larger spatial region than the at least one image frame.

Continuous scanning for localization microscopy

Methods and systems for fluorescence imaging are described herein. The method can include: receiving a fluorescence signal including an excitation signal, a first emission signal for a first fluorophore having a first emission spectra, and a second emission signal for a second fluorophore having a second emission spectra; filtering the fluorescence signal to: isolate a first channel encompassing at least one of: a bandwidth of at least 1 nm within which an emission intensity of the first emission spectra is at least twice an emission intensity of the second emission spectra, a bandwidth having a rising edge of the first emission spectra; and at least 10% by height of a rising edge of the first emission spectra, and produce a channel including the fluorescence signal less the first channel; and directing the first channel and the resulting channel to different regions of one or more cameras for collecting fluorescence emissions.

Multi-color imaging using salvaged fluorescence

One aspect of the invention provides a method for drift correction to correct a 3D point collection dataset to compensate for drift over time. The method includes: (a) separating the 3D dataset into n segments, wherein n&#x3e;1; (b) for each of the n segments, reconstructing a volume image as a 3D histogram in which a count for each voxel in the histogram equals a number of localization estimates falling within the voxel; (c) performing 3D cross-correlation between pairs of the n segments; (d) identifying a correlation peak in a result of the 3D cross-correlation to determine a shift distance between pairs of the n segments; (e) solving an overdetermined system of shift distances to determine independent shifts; and (f) offsetting positions from a plurality of segments in the 3D point collection dataset with the independent shifts calculated in step (e) to correct for drift.

Systems, methods, and computer-readable media for ultra-high resolution 3D imaging of whole cells

Techniques for processing imaging data contaminated by sensor-dependent noise. An imaging method is described. In the imaging method, imaging data corresponding to an imaged region and acquired by at least first and second sensor elements is obtained. A parameterized model is fitted to the imaging data. The parameterized model includes a first sensor-dependent model of noise generated by the first sensor element in a first portion of the imaging data acquired by the first sensor element, and a second sensor-dependent model of noise generated by a second sensor element in a second portion of the imaging data acquired by the second sensor element. The first sensor-dependent noise model differs, at least in part, from the second sensor-dependent noise model.

Techniques for processing imaging data having sensor-dependent noise

Aberrations in stimulated emission depletion microscopy are corrected using an adaptive optics approach using a metric which combines both image sharpness and brightness. Light modulators () are used to perform aberration correction in one or more of the depletion path (), the excitation path (), or the emission path from sample to detector.

Stimulated emission depletion microscopy

A system () and method for creating three dimensional images using probe molecules is disclosed and described. A sample is mounted on a stage (). The sample has a plurality of probe molecules. The sample is illuminated with light, causing the probe molecules to luminesce. The probe luminescence can be split into at least four paths corresponding to at least four detection planes corresponding to object planes in the sample. The at least four detection planes are detected via a camera (). Object planes in corresponding recorded regions of interest are recorded in the camera (). A signal from the regions of interest is combined into a three dimensional image.

Microscopy system and method for creating three dimensional images using probe molecules

A primary beam splitter () of an optical apparatus () can be used to split an incident light beam () into a primary plurality of light beams and to direct a first beam therefrom in a first direction and a second beam therefrom in a second direction orthogonal to the first direction. Secondary beam splitters () positioned in beam paths of the first and second beams can be used to split the first and second beams of the primary plurality of light beams into a secondary plurality of light beams () and to split the same into a tertiary plurality of light beams (). A primary plurality of beam reflectors () can be positioned and used to redirect the secondary and tertiary plurality of light beams toward a common detector ().

Beam splitter module having a secondary beam splitter for providing orthogonal light beams and parallel light beams

A method of performing 3D photoactivation microscope imaging includes providing a sample having a plurality of probes, each of the plurality of probes including a photo-activatable material. Probes from the plurality of probes are activated to form a sparse subset of probes, the sparse subset of probes having probes that are spatially separated by at least a microscope resolution. The sample is illuminated with a readout light source, and light emitted from activated probes is detected. Based on the light emission detected from the activated probes, localized three-dimensional positions of the activated probes are obtained.

Microscopic imaging techniques

A microscopy system is configured for creating 3D images from individually localized probe molecules. The microscopy system includes a sample stage, an activation light source, a readout light source, a beam splitting device, at least one camera, and a controller. The activation light source activates probes of at least one probe subset of photo-sensitive luminescent probes, and the readout light source causes luminescence light from the activated probes. The beam splitting device splits the luminescence light into at least two paths to create at least two detection planes that correspond to the same or different number of object planes of the sample. The camera detects simultaneously the at least two detection planes, the number of object planes being represented in the camera by the same number of recorded regions of interest. The controller is programmable to combine a signal from the regions of interest into a 3D data.

Growing community of inventors

Branford, CT, United States of America

Joerg Bewersdorf