Systems and methods for instant total internal reflection fluorescence/ structured illumination microscopy
Inventors
Shroff, Hari • Taraska, Justin • Giannini, John • Wu, Yicong • Kumar, Abhishek • Guo, Min
Assignees
US Department of Health and Human Services
Publication Number
US-10914933-B2
Publication Date
2021-02-09
Expiration Date
2037-08-23
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Abstract
Various embodiments related to systems and methods for instant structured microscopy where total internal reflection fluorescence techniques are used to improve optical sectioning and signal-to-noise ratio of structured illumination microscopy are herein disclosed.
Core Innovation
The invention relates to systems and methods for instant structured illumination microscopy (SIM) where total internal reflection fluorescence (TIRF) techniques are used to improve optical sectioning and signal-to-noise ratio. The systems employ high-angle marginal annular light beams to excite a sample, enabling evanescent wave excitation without light propagation into the sample, thereby enhancing image contrast and resolution near the sample surface.
The invention addresses the problem in traditional SIM combined with TIRF where the requirement of multiple raw images (e.g., nine) slows acquisition relative to conventional TIRF microscopy. The invention provides a method combining SIM with TIRF without loss of speed, enabling high-speed, super-resolution microscopy at very high signal-to-noise ratios, particularly within a 200 nm distance from a coverslip surface.
Claims Coverage
The patent claims include one independent system claim and one independent method claim, both centered on structured illumination microscopy systems that employ a spinning disk arrangement with converging microlenses and a radial aperture block to achieve TIRF-based super-resolution imaging.
Spinning disk system with radial aperture block for TIRF structured illumination microscopy
A system comprising: a light source generating excitation light; a first spinning disk with converging microlenses converting the excitation light into multiple excitation foci covering the sample field; a radial aperture block positioned between lenses in a telescopic arrangement to block low-angle central rays and allow high-angle marginal rays; a dichroic mirror directing the high-angle rays through a high numerical aperture objective focusing on the sample to generate fluorescent emissions; a spinning pinhole disk with pinholes matching the pitch of the first spinning disk to block out-of-focus emissions; a second spinning disk with converging microlenses positioned between the pinhole disk and sample to locally contract fluorescent emissions into contracted, non-inverted emissions; and a detector capturing these contracted emissions.
Method for TIRF structured illumination microscopy using spinning disks and radial aperture block
A method including: generating excitation light; converting this into multiple excitation foci covering the sample via a first spinning disk with converging microlenses; relaying these foci to an intermediate image plane; blocking low-angle central rays while allowing high-angle marginal rays through a radial aperture block; redirecting high-angle rays through an objective to excite the sample and produce fluorescent emissions; blocking out-of-focus or scattered emissions; and locally contracting the fluorescent emissions into contracted, non-inverted emissions for detection.
The independent claims disclose systems and methods employing a spinning disk arrangement with converging microlenses and a radial aperture block that selectively transmits high-angle marginal rays to produce evanescent wave excitation for total internal reflection fluorescence microscopy combined with structured illumination, enabling high-speed, super-resolution imaging with improved optical sectioning and reduced out-of-focus light.
Stated Advantages
The invention provides fundamentally faster operation than classic TIRF-SIM systems by requiring only one image acquisition instead of multiple, enhancing speed.
Reduced read noise due to fewer images being acquired improves image quality.
Less computational processing is needed, requiring only simple deconvolution rather than extensive Fourier space processing.
Achieves high spatial resolution (~115 nm) comparable to conventional instant SIM without degrading resolution in TIRF mode.
Enables high-speed imaging up to 100 Hz, suitable for live-cell biological applications with high spatiotemporal resolution.
Documented Applications
High-speed, super-resolution imaging of fluorescently labeled biological samples at or near coverslip surfaces within approximately 200 nm, including live-cell imaging.
Imaging dynamics of proteins such as microtubules, GTPases like HRas, and myosin IIA at the plasma membrane.
Dual-color live-cell imaging of protein distributions, for example imaging Halotag-HRAS with GFP-VSVG or GFP-HRAS with ER markers.
Tracking of intracellular particles such as Rab11 decorated vesicles at high frame rates for analysis of cellular transport dynamics.
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