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-10520714-B2
Publication Date
2019-12-31
Expiration Date
2037-08-23
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Abstract
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 disclosed.
Core Innovation
The invention relates to systems and methods for instant structured illumination microscopy (SIM) where total internal reflection fluorescence (TIRF) techniques are employed to improve optical sectioning and the signal-to-noise ratio of structured illumination microscopy. The systems use a radial aperture block, a digital micromirror device, or a spatial light modulator positioned at a plane conjugate to the back focal plane of a high numerical aperture objective lens to allow only high-angle marginal rays to excite the sample, thereby producing evanescent, patterned illumination.
The problem being addressed is the limitation in speed and image acquisition efficiency in combining TIRF with structured illumination microscopy. Traditional TIRF SIM requires multiple raw images (e.g., nine) to be acquired and computationally processed to achieve enhanced resolution, which slows acquisition relative to conventional TIRF microscopy. There is a need for a method that combines SIM with TIRF conditions without resulting in slower speed or excessive computational complexity.
Claims Coverage
The patent contains multiple independent claims that disclose systems for structured illumination microscopy with different configurations of excitation and beam manipulation.
Radial aperture block to enable high-angle excitation with local contraction of fluorescence emissions
A system employing a radial aperture block positioned at the front focal plane of a first lens that blocks low-angle central rays and allows high-angle marginal rays through. These rays are scanned via a galvanometric mirror through an objective lens to evanescently excite the sample. The resulting fluorescence emissions are descanned and focused onto a second microlens array that locally contracts the emissions into a contracted pattern before detection.
Digital micro-mirror device for selective reflection of low-angle rays to modulate evanescent field thickness
A system that replaces the radial aperture block with a digital micro-mirror device (DMD) positioned at the front focal plane of the first lens. The DMD reflects low-angle central rays off axis, allowing only high-angle marginal rays to illuminate the sample. The reflective zone of pixels on the DMD can be varied to modulate the thickness of the evanescent field at the sample. The fluorescence emissions are rescanned and locally contracted by a microlens array before detection.
Spatial light modulator for phase-controlled generation of high-contrast excitation foci with evanescent illumination
A system using a spatial light modulator (SLM) to control the phase of the excitation beam to generate an array of excitation foci with minimal interference. A radial aperture block blocks low-angle rays, allowing only high-angle marginal rays through for evanescent excitation of the sample. The fluorescence emissions are descanned, locally contracted via a microlens array, and rescanned for imaging.
These inventive features together describe novel springing illumination arrangements using radial aperture blocks, digital micro-mirror devices, or spatial light modulators placed at conjugate back focal planes to selectively allow high-angle rays to evanescently excite the sample. The systems employ descanning, local contraction of fluorescence emissions via microlens arrays or spinning disk arrangements, and rescanning onto detectors. These arrangements facilitate high-speed, high-resolution structured illumination microscopy with improved signal-to-noise ratio and optical sectioning.
Stated Advantages
Provides fundamentally faster operation than classic TIRF-SIM systems by requiring acquisition of only one image versus multiple images, enabling a ˜50 fold increase in imaging speed.
Reduces read noise and computational processing needs by acquiring fewer images and requiring only simple deconvolution rather than extensive computational image processing in Fourier space.
Maintains or improves spatial resolution (~115 nm) comparable to conventional instant SIM despite the use of high-angle evanescent illumination.
Offers high signal-to-noise ratios and improved optical sectioning through use of evanescent wave excitation and localized contraction of fluorescence emissions.
Enables high-speed super-resolution microscopy suitable for biological imaging within the evanescent wave decay length (~200 nm of the coverslip surface).
Allows modulation of the evanescent field thickness via a digital micromirror device for nanometer-scale axial localization.
Documented Applications
Cell biology applications requiring high contrast, super-resolution imaging at or near the coverslip boundary such as imaging of cell membranes and plasma membrane protein dynamics.
Imaging dynamics of microtubules, small GTPases like HRas, and intracellular transport proteins such as Rab11 in live cells.
Dual-color live cell imaging combining fluorescently tagged proteins to study spatial and temporal distributions at the plasma membrane.
Tracking fast intracellular particle motion at high temporal resolution, such as Rab11 at 100 Hz frame rates.
Visualization of intracellular calcium flux, actin, myosin IIA, endoplasmic reticulum structures, and other sub-cellular features well matched to high spatiotemporal resolution imaging needs.
Super-resolution imaging enabling the observation of nanometer-scale features such as protein microdomains and microtubules spaced less than 150 nm apart.
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