Abstract: Described herein are 3D single-molecule super-resolution imaging systems and methods. The provided systems and methods use modulation interferometry and phase-sensitive detection techniques that achieve less than 2 nanometer axial localization precision, which is well below the 5-10-nanometer-sized individual protein components. To illustrate the capability of this technique in probing the dynamics of complex macromolecular machines, (1) movement of individual multi-subunit E. coli RNA Polymerases were visualized through the complete transcription cycle, (2) kinetics of the initiation-elongation transition were dissected, (3) the conformational changes from the open initiation complex to the elongation complex were analyzed, and (4) the fate of ?70 initiation factors during promoter escape were determined.

Abstract: Disclosed is three-dimensional interferometric lattice light-sheet (3D-iLLS) imaging, an approach that overcomes limitations of prior microscopy techniques. 3D-iLLS provides, by virtue of selective-plane illumination (SPIM), low light levels and photobleaching, while providing increased background suppression and significantly improved volumetric imaging/sectioning capabilities through 4Pi interferometry. An example setup demonstrated 3D-iLLS with axial resolution and single-particle localization precision down to <100 nm (FWHM) and <10 nm (1?), respectively. 3D-iLLS enables a fuller elucidation of sub-cellular phenomena by enhanced 4D resolution and improved SNR during live imaging.

Abstract: A target may be tagged, and its position tracked to maintain the position of the target at the center (e.g., zero region) of a suppression beam, where suppression is minimal, so the signal from the target is not suppressed relative to signal from background molecules. Point scanning is not needed, as a pixelated detector of a camera can be used to acquire the position of the target in one shot. Images of the object from the pixelated detector can be analyzed to acquire the position of the target. To maintain the target in the zero, the beams may be steered onto the target. Alternatively or additionally, the sample may be moved to place the sample in the center. A correction signal may be sent to cause the sample and/or beams to be moved to maintain the target in position.

Abstract: An embodiment of the present invention is an imaging arrangement that includes imaging optics, a fiducial light source, and a control system. In operation, the imaging optics separate light into first and second tight by wavelength and project the first and second light onto first and second areas within first and second detector regions, respectively. The imaging optics separate fiducial light from the fiducial light source into first and second fiducial light and project the first and second fiducial light onto third and fourth areas within the first and second detector regions, respectively. The control system adjusts alignment of the imaging optics so that the first and second fiducial light projected onto the first and second detector regions maintain relatively constant positions within the first and second detector regions, respectively. Another embodiment of the present invention is a microscope that includes the imaging arrangement.

Abstract: An embodiment of the present invention is an imaging arrangement that includes imaging optics, a fiducial light source, and a control system. In operation, the imaging optics separate light into first and second tight by wavelength and project the first and second light onto first and second areas within first and second detector regions, respectively. The imaging optics separate fiducial light from the fiducial light source into first and second fiducial light and project the first and second fiducial light onto third and fourth areas within the first and second detector regions, respectively. The control system adjusts alignment of the imaging optics so that the first and second fiducial light projected onto the first and second detector regions maintain relatively constant positions within the first and second detector regions, respectively. Another embodiment of the present invention is a microscope that includes the imaging arrangement.