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Systems and methods for improved axial resolution in microscopy using photoswitching and standing wave illumination techniques

A microscope system and standing wave technology, applied in microscopes, material analysis through optical means, optics, etc., can solve problems such as difficult alignment and construction of the system, difficulty in adapting to confocal geometry, and slowing down of the imaging process

Pending Publication Date: 2021-04-09
UNITED STATES OF AMERICA
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Problems solved by technology

First, I5S systems have hitherto required dual-objective interferometry and complex beam setups, which have made said systems difficult to align and construct due to the need to keep optical elements aligned along two separate paths (one for each objective) to achieve much better than spatial precision
Second, the I5S system requires 15 images per focal plane to achieve axial resolution improvement, which significantly slows down the imaging process and thus currently limits imaging of fixed cells
Third, the I5S system does not employ a confocal pinhole such that in densely labeled samples, Poisson noise from out-of-focus light will limit the focal plane contrast
Since the resolution improvements are coupled, this method is not easily adaptable to confocal geometries

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  • Systems and methods for improved axial resolution in microscopy using photoswitching and standing wave illumination techniques
  • Systems and methods for improved axial resolution in microscopy using photoswitching and standing wave illumination techniques
  • Systems and methods for improved axial resolution in microscopy using photoswitching and standing wave illumination techniques

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Embodiment Construction

[0018] It is well known that the axial resolution of conventional widefield fluorescence microscopy is limited to the range between ~500-700 nm. In fluorescence microscopy, systems and methods that can further improve the axial resolution are of great interest, because such improvements can observe more details in biological samples. Disclosed herein are various embodiments related to the systems and methods by acquiring only four additional images at each focal plane (for a total of five images per focal plane instead of one image) Axial resolution down to ~100nm to resolve these defects. Given the modest number of additional images required to increase the axial resolution of images, embodiments of the present systems and methods can be applied to persistent volumetric imaging ("4D imaging") in living cells, which is currently not possible with other microscopy techniques Achieved. Furthermore, the systems and methods of the present invention are flexible and can be combin...

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Abstract

Various embodiments for systems and methods for improved axial resolution in a microscopy using photoswitching and standing-wave illumination techniques are described.

Description

technical field [0001] This document relates generally to improving axial resolution in microscopy and, in particular, to systems and methods for improving axial resolution in instant structured illumination microscopy using optical switching and standing wave illumination techniques. Background technique [0002] In conventional widefield fluorescence microscopy, a known method of increasing the available axial spatial frequency (and thus resolution) is to use standing wave illumination. In this approach, two counterpropagating coherent beams are superimposed on the imaging focal plane. Interference between the beams produces clear periodic illumination fringes with a period given by λ / (2n cos θ), where λ is the illumination wavelength, n is the refractive index (index) of the medium, and θ is the "crossing angle" of the beams , that is, the angle relative to the vertical direction shown in Figure 1A. As shown, the two beams intersect the vertical axis (dotted line, also ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/64
CPCG01N21/6428G02B21/00G01N21/6458G02B21/082G02B21/367G02B21/16G02B21/0056G02B27/58G02B21/008G01N33/582G01N2021/6439G01N2021/6478G02B21/0032G02B21/0048G02B21/0076
Inventor H·施罗夫J·P·詹尼尼吴一聪P·J·拉里维尔郭敏陈记稷H·维什瓦斯劳李雪松
Owner UNITED STATES OF AMERICA