Aberrations in 4Pi Microscopy

Hao, Xiang; Antonello, Jacopo; Allgeyer, Edward S.; Bewersdorf, Joerg; Booth, Martin J.

doi:10.1364/oe.25.014049

Cited by 25 publications

(21 citation statements)

References 22 publications

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“…One approach is multiplane imaging [4][5][6][7] , which requires simultaneous acquisition of multiple images, and was reported to be applicable to an axial range of about 4 μm by scanning of the focal plane through the sample. A second approach is to use interferometry [8][9][10] , which can result in very high localization precision at the expense of optical complexity and limited axial range per slice. In this work, we use the powerful approach of point spread function (PSF) engineering (for a review, see 11 ) that allows for scan-free wide field SR imaging over a several µm axial range per slice.…”

Section: Tilted Light Sheet Microscopy With 3d Point Spread Functionsmentioning

confidence: 99%

3D single-molecule super-resolution microscopy with a tilted light sheet

Gustavsson

Petrov

Lee

et al. 2017

Preprint

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Tilted light sheet microscopy with 3D point spread functions (TILT3D) combines a tilted light sheet illumination scheme and engineered point spread functions with axial ranges up to ten µm for lowbackground, 3D super-localization of single molecules. This system is built upon a standard inverted microscope and has minimal custom parts. The result is simple and flexible 3D super-resolution imaging with tens of nm localization precision throughout thick mammalian cells.Several methods have been developed to extend the imaging capability of single-molecule super-resolution (SR) microscopy 1-3 to three dimensions (3D), including multiplane imaging, interferometric approaches, and point spread function (PSF) engineering (for a review, see 4 ). In the case of PSF engineering, the axial position of a molecule is encoded in the shape of the PSF, and strategies include astigmatism 5 and the bisected pupil PSF 6 with axial ranges of 1-2 µm, self-bending 7 and double-helix (DH) PSFs 8-11 with axial ranges of ~2-3 µm, and the recently developed Tetrapod PSFs 12, 13 which have a tunable range of up to 20 µm. PSF engineering typically only requires the addition of a small number of optical elements to a standard microscope, making the method relatively simple to implement while exhibiting high precision for 3D single-molecule localization.One method to improve the precision of single-molecule localization is to reduce the background coming from out-of-focus fluorophores by using light sheet illumination 14 , where the sample is excited by a thin sheet of light orthogonal to the detection axis. Numerous light sheet designs have been implemented for SR imaging [15][16][17][18][19][20] , but these designs have certain drawbacks such as being incompatible with imaging of fluorophores close to the coverslip using high numerical aperture (NA) imaging objectives 17, 18 or require complicated optical and electronic designs or many custom-made parts which are often expensive and difficult to build and operate, and thus may not be easily accessible to the general research community.Here, we present TILT3D, an imaging platform that combines a novel, tilted light sheet illumination strategy with long axial range PSFs. We alleviate many of the aforementioned difficulties in existing light sheet designs by tilting the illumination plane; for example, two perpendicular objectives in close proximity are not required. TILT3D: (a) yields high localization precision of single molecules in 3D over the entire axial range of a mammalian cell via scanning of the light sheet combined with imaging with engineered PSFs, (b) has the usual light sheet advantages of reduced photobleaching and photodamage of the sample, but most importantly (c) is easy and cost-efficient to implement and operate. We validated TILT3D for 3D SR imaging in mammalian cells by imaging microtubules, mitochondria, and the full nuclear lamina using both DH and Tetrapod PSFs.. CC-BY-NC-ND 4.0 International license peer-reviewed) is the author/funder. It is made available under aTh...

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Section: Tilted Light Sheet Microscopy With 3d Point Spread Functionsmentioning

confidence: 99%

3D single-molecule super-resolution microscopy with a tilted light sheet

Gustavsson

Petrov

Lee

et al. 2017

Preprint

View full text Add to dashboard Cite

show abstract

“…Furthermore, by combining 4Pi microscopy with a multi‐focal multi‐photon imaging technique, the recording speed can be increased . Combination of this microscopy with an optical phase conjugation system allows alignments simplification, while combination with adaptive optics facilitates the image quality improvement . 4Pi microscopy can also be incorporated into other super‐resolution imaging techniques, yielding setups such as 4Pi‐STED, 4Pi‐SMLM, and 4Pi‐RESOLFT, which provide multi‐color, live‐cell, 3D super‐resolution, real‐time, whole‐cell or SPT imaging for biological applications including characterization of actin cytoskeleton, transcription cycle, endoplasmic reticulum, bacteriophages, mitochondria, nuclear pore complexes, primary cilia, Golgi‐apparatus‐associated COPI vesicles, and mouse spermatocyte synaptonemal complexes .…”

Section: Methods For Axial Srfmmentioning

confidence: 99%

“…[68] Combination of this microscopy with an optical phase conjugation system allows alignments simplification, [69] while combination with adaptive optics facilitates the image quality improvement. [70] 4Pi microscopy can also be incorporated into other super-resolution imaging techniques, yielding setups such as 4Pi-STED, [71,72] 4Pi-SMLM, [73][74][75] and 4Pi-RESOLFT, [76] which provide multi-color, [74] live-cell, [66,68,77,78] 3D super-resolution, [73,79] real-time, [80] whole-cell [75] or SPT [80,81] imaging for biological applications including characterization of actin cytoskeleton, [79] transcription cycle, [80] endoplasmic Interference-based axial SRFM. a) Both the incident light and fluorescence from two opposite optical paths will interfere in Type-C 4Pi to increase the NA of the optical system.…”

Section: Pi Microscopymentioning

confidence: 99%

Breaking the Axial Diffraction Limit: A Guide to Axial Super‐Resolution Fluorescence Microscopy

Liu

Toussaint

Okoro

et al. 2018

Laser & Photonics Reviews

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Optical microscopy is a powerful tool for understanding the fundamentals of the microscopic world. However, for centuries its resolving ability remained limited by the optical diffraction limit. Super‐resolution fluorescence microscopy (SRFM) has been introduced to break the diffraction limit and significantly expand the fields in which optical microscopy can be applied. Unfortunately, SRFM contributes little towards axial resolution enhancement, rendering observation of the axial and three‐dimensional structures of biological tissues difficult; this may yield a misunderstanding of intracellular interactions. Based on the existing literature, the development of axial SRFM is still behind that of lateral SRFM. In light of this, this Review presents a comprehensive summary of the principles, development, characteristics, and applications of existing techniques for improving the axial resolution. This Review will provide a guide to researchers and promote further development of related technology.

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“…One approach is multiplane imaging 4 – 7 , which requires simultaneous acquisition of multiple images, and was reported to be applicable to an axial range of about 4 μm. A second approach is to use interferometry 8 – 10 , which can result in very high localization precision at the expense of optical complexity and limited axial range per slice. In this work, we use the powerful approach of point spread function (PSF) engineering (for a review, see ref.…”

Section: Introductionmentioning

confidence: 99%

3D single-molecule super-resolution microscopy with a tilted light sheet

et al. 2018

View full text Add to dashboard Cite

Tilted light sheet microscopy with 3D point spread functions (TILT3D) combines a novel, tilted light sheet illumination strategy with long axial range point spread functions (PSFs) for low-background, 3D super-localization of single molecules as well as 3D super-resolution imaging in thick cells. Because the axial positions of the single emitters are encoded in the shape of each single-molecule image rather than in the position or thickness of the light sheet, the light sheet need not be extremely thin. TILT3D is built upon a standard inverted microscope and has minimal custom parts. The result is simple and flexible 3D superresolution imaging with tens of nm localization precision throughout thick mammalian cells. We validate TILT3D for 3D super-resolution imaging in mammalian cells by imaging mitochondria and the full nuclear lamina using the double-helix PSF for single-molecule detection and the recently developed tetrapod PSFs for fiducial bead tracking and live axial drift correction.

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Aberrations in 4Pi Microscopy

Cited by 25 publications

References 22 publications

3D single-molecule super-resolution microscopy with a tilted light sheet

3D single-molecule super-resolution microscopy with a tilted light sheet

Breaking the Axial Diffraction Limit: A Guide to Axial Super‐Resolution Fluorescence Microscopy

3D single-molecule super-resolution microscopy with a tilted light sheet

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