Expand this Topic clickable element to expand a topic
Skip to content
Optica Publishing Group

Image scanning microscopy with multiphoton excitation or Bessel beam illumination

Not Accessible

Your library or personal account may give you access

Abstract

Image scanning microscopy is a technique of confocal microscopy in which the confocal pinhole is replaced by a detector array, and the image is reconstructed most straightforwardly by pixel reassignment. In the fluorescence mode, the detector array collects most of the fluorescent light, so the signal-to-noise ratio is much improved compared with confocal microscopy with a small pinhole, while the resolution is improved compared with conventional fluorescence microscopy. Here we consider two cases in which the illumination and detection point spread functions are dissimilar: illumination with a Bessel beam and multiphoton microscopy. It has been shown previously that for Bessel beam illumination in image scanning microscopy with a large array, the imaging performance is degraded. On the other hand, it is also known that the resolution of confocal microscopy is improved by Bessel beam illumination. Here we analyze image scanning microscopy with Bessel beam illumination together with a small array and show that an improvement in transverse resolution (width of the point spread function) by a factor of 1.78 compared with a conventional fluorescence microscope can be obtained. We also examine the behavior of image scanning microscopy in two- or three-photon fluorescence and for two-photon excitation also with Bessel beam illumination. The combination of the optical sectioning effect of image scanning microscopy with multiphoton microscopy reduces background from the sample surface, which can increase penetration depth. For a detector array size of two Airy units, the resolution of two-photon image scanning microscopy is a factor 1.85 better and the peak of the point spread function 2.84 times higher than in nonconfocal two-photon fluorescence. The resolution of three-photon image scanning microscopy is a factor 2.10 better, and the peak of the point spread function is 3.77 times higher than in nonconfocal three-photon fluorescence. The resolution of two-photon image scanning microscopy with Bessel beam illumination is a factor 2.13 better than in standard two-photon fluorescence. Axial resolution and optical sectioning in two-photon or three-photon fluorescence are also improved by using the image scanning modality.

© 2020 Optical Society of America

Full Article  |  PDF Article
More Like This
Pixel reassignment in image scanning microscopy: a re-evaluation

Colin J. R. Sheppard, Marco Castello, Giorgio Tortarolo, Takahiro Deguchi, Sami V. Koho, Giuseppe Vicidomini, and Alberto Diaspro
J. Opt. Soc. Am. A 37(1) 154-162 (2020)

Image formation in image scanning microscopy, including the case of two-photon excitation

Colin J. R. Sheppard, Marco Castello, Giorgio Tortarolo, Giuseppe Vicidomini, and Alberto Diaspro
J. Opt. Soc. Am. A 34(8) 1339-1350 (2017)

Pixel reassignment in image scanning microscopy with a doughnut beam: example of maximum likelihood restoration

Colin J. R. Sheppard, Marco Castello, Giorgio Tortarolo, Eli Slenders, Takahiro Deguchi, Sami V. Koho, Paolo Bianchini, Giuseppe Vicidomini, and Alberto Diaspro
J. Opt. Soc. Am. A 38(7) 1075-1084 (2021)

Cited By

You do not have subscription access to this journal. Cited by links are available to subscribers only. You may subscribe either as an Optica member, or as an authorized user of your institution.

Contact your librarian or system administrator
or
Login to access Optica Member Subscription

Figures (22)

You do not have subscription access to this journal. Figure files are available to subscribers only. You may subscribe either as an Optica member, or as an authorized user of your institution.

Contact your librarian or system administrator
or
Login to access Optica Member Subscription

Tables (1)

You do not have subscription access to this journal. Article tables are available to subscribers only. You may subscribe either as an Optica member, or as an authorized user of your institution.

Contact your librarian or system administrator
or
Login to access Optica Member Subscription

Equations (7)

You do not have subscription access to this journal. Equations are available to subscribers only. You may subscribe either as an Optica member, or as an authorized user of your institution.

Contact your librarian or system administrator
or
Login to access Optica Member Subscription

Select as filters


Select Topics Cancel
© Copyright 2024 | Optica Publishing Group. All Rights Reserved