Optimising performance of a confocal fluorescence microscope with a differential pinhole

Kakade, Rohan; Walker, John; Phillips, A.J.

doi:10.1088/0957-0233/27/1/015401

Cited by 7 publications

(2 citation statements)

References 35 publications

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“…We note that the advantage of using information from differences in confocal pinhole size has long been recognized (Heintzmann et al, 2003;Kakade et al, 2015;Martinez-Corral et al, 2003;Wang et al, 2013) and the virtual adaptable aperture system (VAAS) from Nikon (Okugawa, 2008) represents a commercial implementation based on the same principle. However, most of the proposed techniques are based on the weighted subtraction of two images.…”

Section: Split-pin Imaging Of Subcellular Structuresmentioning

confidence: 99%

A phasor‐based approach to improve optical sectioning in any confocal microscope with a tunable pinhole

D'Amico

Franco

Cerutti

et al. 2022

Microscopy Res & Technique

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Confocal fluorescence microscopy is a well‐established imaging technique capable of generating thin optical sections of biological specimens. Optical sectioning in confocal microscopy is mainly determined by the size of the pinhole, a small aperture placed in front of a point detector. In principle, imaging with a closed pinhole provides the highest degree of optical sectioning. In practice, the dramatic reduction of signal‐to‐noise ratio (SNR) at smaller pinhole sizes makes challenging the use of pinhole sizes significantly smaller than 1 Airy Unit (AU). Here, we introduce a simple method to “virtually” perform confocal imaging at smaller pinhole sizes without the dramatic reduction of SNR. The method is based on the sequential acquisition of multiple confocal images acquired at different pinhole aperture sizes and image processing based on a phasor analysis. The implementation is conceptually similar to separation of photons by lifetime tuning (SPLIT), a technique that exploits the phasor analysis to achieve super‐resolution, and for this reason we call this method SPLIT‐pinhole (SPLIT‐PIN). We show with simulated data that the SPLIT‐PIN image can provide improved optical sectioning (i.e., virtually smaller pinhole size) but better SNR with respect to an image obtained with closed pinhole. For instance, two images acquired at 2 and 1 AU can be combined to obtain a SPLIT‐PIN image with a virtual pinhole size of 0.2 AU but with better SNR. As an example of application to biological imaging, we show that SPLIT‐PIN improves confocal imaging of the apical membrane in an in vitro model of the intestinal epithelium. Research Highlights We describe a method to boost the optical sectioning power of any confocal microscope. The method is based on the sequential acquisition of multiple confocal images acquired at different pinhole aperture sizes. The resulting image series is analyzed using the phasor‐based separation of photons by lifetime tuning (SPLIT) algorithm. The SPLIT‐pinhole (SPLIT‐PIN) method produces images with improved optical sectioning but preserved SNR. This is the first time that the phasor analysis and SPLIT algorithms are used to exploit the spatial information encoded in a tunable pinhole size and to improve optical sectioning of the confocal microscope.

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Section: Split-pin Imaging Of Subcellular Structuresmentioning

confidence: 99%

A phasor‐based approach to improve optical sectioning in any confocal microscope with a tunable pinhole

D'Amico

Franco

Cerutti

et al. 2022

Microscopy Res & Technique

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“…It achieves this by rejecting light that does not pass through the detection pinhole; thus this detection arrange ment further limits the number of photons that are collected with a subsequent impact on the signal tonoise ratio (SNR). By using alternative pinhole geometries such as in subtractive imaging [4][5][6][7][8][9] it is possible to improve the 3D resolution of a CFM system. These approaches however achieve the same at the expense of SNR.…”

Section: Introductionmentioning

confidence: 99%

Resolution and signal-to-noise ratio improvement in confocal fluorescence microscopy using array detection and maximum-likelihood processing

Kakade¹,

Walker²,

Phillips³

2016

Meas. Sci. Technol.

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Confocal fluorescence microscopy (CFM) is widely used in biological sciences because of its enhanced 3D resolution that allows image sectioning and removal of out-of-focus blur. This is achieved by rejection of the light outside a detection pinhole in a plane confocal with the illuminated object. In this paper, an alternative detection arrangement is examined in which the entire detection/image plane is recorded using an array detector rather than a pinhole detector. Using this recorded data an attempt is then made to recover the object from the whole set of recorded photon array data; in this paper maximum-likelihood estimation has been applied. The recovered object estimates are shown (through computer simulation) to have good resolution, image sectioning and signal-to-noise ratio compared with conventional pinhole CFM images.

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SPLIT-PIN software enabling confocal and super-resolution imaging with a virtually closed pinhole

Franco

Costantino

Cerutti

et al. 2023

Sci Rep

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In point-scanning microscopy, optical sectioning is achieved using a small aperture placed in front of the detector, i.e. the detection pinhole, which rejects the out-of-focus background. The maximum level of optical sectioning is theoretically obtained for the minimum size of the pinhole aperture, but this is normally prevented by the dramatic reduction of the detected signal when the pinhole is closed, leading to a compromise between axial resolution and signal-to-noise ratio. We have recently demonstrated that, instead of closing the pinhole, one can reach a similar level of optical sectioning by tuning the pinhole size in a confocal microscope and by analyzing the resulting image series. The method, consisting in the application of the separation of photons by lifetime tuning (SPLIT) algorithm to series of images acquired with tunable pinhole size, is called SPLIT-pinhole (SPLIT-PIN). Here, we share and describe a SPLIT-PIN software for the processing of series of images acquired at tunable pinhole size, which generates images with reduced out-of-focus background. The software can be used on series of at least two images acquired on available commercial microscopes equipped with a tunable pinhole, including confocal and stimulated emission depletion (STED) microscopes. We demonstrate applicability on different types of imaging modalities: (1) confocal imaging of DNA in a non-adherent cell line; (2) removal of out-of-focus background in super-resolved STED microscopy; (3) imaging of live intestinal organoids stained with a membrane dye.

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Optimising performance of a confocal fluorescence microscope with a differential pinhole

Cited by 7 publications

References 35 publications

A phasor‐based approach to improve optical sectioning in any confocal microscope with a tunable pinhole

A phasor‐based approach to improve optical sectioning in any confocal microscope with a tunable pinhole

Resolution and signal-to-noise ratio improvement in confocal fluorescence microscopy using array detection and maximum-likelihood processing

SPLIT-PIN software enabling confocal and super-resolution imaging with a virtually closed pinhole

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