EVALUATION OF STED SUPER-RESOLUTION IMAGE QUALITY BY IMAGE CORRELATION SPECTROSCOPY (QuICS)

Cerutti, Elena; D'Amico, Morgana; Cainero, Isotta; Dellino, Gaetano Ivan; Faretta, Mario; Vicidomini, Giuseppe; Pelicci, Pier Giuseppe; Bianchini, Paolo; Diaspro, Alberto; Lanzanò, Luca

doi:10.1101/2021.08.30.457899

Cited by 1 publication

(6 citation statements)

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“…The generated SPLIT‐PIN images were analyzed using a recently introduced algorithm that evaluates the image quality by image correlation spectroscopy (QuICS) (Cerutti et al, 2021). The QuICS analysis was performed in MATLAB using the code QuICS_v2.m available at https://github.com/llanzano/QuICS.…”

Section: Methodsmentioning

confidence: 99%

“…Finally, the parameters R , B , and N have been calculated as follows:

\begin{matrix} R goodbreak= \sqrt{2 italicln 2} w, \\ B goodbreak= G_{NF} ((), 0) I_{italicav,} \\ N goodbreak= \frac{G ((), 0) - G_{NF} ((), 0)}{G_{NF} ((), 0)}, \end{matrix}

where we have indicated I av as the average intensity value over all the pixels of the image. R is the width of the autocorrelation function, related to the spatial resolution; B is the brightness, related to the image contrast; and N is the relative noise variance, related to the SNR of the image (Cerutti et al, 2021).…”

Section: Methodsmentioning

confidence: 99%

“…To evaluate the quality of the images provided by SPLIT-PIN, we use the recently introduced QuICS algorithm, a tool based on image correlation spectroscopy (Cerutti et al, 2021). QuICS allows extracting three parameters related to the resolution, contrast e SNR of the image.…”

Section: Introductionmentioning

confidence: 99%

“…In STED microscopy, this additional channel can be represented by the fluorescence lifetime variations induced by a STED beam (Coto Hernandez et al, 2019; Lanzano et al, 2015; Lanzano et al, 2017; Tortarolo et al, 2019; Wang et al, 2018) or a tunable depletion power (Pelicci et al, 2020; Sarmento et al, 2018). In STED microscopy, the application of SPLIT produces images that have higher resolution and better contrast, compared to their counterpart STED images (Cerutti et al, 2021). Recently, we have shown that SPLIT can be applied also to structured illumination microscopy (SIM), using as the additional channel the illumination pattern translation step (Cainero et al, 2021).…”

Section: Introductionmentioning

confidence: 99%

“…Notably, our method can be implemented on any confocal microscope equipped with a tunable pinhole size (to the best of our knowledge, most of the commercially available confocal laser scanning microscopes allow the users to tune the size of the pinhole). To evaluate the quality of the images provided by SPLIT‐PIN, we use the recently introduced QuICS algorithm, a tool based on image correlation spectroscopy (Cerutti et al, 2021). QuICS allows extracting three parameters related to the resolution, contrast e SNR of the image.…”

Section: Introductionmentioning

confidence: 99%

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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: Methodsmentioning

confidence: 99%

“…Finally, the parameters R , B , and N have been calculated as follows: