Focus image scanning microscopy for sharp and gentle super-resolved microscopy

Abstract

To date, the feasibility of super-resolution microscopy for imaging live and thick samples is still limited. Stimulated emission depletion (STED) microscopy requires high-intensity illumination to achieve sub-diffraction resolution, potentially introducing photodamage to live specimens. Moreover, the out-of-focus background may degrade the signal stemming from the focal plane. Here, we propose a new method to mitigate these limitations without drawbacks. First, we enhance a STED microscope with a detector array, enabling image scanning microscopy (ISM). Therefore, we implement STED-ISM, a method that exploits the working principle of ISM to reduce the depletion intensity and achieve a target resolution. Later, we develop Focus-ISM, a strategy to improve the optical sectioning and remove the background of any ISM-based imaging technique, with or without a STED beam. The proposed approach requires minimal architectural changes to a conventional microscope but provides substantial advantages for live and thick sample imaging.

Tracking the Dissolution Surface Kinetics of a Single Fluorescent Cyclodextrin Metal–Organic Framework by Confocal Laser Scanning Microscopy

“…It was found that (i) three processes, including nucleation, germination growth, and saturation stages, were found in the growth of CD-MOF⊃FL and the growth kinetics followed Avrami’s model, (ii) the dissolution rate at the face of a single CD-MOF⊃FL crystal was slower than that of its arris and the dissolution rate of the CD-MOF⊃FL crystal was increased with the increase of the water amount in methanol solution, and (iii) the dissolution process of the CD-MOF⊃FL crystal is a competitive process of erosion and diffusion in different methanol aqueous solutions and the dissolution kinetics followed the Korsmeyer–Peppas model. In this work, the spatial resolution of CLSM is limited at the micrometer scale, which can be improved by the employment of super-resolution reconstruction technology at the nanometer scale . These results offer new insights into the nature of dissolution kinetics of CD-MOF and provide new venues for the quantitative analysis of solid dissolution and growth at the single-particle level.…”

“…In this work, the spatial resolution of CLSM is limited at the micrometer scale, which can be improved by the employment of super-resolution reconstruction technology at the nanometer scale. 54 These results offer new insights into the nature of dissolution kinetics of CD-MOF and provide new venues for the quantitative analysis of solid dissolution and growth at the single-particle level.…”

Photon-Weight-Reallocated Stimulated Emission Depletion Nanoscopy

“…However, in general, the signal collection capabilities of both central and edge detectors tend to become more similar as the defocus increases. The f-STED-ISM method removes the background of the STED using a square array detector. It supplied an adaptive background suppression method based on the linear ratio relationship between in-focus and out-of-focus Gaussian standard deviations.…”

“…Nonetheless, these methods require optically complex and cumbersome imaging processes, making them unfriendly to users and samples. A method termed as focus-image-scanning STED (f-STED-ISM) utilizes a cellular array detector to record the defocus information and remove the background disturbances of the image. However, this method does not work well when removing the peripheral inadequate-depletion fluorescence.…”

Compact and effective photon-resolved image scanning microscope

“…The combination of tailored data acquisition (DAQ) systems with these SPAD array detectors enables photon-resolved measurement of the fluorescence emission: every photon can be tagged with (i) spatial signatures, namely, the emission and detection coordinates of the photon in the detector and sample plane, respectively, and (ii) temporal signatures, namely, the time delay of the photon with respect to specific reference events. A series of recent publications 5 – 10 have demonstrated the benefits of leveraging the unique information provided by the SPAD arrays. The photon-resolved spatial and temporal information improves the performance of most advanced LSM-based techniques and paves the way for new ones.…”