Synergic Combination of Stimulated Emission Depletion Microscopy with Image Scanning Microscopy to Reduce Light Dosage

Tortarolo, Giorgio; Castello, Marco; Koho, Sami; Vicidomini, Giuseppe

doi:10.1101/741389

Cited by 6 publications

(5 citation statements)

References 35 publications

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“…It is readily implementable without any hardware changes. When combined with other concepts like event-triggered imaging 24 and/or single-photon avalanche diode (SPAD) array detector 25 , our method can further reduce the phototoxic effects of live-cell STED to a bare minimum. Similarly, our concept could be combined with an ultrafast scanning system 26 to enable gentle live-cell nanoscopy at maximum speed.…”

Section: Resultsmentioning

confidence: 99%

Deep learning enables fast, gentle STED microscopy

et al. 2023

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STED microscopy is widely used to image subcellular structures with super-resolution. Here, we report that restoring STED images with deep learning can mitigate photobleaching and photodamage by reducing the pixel dwell time by one or two orders of magnitude. Our method allows for efficient and robust restoration of noisy 2D and 3D STED images with multiple targets and facilitates long-term imaging of mitochondrial dynamics.

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Section: Resultsmentioning

confidence: 99%

Deep learning enables fast, gentle STED microscopy

et al. 2023

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“…The point-spread function (PSF) of the scanned-image generated by the element at position x d of a detector array is given by the PSF of a laser scanning microscope, just with a shifted pinhole aperture [8, 9]. The equation is where x s = ( x s , y s ) are the scan coordinates on the sample plane in focus, p is the pinhole aperture that describes the sensitive area of the element of the detector array located at x d = ( x d , y d ), and h exc and h det are, respectively, the excitation and detection normalized PSFs .…”

Section: Supplementary Materialsmentioning

confidence: 99%

Focus-ISM for Sharp and Gentle Super-Resolved Microscopy

Tortarolo

Zunino

Fersini

et al. 2022

Preprint

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Super-resolution microscopy is routinely used for fixed and thin samples, while its feasibility for imaging live and thick samples is still limited. In the case of stimulated emission depletion (STED) microscopy, the high-intensity illumination required to achieve effective sub-diffraction resolution can introduce photo-damage, thus reducing the compatibility of the technique with live-cell imaging. Moreover, the out-of-focus fluorescence background may overcome the often faint signal stemming from the focal point, thus constraining imaging to thin samples. Here, we combined STED microscopy with image-scanning microscopy (ISM) to mitigate these limitations without any practical disadvantages. We first enhanced a laser scanning microscope (LSM) by introducing a detector array, hence providing access to a set of additional spatial information that is not available with a typical single-element detector. Then, we exploited this extended dataset to implement focus-ISM, a novel method that relaxes the high-intensity requirement of STED microscopy and removes the out-of-focus background. Additionally, we generalized the focus-ISM method to conventional LSM, namely without a STED beam. The proposed approach requires minimal architectural changes compared with conventional STED microscopes but provides substantial advantages for live and thick sample imaging while maintaining all compatibility with all recent advances in STED and confocal microscopy. As such, focus-ISM represents an essential step towards a universal super-resolved LSM technique for subcellular imaging.

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“…Our imagers have been successfully integrated into ISM experiments, to demonstrate the advantages of a SPAD-based detector and to compare the lower noise of 0.35 µm-HVCMOS device against the higher PDE of 0.16 µm-BCD one. Both SPAD arrays have been integrated into a custom confocal laser-scanning microscope, replacing its single-point detector, as described in detail in [47,48,27]. Output lines from the sensor array have been connected to an FPGA-based board (NI-USB-7856R from National Instruments) for counting photons detected in each laser spot position and for managing the entire microscope system (including the synchronization with scanning devices).…”

Section: Image Scanning Microscopy Experimentsmentioning

confidence: 99%

“…In a nutshell, image scanning microscopy requires to collect the image of excitation/detection region for each scanning position of the sample. Since the excitation region is diffraction-limited, its size is typically in the range of few hundreds nanometer (the size reduces when ISM is combined with STED microscopy [27]) and the pixel dwell-time is in the microseconds range (few tens of nanoseconds when ISM is combined with resonant scanners). Thus, it is clear the importance of having a detector array with a limited number of pixel but with asynchronous readout (no frame-rate).…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

SPAD-based asynchronous-readout array detectors for image-scanning microscopy

Buttafava¹,

Villa²,

Castello³

et al. 2020

Optica

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Fluorescence microscopy and derived techniques are continuously looking for photodetectors able to guarantee increased sensitivity, high spatial and temporal resolution and ease of integration into modern microscopy architectures. Recent advances in single-photon avalanche diodes (SPADs) fabricated with industry-standard microelectronic processes allow the development of new detection systems tailored to address the requirements of advanced imaging techniques (such as image-scanning microscopy). To this aim, we present the complete design and characterization of two bidimensional SPAD arrays composed of 25 fully independent and asynchronously-operated pixels, both having fill-factor of about 50% and specifically designed for being integrated into existing laser scanning microscopes. We used two different microelectronics technologies to fabricate our detectors: the first technology exhibiting very low noise (roughly 200 dark counts per second at room temperature), and the second one showing enhanced detection efficiency (more than 60% at a wavelength of 500 nm). Starting from the silicon-level device structures and moving towards the in-pixel and readout electronics description, we present performance assessments and comparisons between the two detectors. Images of a biological sample acquired after their integration into our custom imagescanning microscope finally demonstrate their exquisite on-field performance in terms of spatial resolution and contrast enhancement. We envisage that this work can trigger the development of a new class of SPAD-based detector arrays able to substitute the typical singleelement sensor used in fluorescence laser scanning microscopy.

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Synergic Combination of Stimulated Emission Depletion Microscopy with Image Scanning Microscopy to Reduce Light Dosage

Cited by 6 publications

References 35 publications

Deep learning enables fast, gentle STED microscopy

Deep learning enables fast, gentle STED microscopy

Focus-ISM for Sharp and Gentle Super-Resolved Microscopy

SPAD-based asynchronous-readout array detectors for image-scanning microscopy

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