A robust and versatile platform for image scanning microscopy enabling super-resolution FLIM

Castello, Marco; Tortarolo, Giorgio; Buttafava, Mauro; Deguchi, Takahiro; Villa, Federica; Koho, Sami; Pesce, Luca; Oneto, Michele; Pelicci, Simone; Lanzanò, Luca; Bianchini, Paolo; Sheppard, Colin J. R.; Diaspro, Alberto; Tosi, Alberto; Vicidomini, Giuseppe

doi:10.1038/s41592-018-0291-9

Cited by 170 publications

(173 citation statements)

References 33 publications

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“…Another interesting perspective is the combination of our FRET method with super‐resolution microscopy. For instance, super‐resolved FLIM‐FRET measurements could be performed, thanks to the recent integration of FLIM with image scanning microscopy , a technique similar to SIM that does not require the use of special fluorophores. Finally, we believe that our method could be further improved with the use of novel fluorogenic dyes recently developed for a more efficient staining of chromatin .…”

Section: Discussionmentioning

confidence: 99%

Chromatin nanoscale compaction in live cells visualized by acceptor‐to‐donor ratio corrected Förster resonance energy transfer between DNA dyes

Pelicci

Diaspro

Lanzanò

2019

Journal of Biophotonics

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@Chromatin nanoscale architecture in live cells can be studied by Förster resonance energy transfer (FRET) between fluorescently labeled chromatin components, such as histones. A higher degree of nanoscale compaction is detected as a higher FRET level, since this corresponds to a higher degree of proximity between donor and acceptor molecules. However, in such a system, the stoichiometry of the donors and acceptors engaged in the FRET process is not well defined and, in principle, FRET variations could be caused by variations in the acceptor‐to‐donor ratio rather than distance. Here, to get a FRET level independent of the acceptor‐to‐donor ratio, we combine fluorescence lifetime imaging detection of FRET with a normalization of the FRET level to a pixel‐wise estimation of the acceptor‐to‐donor ratio. We use this method to study FRET between two DNA binding dyes staining the nuclei of live cells. We show that this acceptor‐to‐donor ratio corrected FRET imaging reveals variations of nanoscale compaction in different chromatin environments. As an application, we monitor the rearrangement of chromatin in response to laser‐induced microirradiation and reveal that DNA is rapidly decompacted, at the nanoscale, in response to DNA damage induction.

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Section: Discussionmentioning

confidence: 99%

Chromatin nanoscale compaction in live cells visualized by acceptor‐to‐donor ratio corrected Förster resonance energy transfer between DNA dyes

Pelicci

Diaspro

Lanzanò

2019

Journal of Biophotonics

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“…The new microscope opens the door to monitoring rapidly evolving processes or events that occur over temporally or spatially varying scales. We anticipate that coupling the technique with parallelized detection methods [9] or nonlinear excitation [15,29] will lead to improved signal-to-noise ratio, spatial resolution or penetration depth, helping to reconstruct complex 3D phenomena with maximal detail.…”

Section: Discussionmentioning

confidence: 99%

“…Based on the raster scanning of a laser beam for illumination CLSM provides optical sectioning with synchronous multi-channel imaging. In addition, these confocal architectures are compatible with super-resolution microscopy and advanced spectroscopic techniques such as fluorescence correlation spectroscopy and fluorescence lifetime imaging [5][6][7][8][9]. However, despite the complete toolset that CLSM represents, a central question in this imaging modality is how to select the main scanning parameters (number of pixels, pixel dwell time and scanned volume) to maximize the spatiotemporal information retrieved from a sample.…”

Section: Introductionmentioning

confidence: 99%

Volumetric Lissajous Confocal Microscopy

Deguchi

Bianchini

Palazzolo

et al. 2019

Preprint

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Dynamic biological systems present challenges to existing three-dimensional (3D) optical microscopes because of their continuous temporal and spatial changes. Most techniques are based on rigid architectures, as in confocal microscopy, where a laser beam is sequentially scanned at a predefined spatial sampling rate and pixel dwell time. Here, we developed volumetric Lissajous confocal microscopy to achieve unsurpassed 3D scanning speed with a tunable sampling rate. The system combines an acoustic liquid lens for continuous axial focus translation with a resonant scanning mirror. Accordingly, the excitation beam follows a dynamic Lissajous trajectory enabling sub-millisecond acquisitions of image series containing 3D information at a sub-Nyquist sampling rate. By temporal accumulation and/or advanced interpolation algorithms, volumetric imaging rate is selectable using a post-processing step at the desired spatiotemporal resolution for events of interest. We demonstrate multicolor and calcium imaging over volumes of tens of cubic microns with acquisition speeds up to 5 kHz.

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“…Later, Zeiss made the imaging scanning microscopy commercial with the presence of Airyscan system (Huff et al, 2015), which uses gallium arsenide phosphide photomultiplier tube (GaAsP-PMT) area detector instead of former CCD. Recently, the ISM technique was combined with I M A G E S C A N N I N G D I F F E R E N C E M I C R O S C O P Y 9 9 many other superresolved methods (Castello et al, 2019;Tenne et al, 2019) and has been applied to various research areas.…”

Section: Introductionmentioning

confidence: 99%

Image scanning difference microscopy

Chen

Liu

Zhang

et al. 2019

Journal of Microscopy

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Summary Here, we propose a novel imaging method, which is called image scanning difference microscopy (ISDM), for superresolution imaging. In ISDM, we implement a detector array composed of 19 avalanche photodiodes (APD) rather than single‐point detector in standard confocal microscopy for reconstructing superresolved images with higher signal‐to‐noise ratio (SNR). Combining with our former proposed fluorescence emission difference (FED) method, we have achieved a lateral resolution of 111 nm (∼λ/6) without the damage of image quality, the highest FED resolution to the best of our knowledge. With its simple setup and remarkable performance, we believe that ISDM can become a versatile observation tool in biology and other fundamental studies. Lay Description Fluorescence emission difference (FED) microscopy is a really simple and generalisable superresolved fluorescence microscopy method based on PSF engineering and difference algorithm recently. Compared to stimulated‐emission‐depletion fluorescence microscopy (STED), FED don't need complicated system or precise alignment and polarisation, available for wide variety of dyes and has low photobleaching and phototoxicity for living cells. However, the distortion caused by negative value is one of the biggest obstacles to the further development of FED. In light of this, we propose a novel superresolution imaging method based on the FED method with parallel detection system, which is called image scanning difference microscopy (ISDM). Our method has achieved a significant breakthrough in FED, increasing the resolution further while reducing artefacts generated by negative values, which cannot be accomplished through combining other methods. In addition, ISDM does not require complex setup and optical alignment, long time imaging and imposing no constraint on dyes. Importantly, we realised a transverse resolution of ∼λ/6 (triple diffraction limit) with single wavelength, single incident path and low light intensity, which has never been achieved in any other far‐field superresolution microscopy.

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A robust and versatile platform for image scanning microscopy enabling super-resolution FLIM

Cited by 170 publications

References 33 publications

Chromatin nanoscale compaction in live cells visualized by acceptor‐to‐donor ratio corrected Förster resonance energy transfer between DNA dyes

Chromatin nanoscale compaction in live cells visualized by acceptor‐to‐donor ratio corrected Förster resonance energy transfer between DNA dyes

Volumetric Lissajous Confocal Microscopy

Image scanning difference microscopy

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