Compact and effective photon-resolved image scanning microscope

Tortarolo, Giorgio; Zunino, Alessandro; Piazza, Simonluca; Donato, Mattia; Zappone, Sabrina; Pierzyńska-Mach, Agnieszka; Castello, Marco; Vicidomini, Giuseppe

doi:10.1117/1.ap.6.1.016003

Cited by 8 publications

(1 citation statement)

References 42 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Despite its maturity, ISM is still a dynamic and quickly evolving research field, thanks to the recent introduction of innovative single photon avalanche diode (SPAD) array detectors, which add further versatility to the microscopes. Indeed, SPAD arrays can detect single photons with tens of picosecond temporal precision [6], enabling fluoresce lifetime ISM [7], quantum ISM (Q-ISM) [8], and super-resolution optical fluctuation ISM (SOFISM) [9]. Furthermore, ISM can be synergically combined with other scanning techniques, such as stimulated emission depletion (STED) [10] and multi-photon excitation microscopy [11].…”

Section: Introductionmentioning

confidence: 99%

Image scanning microscopy reconstruction by autocorrelation inversion

Ancora,

Zunino,

Vicidomini

et al. 2024

J. Phys. Photonics

View full text Add to dashboard Cite

Confocal laser scanning microscopy (CLSM) stands out as one of the most widely used microscopy techniques thanks to its three-dimensional imaging capability and its sub-diffraction spatial resolution, achieved through the closure of a pinhole in front of a single-element detector. However, the pinhole also rejects useful photons, and beating the diffraction limit comes at the price of irremediably compromising the signal-to-noise ratio (SNR) of the data. Image scanning microscopy (ISM) emerged as the rational evolution of CLSM, exploiting a small array detector in place of the pinhole and the single-element detector. Each sensitive element is small enough to achieve sub-diffraction resolution through the confocal effect, but the size of the whole detector is large enough to guarantee excellent collection efficiency and SNR. However, the raw data produced by an ISM setup consists of a 4D dataset, which can be seen as a set of confocal-like images. Thus, fusing the dataset into a single super-resolved image requires a dedicated reconstruction algorithm. Conventional methods are multi-image deconvolution, which requires prior knowledge of the system point spread functions (PSF), or adaptive pixel reassignment (APR), which is effective only on a limited range of experimental conditions. In this work, we describe and validate a novel concept for ISM image reconstruction based on autocorrelation inversion. We leverage unique properties of the autocorrelation to discard low-frequency components and maximize the resolution of the reconstructed image without any assumption on the image or any knowledge of the PSF. Our results push the quality of the ISM reconstruction beyond the level provided by APR and open new perspectives for multi-dimensional image processing.

show abstract

Section: Introductionmentioning

confidence: 99%

Image scanning microscopy reconstruction by autocorrelation inversion

Ancora,

Zunino,

Vicidomini

et al. 2024

J. Phys. Photonics

View full text Add to dashboard Cite

show abstract

4D Single-particle tracking with asynchronous read-out single-photon avalanche diode array detector

Bucci,

Tortarolo,

Held

et al. 2024

Nat Commun

View full text Add to dashboard Cite

Single-particle tracking techniques enable investigation of the complex functions and interactions of individual particles in biological environments. Many such techniques exist, each demonstrating trade-offs between spatiotemporal resolution, spatial and temporal range, technical complexity, and information content. To mitigate these trade-offs, we enhanced a confocal laser scanning microscope with an asynchronous read-out single-photon avalanche diode array detector. This detector provides an image of the particle’s emission, precisely reflecting its position within the excitation volume. This localization is utilized in a real-time feedback system to drive the microscope scanning mechanism and ensure the particle remains centered inside the excitation volume. As each pixel is an independent single-photon detector, single-particle tracking is combined with fluorescence lifetime measurement. Our system achieves 40 nm lateral and 60 nm axial localization precision with 100 photons and sub-millisecond temporal sampling for real-time tracking. Offline tracking can refine this precision to the microsecond scale. We validated the system’s spatiotemporal resolution by tracking fluorescent beads with diffusion coefficients up to 10 μm2/s. Additionally, we investigated the movement of lysosomes in living SK-N-BE cells and measured the fluorescence lifetime of the marker expressed on a membrane protein. We expect that this implementation will open other correlative imaging and tracking studies.

show abstract