Lorenzo Scipioni

Measurement of nanoscale three-dimensional diffusion in the interior of living cells by STED-FCS

The observation of molecular diffusion at different spatial scales, and in particular below the optical diffraction limit (<200 nm), can reveal details of the subcellular topology and its functional organization. Stimulated-emission depletion microscopy (STED) has been previously combined with fluorescence correlation spectroscopy (FCS) to investigate nanoscale diffusion (STED-FCS). However, stimulated-emission depletion fluorescence correlation spectroscopy has only been used successfully to reveal functional organization in two-dimensional space, such as the plasma membrane, while, an efficient implementation for measurements in three-dimensional space, such as the cellular interior, is still lacking. Here we integrate the STED-FCS method with two analytical approaches, the recent separation of photons by lifetime tuning and the fluorescence lifetime correlation spectroscopy, to simultaneously probe diffusion in three dimensions at different sub-diffraction scales. We demonstrate that this method efficiently provides measurement of the diffusion of EGFP at spatial scales tunable from the diffraction size down to ∼80 nm in the cytoplasm of living cells.The measurement of molecular diffusion at sub-diffraction scales has been achieved in 2D space using STED-FCS, but an implementation for 3D diffusion is lacking. Here the authors present an analytical approach to probe diffusion in 3D space using STED-FCS and measure the diffusion of EGFP at different spatial scales.

Phasor Analysis of Local ICS Detects Heterogeneity in Size and Number of Intracellular Vesicles

Organelles represent the scale of organization immediately below that of the cell itself, and their composition, size, and number are tailored to their function. Monitoring the size and number of organelles in live cells is relevant for many applications but can be challenging due to their highly heterogeneous properties. Image correlation spectroscopy is a well-established analysis method capable of extracting the average size and number of particles in images. However, when image correlation spectroscopy is applied to a highly heterogeneous system, it can fail to retrieve, from a single correlation function, the characteristic size and the relative amount associated to each subspecies. Here, we describe a fast, unbiased, and fit-free algorithm based on the phasor analysis of multiple local image correlation functions, capable of mapping the sizes of elements contained in a heterogeneous system. The method correctly provides the size and number of separate subspecies, which otherwise would be hidden in the average properties of a single correlation function. We apply the method to quantify the spatial and temporal heterogeneity in the size and number of intracellular vesicles formed after endocytosis in live cells.

Exploiting the tunability of stimulated emission depletion microscopy for super-resolution imaging of nuclear structures

Imaging of nuclear structures within intact eukaryotic nuclei is imperative to understand the effect of chromatin folding on genome function. Recent developments of super-resolution fluorescence microscopy techniques combine high specificity, sensitivity, and less-invasive sample preparation procedures with the sub-diffraction spatial resolution required to image chromatin at the nanoscale. Here, we present a method to enhance the spatial resolution of a stimulated-emission depletion (STED) microscope based only on the modulation of the STED intensity during the acquisition of a STED image. This modulation induces spatially encoded variations of the fluorescence emission that can be visualized in the phasor plot and used to improve and quantify the effective spatial resolution of the STED image. We show that the method can be used to remove direct excitation by the STED beam and perform dual color imaging. We apply this method to the visualization of transcription and replication foci within intact nuclei of eukaryotic cells.A known limitation of super-resolution STED microscopy is the need of high laser power which can cause photobleaching and phototoxicity. Here the authors further optimize this method and show that modulating STED intensity during acquisition results in an enhanced resolution and reduced background.

Local raster image correlation spectroscopy generates high-resolution intracellular diffusion maps

Raster image correlation spectroscopy (RICS) is a powerful method for measuring molecular diffusion in live cells directly from images acquired on a laser scanning microscope. However, RICS only provides single average diffusion coefficients from regions with a lateral size on the order of few micrometers, which means that its spatial resolution is mainly limited to the cellular level. Here we introduce the local RICS (L-RICS), an easy-to-use tool that generates high resolution maps of diffusion coefficients from images acquired on a laser scanning microscope. As an application we show diffusion maps of a green fluorescent protein (GFP) within the nucleus and within the nucleolus of live cells at an effective spatial resolution of 500 nm. We find not only that diffusion in the nucleolus is slowed down compared to diffusion in the nucleoplasm, but also that diffusion in the nucleolus is highly heterogeneous.Lorenzo Scipioni et al. present Local Raster Image Correlation Spectroscopy (L-RICS), a method for generating sub-micrometer diffusion maps. They apply L-RICS to GFP in live cells and find that diffusion coefficients differ between the nucleus and nucleolus and are highly heterogeneous within compartments.

Role of the Pico- Nano-Second Temporal Dimension in STED Microscopy

In the last decades, several techniques have been developed to push the spatial resolution of far-field fluorescence microscopy beyond the diffraction limit. Stimulated emission depletion (STED) microscopy is a super-resolution technique in which the targeted switching off of the fluorophores by a secondary laser beam results in an effective increase in optical resolution. However, to fully exploit the maximum performances of a STED microscope (effective spatial resolution achievable for a given STED beam’s intensity, versatility, live-cell imaging capability, etc.) several experimental precautions have to be considered. In this respect, the temporal dimension (at the pico- and nanosecond scale) has often a central role on the overall efficiency and versatility of a STED microscope, working in pulsed or continuous-wave mode.