Mickaël Lelek

QuickPALM: 3D real-time photoactivation nanoscopy image processing in ImageJ

To the Editor: Although conventional microscopes have a reso-lution limited by diffraction to about half the wavelength of light, several recent advances have led to microscopy methods that achieve roughly tenfold improvements in resolution. Among them, photoactivated light microscopy (PALM) and stochastic optical resolution microscopy (STORM) have become particularly popular, as they only require relatively simple and affordable modifications to a standard total internal reflection fluorescence (TIRF) microscope and have been extended to three-dimensional (3D) super-resolution and multicolor imaging.

Superresolution imaging of HIV in infected cells with FlAsH-PALM

Imaging protein assemblies at molecular resolution without affecting biological function is a long-standing goal. The diffraction-limited resolution of conventional light microscopy (∼200–300 nm) has been overcome by recent superresolution (SR) methods including techniques based on accurate localization of molecules exhibiting stochastic fluorescence; however, SR methods still suffer important restrictions inherent to the protein labeling strategies. Antibody labels are encumbered by variable specificity, limited commercial availability and affinity, and are mostly restricted to fixed cells. Fluorescent protein fusions, though compatible with live cell imaging, substantially increase protein size and can interfere with their biological activity. We demonstrate SR imaging of proteins tagged with small tetracysteine motifs and the fluorescein arsenical helix binder (FlAsH-PALM). We applied FlAsH-PALM to image the integrase enzyme (IN) of HIV in fixed and living cells under experimental conditions that fully preserved HIV infectivity. The obtained resolution (∼30 nm) allowed us to characterize the distribution of IN within virions and intracellular complexes and to distinguish different HIV structural populations based on their morphology. We could thus discriminate ∼100 nm long mature conical cores from immature Gag shells and observe that in infected cells cytoplasmic (but not nuclear) IN complexes display a morphology similar to the conical capsid. Together with the presence of capsid proteins, our data suggest that cytoplasmic IN is largely present in intact capsids and that these can be found deep within the cytoplasm. FlAsH-PALM opens the door to in vivo SR studies of microbial complexes within host cells and may help achieve truly molecular resolution.

Chromatin organization at the nuclear pore favours HIV replication

The molecular mechanisms that allow HIV to integrate into particular sites of the host genome are poorly understood. Here we tested if the nuclear pore complex (NPC) facilitates the targeting of HIV integration by acting on chromatin topology. We show that the integrity of the nuclear side of the NPC, which is mainly composed of Tpr, is not required for HIV nuclear import, but that Nup153 is essential. Depletion of Tpr markedly reduces HIV infectivity, but not the level of integration. HIV integration sites in Tpr-depleted cells are less associated with marks of active genes, consistent with the state of chromatin proximal to the NPC, as analysed by super-resolution microscopy. LEDGF/p75, which promotes viral integration into active genes, stabilizes Tpr at the nuclear periphery and vice versa. Our data support a model in which HIV nuclear import and integration are concerted steps, and where Tpr maintains a chromatin environment favourable for HIV replication.

Pulsed blue laser at 491nm by Nonlinear Cavity Dumping

A nonlinear cavity dumping process is applied for the first time to generate kW peak power pulses at 491 nm. The system is based on efficient sum-frequency mixing of 1063 nm and 912 nm radiations in a BiBO nonlinear crystal placed inside a Nd:GdVO4 laser oscillator with a high finesse cavity at 912 nm. The nonlinear cavity dumping process is triggered by high peak power nanosecond pulses from a 1063 nm Q-switched Nd:GdVO4 laser operating at 10 kHz. To reach the kW range at 491 nm a key point is to Q-switch the high finesse 912 nm cavity instead of continuous wave operation. Thus, the peak power (9.3 kW for 3 ns pulses) and the average power (280 mW) obtained at 491 nm are 14 times higher than the one obtained when the 912 nm laser operated in continuous wave.

Deep learning massively accelerates super-resolution localization microscopy

The speed of super-resolution microscopy methods based on single-molecule localization, for example, PALM and STORM, is limited by the need to record many thousands of frames with a small number of observed molecules in each. Here, we present ANNA-PALM, a computational strategy that uses artificial neural networks to reconstruct super-resolution views from sparse, rapidly acquired localization images and/or widefield images. Simulations and experimental imaging of microtubules, nuclear pores, and mitochondria show that high-quality, super-resolution images can be reconstructed from up to two orders of magnitude fewer frames than usually needed, without compromising spatial resolution. Super-resolution reconstructions are even possible from widefield images alone, though adding localization data improves image quality. We demonstrate super-resolution imaging of >1,000 fields of view containing >1,000 cells in ∼3 h, yielding an image spanning spatial scales from ∼20 nm to ∼2 mm. The drastic reduction in acquisition time and sample irradiation afforded by ANNA-PALM enables faster and gentler high-throughput and live-cell super-resolution imaging.

Single-shot SPectral interferometry resolved in time

Shearing interferometry applied to the spectral domain is used for femtoseconde pulse single-shot measurement. An innovating configuration delivering a bidimensional experimental direct representation of the spectral phase of the pulse is also presented.

Two-photon fluorescence microendoscope using a flexible multicore image guide

A flexible image guide is excited by femtoseconde pulses. Precompensation of dispersion and nonlinear effects encountered in the waveguide allows the recording of two photon fluorescence endoscopic images of colon cells.

Spectral shearing interferometry applied to real time femtosecond pulse field reconstruction

Real time ultra-short pulse coherent characterization is experimentally demonstrated using a new configuration of spectral interferometry resolved in time (SPIRIT). Complex pulse reconstruction is studied numerically.

Single shot method for real time femtosecond pulse field reconstruction

We have experimentally demonstrated a new configuration of Single-Shot SPIRIT was validated during the characterization of nearly gaussian femtosecond pulses exiting of a Ti:Sapphire laser source, which delivers pulses at 75 MHz repetition rate at 830 nm. This technique permits to have fast and direct access to phase and amplitude of short optical pulses. The extension to low repetition rate amplified femtosecond system is under way. A detectivity threshold in the range of 100 nanojoules has been estimated in the case of single femtosecond pulse measurement. The numerical model will enable us to foresee the limitation of the technique. In the output plane of the setup, this gate pulse is spatially broadened by a cylindrical lens. The remaining fraction of the initial pulse enters a Mach-Zehnder interferometer which will generate a pair of twin pulses. After dispersion by a diffraction grating, the twin spectra are displayed in the output.