STED-SPIM made simple

This work has been partially funded by the Agence Nationale de la Recherche under the program. ANR-13-NANO-0004-01. Teodora Scheul acknowledges a doctoral fellowship from the Nanosciences Foundation (Grenoble, France). We thank the company TEEMPhotonics (Meylan, France) for the loan of a laser.International audienceWe report the development of a stimulated emission depletion (STED) selective plane illumination (SPIM) microscope based on a single diode-pumped solid state (DPSS) laser that simultaneously delivers nanosecond-pulses at two wavelengths. The two wavelengths, 355 nm and 532 nm, are generated by harmonic conversion and they are used to induce respectively excitation and stimulated emission depletion. This source should allow a low-cost, compact, very efficient and simplified STED scheme since the two beams are intrinsically aligned and synchronized. Using a chromatic beam shaping device which leaves the excitation beam unaffected and produces a donut-shaped STED beam, we demonstrate a 300% reduction of the light sheet thickness, together with an enhancement of the sheet uniformity over larger field of view, at low STED power, in Coumarin dye solution. OCIS codes: (180.2520) Fluorescence microscopy; (180.0180) Microscopy. References and links 1. S. W. Hell and J. Wichmann, "Breaking the diffraction resolution limit by stimulated emission: stimulated-emission-depletion fluorescence microscopy," Opt. Lett. 19(11), 780–782 (1994). 2. M. Dyba and S. W. Hell, "Focal spots of size λ/23 open up far-field fluorescence microscopy at 33 nm axial resolution," Phys., "High-resolution three-dimensional imaging of large specimens with light sheet-based microscopy," Nat. Methods 4(4), 311–313 (2007). 8

Correction of cell-induced optical aberrations in a fluorescence fluctuation microscope

We describe the effect of optical aberrations on fluorescence fluctuations microscopy (FFM), when focusing through a single living cell. FFM measurements are performed in an aqueous fluorescent solution and prove to be a highly sensitive tool to assess the optical aberrations introduced by the cell. We demonstrate an adaptive optics (AO) system to remove the aberration-related bias in the FFM measurements. Our data show that AO is not only useful when imaging deep in tissues but also when performing FFM measurements through a single cellular layer. This work paves the way for the application of FFM to complex three-dimensional multicellular samples

Multi-confocal Fluorescence Correlation Spectroscopy : experimental demonstration and potential applications for living cell measurements

We report, for the first time, a multi-confocal Fluorescence Correlation Spectroscopy (mFCS) technique which allows parallel measurements at different locations, by combining a Spatial Light Modulator (SLM), with an Electron Multiplying-CCD camera (EM-CCD). The SLM is used to produce a series of laser spots, while the pixels of the EM-CCD play the roles of virtual pinholes. The phase map addressed to the SLM is calculated by using the spherical wave approximation and makes it possible to produce several diffraction limited laser spots, either aligned or spread over the field of view. To attain fast enough imaging rates, the camera has been used in different acquisition modes, the fastest of which leads to a time resolution of 100 $\mu$s. We qualified the experimental set-up by using solutions of sulforhodamine G in glycerol and demonstrated that the observation volumes are similar to that of a standard confocal set-up. To demonstrate that our mFCS method is suitable for intracellular studies, experiments have been conducted on two stable cell lines: mouse embryonic fibroblasts expressing eGFP-actin and H1299 cells expressing the heat shock factor fusion protein HSF1-eGFP. In the first case we could recover, by analyzing the auto-correlation curves, the diffusion constant of G-actin within the cytoplasm, although we were also sensitive to the complex network of interactions with F-actin. Concerning HSF1, we could clearly observe the modifications of the number of molecules and of the HSF1 dynamics during heat shock

Automatic laser alignment for multifocal microscopy using a LCOS-SLM and a 32x32 pixel CMOS SPAD array

International audienceAlignment of a laser to a point source detector for confocal microscopy can be a time-consuming task. The problem is further exacerbated when multiple laser excitation spots are used in conjunction with a multiple pixel single photon detector; in addition to X, Y and Z positioning, pixels in a 2D array detector can also be misaligned in roll, pitch and yaw with respect to each other, causing magnification, rotation and focus variation across the array. We present a technique for automated multiple point laser alignment to overcome these issues using closed-loop feedback between a laser illuminated computer controlled Liquid Crystal on Silicon Spatial Light Modulator (LCOS-SLM) acting as the excitation source and a 32 32 pixel CMOS Single Photon Avalanche Diode (SPAD) array as the multiple pixel detection element. The alignment procedure is discussed and simulated to prove its feasibility before being implemented and tested in a practical optical system. We show that it is possible to align each independent laser point in a sub-second time scale, significantly simplifying and speeding up experimental set-up times. The approach provides a solution to the difficulties associated with multiple point confocal laser alignment to multiple point detector arrays, paving the way for further advances in applications such as Fluorescence Correlation Spectroscopy (FCS) and Fluorescence Lifetime Imaging Microscopy (FLIM)

Wavelet penalized likelihood estimation in generalized functional models

The paper deals with generalized functional regression. The aim is to estimate the influence of covariates on observations, drawn from an exponential distribution. The link considered has a semiparametric expression: if we are interested in a functional influence of some covariates, we authorize others to be modeled linearly. We thus consider a generalized partially linear regression model with unknown regression coefficients and an unknown nonparametric function. We present a maximum penalized likelihood procedure to estimate the components of the model introducing penalty based wavelet estimators. Asymptotic rates of the estimates of both the parametric and the nonparametric part of the model are given and quasi-minimax optimality is obtained under usual conditions in literature. We establish in particular that the LASSO penalty leads to an adaptive estimation with respect to the regularity of the estimated function. An algorithm based on backfitting and Fisher-scoring is also proposed for implementation. Simulations are used to illustrate the finite sample behaviour, including a comparison with kernel and splines based methods

Comparative transcriptomics of drought responses in Populus: a meta-analysis of genome-wide expression profiling in mature leaves and root apices across two genotypes

<p>Abstract</p> <p>Background</p> <p>Comparative genomics has emerged as a promising means of unravelling the molecular networks underlying complex traits such as drought tolerance. Here we assess the genotype-dependent component of the drought-induced transcriptome response in two poplar genotypes differing in drought tolerance. Drought-induced responses were analysed in leaves and root apices and were compared with available transcriptome data from other <it>Populus </it>species.</p> <p>Results</p> <p>Using a multi-species designed microarray, a genomic DNA-based selection of probesets provided an unambiguous between-genotype comparison. Analyses of functional group enrichment enabled the extraction of processes physiologically relevant to drought response. The drought-driven changes in gene expression occurring in root apices were consistent across treatments and genotypes. For mature leaves, the transcriptome response varied weakly but in accordance with the duration of water deficit. A differential clustering algorithm revealed similar and divergent gene co-expression patterns among the two genotypes. Since moderate stress levels induced similar physiological responses in both genotypes, the genotype-dependent transcriptional responses could be considered as intrinsic divergences in genome functioning. Our meta-analysis detected several candidate genes and processes that are differentially regulated in root and leaf, potentially under developmental control, and preferentially involved in early and long-term responses to drought.</p> <p>Conclusions</p> <p>In poplar, the well-known drought-induced activation of sensing and signalling cascades was specific to the early response in leaves but was found to be general in root apices. Comparing our results to what is known in arabidopsis, we found that transcriptional remodelling included signalling and a response to energy deficit in roots in parallel with transcriptional indices of hampered assimilation in leaves, particularly in the drought-sensitive poplar genotype.</p

The FunGenES Database: A Genomics Resource for Mouse Embryonic Stem Cell Differentiation

Embryonic stem (ES) cells have high self-renewal capacity and the potential to differentiate into a large variety of cell types. To investigate gene networks operating in pluripotent ES cells and their derivatives, the “Functional Genomics in Embryonic Stem Cells” consortium (FunGenES) has analyzed the transcriptome of mouse ES cells in eleven diverse settings representing sixty-seven experimental conditions. To better illustrate gene expression profiles in mouse ES cells, we have organized the results in an interactive database with a number of features and tools. Specifically, we have generated clusters of transcripts that behave the same way under the entire spectrum of the sixty-seven experimental conditions; we have assembled genes in groups according to their time of expression during successive days of ES cell differentiation; we have included expression profiles of specific gene classes such as transcription regulatory factors and Expressed Sequence Tags; transcripts have been arranged in “Expression Waves” and juxtaposed to genes with opposite or complementary expression patterns; we have designed search engines to display the expression profile of any transcript during ES cell differentiation; gene expression data have been organized in animated graphs of KEGG signaling and metabolic pathways; and finally, we have incorporated advanced functional annotations for individual genes or gene clusters of interest and links to microarray and genomic resources. The FunGenES database provides a comprehensive resource for studies into the biology of ES cells