The cell has a three-dimensional (3D) structure and its spatial arrangement is often very important to molecular mechanisms essential for life. In order to visualize 3D morphologies of cells, confocal laser imaging was developed. 1 The method is, however, only applicable to fluorescence-probed molecules, 2 which limits the observable number of molecules, and such artificial probing sometime perturbs normal molecular mechanisms. Cotte et al. applied holographic and tomographic irradiation to microscopy and finally innovated a threedimensional computed holographic and tomographic (HT) laser microscope. 3 The laser beam that penetrates the cell at an angle experiences a delay in the phase of its beam, which is magnified and overlayed with reference beam to make a holographic image. The holograms at various angles then deconvoluted by tomographic algorithms to create a precise 3D cell image. The 3D-HT microscope can visualize 3D morphological aspects by contrasting refractive indexes observed by the laser monochromatic wavelength, making staining unnecessary.We have developed live single-cell mass spectrometry, 4-7 in which the contents of a single cell, usually picoliter level or less, are sucked by a nanospray tip (a sort of glass capillary needle) and fed directly into a mass spectrometer after the addition of an ionization solvent to the rear end of the tip. In this method, the exact amount sucked is unclear because it is such a tiny volume. Furthermore, 3D spatial location and identity of the contents are also ambiguous. Through the combination of these two techniques, 3D-HT microscopy and live single-cell mass spectrometry, greater 3D spatial resolution (X-Y-axis 0.18 μm and Z-axis 0.33 μm) and improved quantitative single-cell analysis is expected. The first trial of this combination and its results are documented in this paper, and we think nextgeneration live single-cell mass spectrometry is quite promising.Human hepatocellular carcinoma cell line (HepG2) was cultured in Dulbecco's modified Eagle medium in addition to 10% fetal calf serum (FBS), 100 mg/mL penicillin, and 100 mg/mL streptomycin G in 35 mm glass bottom dishes at 37 C and 5% CO2. HepG2 cells were positioned under the HT laser microscope, and the HT scan took 2 s to acquire one 3D image. Figure 1 shows the schematic principle of the HT laser microscope (3D Cell Explorer, Nanolive, SA, Switzerland). Fig. 1 Schematic of live single-cell mass spectrometry with quantitation by holographic and tomographic laser microscopy. The laser beam is split into a reference beam (going down to the CCD camera) and an observation beam that irradiates the cell at 45 degree angle. A micromanipulator was setup next to microscope to allow precise suction with a nanospray tip. The sucked cellular matter was then blasted through the mass spectrometer.

In a wider and wider range of research and engineering activities, there is a growing interest for full-field techniques, featuring nanometric sensitivities, and able to be addressed to dynamic behaviors characterization. Speckle interferometry (SI) techniques are acknowledged as good candidates to tackle this challenge. To get rid of the intrinsic correlation length limitation and simplify the un-wrapping step, a straightforward approach lies in the pixel history analysis. The need of increasing performances in terms of accuracy and computation speed is permanently demanding new efficient processing techniques. We propose in this paper a fast implementation of the Empirical Mode Decomposition (EMD) to put the SI pixel signal in an appropriate shape for accurate phase computation. As one of the best way to perform it, the analytic method based on the Hilbert transform (HT) of the so-transformed signal will then be reviewed. For short evaluation, a zero-crossing technique which exploits directly the extrema finding step of the EMD will be presented. We propose moreover a technique to discard the under-modulated pixels which yield wrong phase evaluation. This work is actually an attempt to elaborate a phase extraction procedure which exploits all the reliable information in 3D, - two space and one time coordinates -, to endeavor to make the most of SI raw data.

Holo-tomographic microscopy (HTM) is a label-free non-phototoxic microscopy method reporting the fine changes of a cell's refractive indexes (RI) in 3D. By combining HTM with epifluorescence, we demonstrate that cellular organelles such as Lipid droplets and mitochondria show a specific RI signature that distinguishes them with high resolution and contrast. We further show that HTM allows to follow in unprecedented ways the dynamics of mitochondria, lipid droplets as well as that of endocytic structures in live cells over long period of time, which led us to observe to our knowledge for the first time a global organelle spinning occurring before mitosis.

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Optical full-field techniques have a great importance in modern experimental mechanics.Even if they are reasonably spread among the university laboratories, their diffusion in industrial companies remains very narrow for several reasons, especially a lack of metrological performance assessment. A full-field measurement can be characterized by its resolution, bias, measuring range, and by a specific quantity, the spatial resolution. The present paper proposes an original procedure to estimate in one single step the resolution, bias and spatial resolution for a given operator (decoding algorithms such as image correlation, low-pass filters, derivation tools ...). This procedure is based on the construction of a particular multi-frequential field, and a Bode diagram representation of the results. This analysis is applied to various phase demodulating algorithms suited to estimate in-plane displacements. Keywords Fringe • Error assessment • Spatial resolution • Displacement resolution • Benchmarkextraction from a single fringe patterns. Consequently, they should be used especially with the grid method, but also to many high speed implementations (for example: fringe projection, deflectometry or speckle or moiré interferometry).The proposed methodology is based on a generic synthetic image undergoing sinusoidal displacements. The procedure used to generate original images containing very rich information in terms of frequency and phase distributions is presented and justified in the first section of this paper.Displacements are computed by seven fringe analysis codes for various parameters. The measured displacements are compared to the prescribed ones which serve as a reference through a specific method which is described. The two main interests are i-to rely on a reference phase distribution which is perfectly known a priori and ii-to control any additive noise that can potentially be added to this reference, thus leading to a precise assessment of its influence on the measurements provided by various algorithms. Obtained results are presented, analyzed and discussed.