Thibault Roland

Amplitude and phase images of cellular structures with a scanning surface plasmon microscope

Imaging cellular internal structure at nanometer scale axial resolution with non invasive microscopy techniques has been a major technical challenge since the nineties. We propose here a complement to fluorescence based microscopies with no need of staining the biological samples, based on a Scanning Surface Plasmon Microscope (SSPM). We describe the advantages of this microscope, namely the possibility of both amplitude and phase imaging and, due to evanescent field enhancement by the surface plasmon resonance, a very high resolution in Z scanning (Z being the axis normal to the sample). We show for fibroblast cells (IMR90) that SSPM offers an enhanced detection of index gradient regions, and we conclude it is very well suited to discriminate regions of variable density in biological media such as cell compartments, nucleus, nucleoli and membranes.

Modeling of the scanning surface plasmon microscope

We propose a family of exact solutions of Maxwells equations to model some aspects of the imaging process involved in the scanning surface plasmon microscope (SSPM). More precisely, we compute the SSPM response of a spherical nanoparticle immobilized close to a thin gold layer and illuminated by a tightly focused spot. We discuss the influence of parameters such as the defocus and the width of the gold layer on the image contrast. We show that this microscopy combines a subwavelength spatial resolution together with high sensitivity to small changes in dielectric properties on the nanoparticle.

The Flagellar Motor of Caulobacter Crescentus Generates More Torque When a Cell Swims Backward

Caulobacter crescentus, a monotrichous bacterium, swims by rotating a single right-handed helical filament. CW motor rotation thrusts the cell forward 1, a mode of motility known as the pusher mode; CCW motor rotation pulls the cell backward, a mode of motility referred to as the puller mode 2. The situation is opposite in E. coli, a peritrichous bacterium, where CCW rotation of multiple left-handed filaments drives the cell forward. The flagellar motor in E. coli generates more torque in the CCW direction than the CW direction in swimming cells 3,4. However, monotrichous bacteria including C. crescentus swim forward and backward at similar speeds, prompting the assumption that motor torques in the two modes are the same 5,6. Here, we present evidence that motors in C. crescentus develop higher torques in the puller mode than in the pusher mode, and suggest that the anisotropy in torque-generation is similar in two species, despite the differences in filament handedness and motor bias (probability of CW rotation).

Response thresholds in bacterial chemotaxis

The absence of responses to shallow temporal ramps of chemicals does not appear to be accounted for by motor remodeling. Stimulation of Escherichia coli by exponential ramps of chemoattractants generates step changes in the concentration of the response regulator, CheY-P. Because flagellar motors are ultrasensitive, this should change the fraction of time that motors spin clockwise, the CWbias. However, early work failed to show changes in CWbias when ramps were shallow. This was explained by a model for motor remodeling that predicted plateaus in plots of CWbias versus [CheY-P]. We looked for these plateaus by examining distributions of CWbias in populations of cells with different mean [CheY-P]. We did not find such plateaus. Hence, we repeated the work on shallow ramps and found that motors did indeed respond. These responses were quantitatively described by combining motor remodeling with ultrasensitivity in a model that exhibited high sensitivities over a wide dynamic range.

High resolution surface plasmon microscopy for cell imaging

We introduce a new non-labeling high resolution microscopy method for cellular imaging. This method called SSPM (Scanning Surface Plasmon Microscopy) pushes down the resolution limit of surface plasmon resonance imaging (SPRi) to sub-micronic scales. High resolution SPRi is obtained by the surface plasmon lauching with a high numerical aperture objective lens. The advantages of SPPM compared to other high resolution SPRis rely on three aspects; (i) the interferometric detection of the back reflected light after plasmon excitation, (ii) the twodimensional scanning of the sample for image reconstruction, (iii) the radial polarization of light, enhancing both resolution and sensitivity. This microscope can afford a lateral resolution of - 150 nm in liquid environment and - 200 nm in air. We present in this paper images of IMR90 fibroblasts obtained with SSPM in dried environment. Internal compartments such as nucleus, nucleolus, mitochondria, cellular and nuclear membrane can be recognized without labelling. We propose an interpretation of the ability of SSPM to reveal high index contrast zones by a local decomposition of the V (Z) function describing the response of the SSPM.

The Screw-Like Movement of a Gliding Bacterium Is Powered by Spiral Motion of Cell-Surface Adhesins.

Flavobacterium johnsoniae, a rod-shaped bacterium, glides over surfaces at speeds of ∼2 μm/s. The propulsion of a cell-surface adhesin, SprB, is known to enable gliding. We used cephalexin to generate elongated cells with irregular shapes and followed their displacement in three dimensions. These cells rolled about their long axes as they moved forward, following a right-handed trajectory. We coated gold nanoparticles with an SprB antibody and tracked them in three dimensions in an evanescent field where the nanoparticles appeared brighter when they were closer to the glass. The nanoparticles followed a right-handed spiral trajectory on the surface of the cell. Thus, if SprB were to adhere to the glass rather than to a nanoparticle, the cell would move forward along a right-handed trajectory, as observed, but in a direction opposite to that of the nanoparticle.

Small molecule injection into single-cell C. elegans embryos via carbon-reinforced nanopipettes.

The introduction of chemical inhibitors into living cells at specific times in development is a useful method for investigating the roles of specific proteins or cytoskeletal components in developmental processes. Some embryos, such as those of Caenorhabditis elegans, however, possess a tough eggshell that makes introducing drugs and other molecules into embryonic cells challenging. We have developed a procedure using carbon-reinforced nanopipettes (CRNPs) to deliver molecules into C. elegans embryos with high temporal control. The use of CRNPs allows for cellular manipulation to occur just subsequent to meiosis II with minimal damage to the embryo. We have used our technique to replicate classical experiments using latrunculin A to inhibit microfilaments and assess its effects on early polarity establishment. Our injections of latrunculin A confirm the necessity of microfilaments in establishing anterior-posterior polarity at this early stage, even when microtubules remain intact. Further, we find that latrunculin A treatment does not prevent association of PAR-2 or PAR-6 with the cell cortex. Our experiments demonstrate the application of carbon-reinforced nanopipettes to the study of one temporally-confined developmental event. The use of CRNPs to introduce molecules into the embryo should be applicable to investigations at later developmental stages as well as other cells with tough outer coverings.