Gábor Steinbach

Atomic Force Microscopy Stiffness Tomography on Living Arabidopsis thaliana Cells Reveals the Mechanical Properties of Surface and Deep Cell-Wall Layers during Growth

Cell-wall mechanical properties play a key role in the growth and the protection of plants. However, little is known about genuine wall mechanical properties and their growth-related dynamics at subcellular resolution and in living cells. Here, we used atomic force microscopy (AFM) stiffness tomography to explore stiffness distribution in the cell wall of suspension-cultured Arabidopsis thaliana as a model of primary, growing cell wall. For the first time that we know of, this new imaging technique was performed on living single cells of a higher plant, permitting monitoring of the stiffness distribution in cell-wall layers as a function of the depth and its evolution during the different growth phases. The mechanical measurements were correlated with changes in the composition of the cell wall, which were revealed by Fourier-transform infrared (FTIR) spectroscopy. In the beginning and end of cell growth, the average stiffness of the cell wall was low and the wall was mechanically homogenous, whereas in the exponential growth phase, the average wall stiffness increased, with increasing heterogeneity. In this phase, the difference between the superficial and deep wall stiffness was highest. FTIR spectra revealed a relative increase in the polysaccharide/lignin content.

Anisotropic Organization and Microscopic Manipulation of Self-Assembling Synthetic Porphyrin Microrods That Mimic Chlorosomes: Bacterial Light-Harvesting Systems

Being able to control in time and space the positioning, orientation, movement, and sense of rotation of nano- to microscale objects is currently an active research area in nanoscience, having diverse nanotechnological applications. In this paper, we demonstrate unprecedented control and maneuvering of rod-shaped or tubular nanostructures with high aspect ratios which are formed by self-assembling synthetic porphyrins. The self-assembly algorithm, encoded by appended chemical-recognition groups on the periphery of these porphyrins, is the same as the one operating for chlorosomal bacteriochlorophylls (BChls). Chlorosomes, rod-shaped organelles with relatively long-range molecular order, are the most efficient naturally occurring light-harvesting systems. They are used by green photosynthetic bacteria to trap visible and infrared light of minute intensities even at great depths, e.g., 100 m below water surface or in volcanic vents in the absence of solar radiation. In contrast to most other natural light-harvesting systems, the chlorosomal antennae are devoid of a protein scaffold to orient the BChls; thus, they are an attractive goal for mimicry by synthetic chemists, who are able to engineer more robust chromophores to self-assemble. Functional devices with environmentally friendly chromophores-which should be able to act as photosensitizers within hybrid solar cells, leading to high photon-to-current conversion efficiencies even under low illumination conditions-have yet to be fabricated. The orderly manner in which the BChls and their synthetic counterparts self-assemble imparts strong diamagnetic and optical anisotropies and flow/shear characteristics to their nanostructured assemblies, allowing them to be manipulated by electrical, magnetic, or tribomechanical forces.

Some new faces of membrane microdomains: A complex confocal fluorescence, differential polarization, and FCS imaging study on live immune cells

Lipid rafts are cholesterol‐ and glycosphingolipid‐rich plasma membrane microdomains, which control signal transduction, cellular contacts, pathogen recognition, and internalization processes. Their stability/lifetime, heterogeneity remained still controversial, mostly due to the high diversity of raft markers and cellular models. The correspondence of the rafts of living cells to liquid ordered (Lo) domains of model membranes and the effect of modulating rafts on the structural dynamics of their bulk membrane environment are also yet unresolved questions. Spatial overlap of various lipid and protein raft markers on live cells was studied by confocal laser scanning microscopy, while fluorescence polarization of DiIC18(3) and Bodipy‐phosphatidylcholine was imaged with differential polarization CLSM (DP‐CLSM). Mobility of the diI probe under different conditions was assessed by fluorescence correlation spectroscopic (FCS). GM1 gangliosides highly colocalized with GPI‐linked protein markers of rafts and a new anti‐cholesterol antibody (AC8) in various immune cells. On the same cells, albeit not fully excluded from rafts, diI colocalized much less with raft markers of both lipid and protein nature, suggesting the Lo membrane regions are not equivalents to lipid rafts. The DP‐CLSM technique was capable of imaging probe orientation and heterogeneity of polarization in the plasma membrane of live cells, reflecting differences in lipid order/packing. This property—in accordance with diI mobility assessed by FCS—was sensitive to modulation of rafts either through their lipids or proteins. Our complex imaging analysis demonstrated that two lipid probes—GM1 and a new anti‐cholesterol antibody—equivocally label the membrane rafts on a variety of cell types, while some raft‐associated proteins (MHC‐II, CD48, CD59, or CD90) do not colocalize with each other. This indicates the compositional heterogeneity of rafts. Usefulness of the DP‐CLSM technique in imaging immune cell surface, in terms of lipid order/packing heterogeneities, was also shown together with its sensitivity to monitor biological modulation of lipid rafts.

Imaging Fluorescence Detected Linear Dichroism of Plant Cell Walls in Laser Scanning Confocal Microscope

Anisotropy carries important information on the molecular organization of biological samples. Its determination requires a combination of microscopy and polarization spectroscopy tools. The authors constructed differential polarization (DP) attachments to a laser scanning microscope in order to determine physical quantities related to the anisotropic distribution of molecules in microscopic samples; here the authors focus on fluorescence‐detected linear dichroism (FDLD). By modulating the linear polarization of the laser beam between two orthogonally polarized states and by using a demodulation circuit, the authors determine the associated transmitted and fluorescence intensity‐difference signals, which serve the basis for LD (linear dichroism) and FDLD, respectively. The authors demonstrate on sections of Convallaria majalis root tissue stained with Acridin Orange that while (nonconfocal) LD images remain smeared and weak, FDLD images recorded in confocal mode reveal strong anisotropy of the cell wall. FDLD imaging is suitable for mapping the anisotropic distribution of transition dipoles in 3 dimensions. A mathematical model is proposed to account for the fiber‐laminate ultrastructure of the cell wall and for the intercalation of the dye molecules in complex, highly anisotropic architecture.

Imaging anisotropy using differential polarization laser scanning confocal microscopy

We have constructed differential polarization (DP) attachments to a laser scanning microscope (LSM) for imaging the main DP quantities of anisotropic microscopic objects. The DP-LSM operates with high-frequency modulation and subsequent demodulation and displays the main DP quantities pixel by pixel. These, for linearly polarized light, include: (i) linear birefringence (LB), which is exhibited by structurally and/or optically anisotropic material; (ii) linear dichroism (LD), which carries information on the anisotropic distribution of the molecules, i.e. of their absorbance transition dipole vectors, in the sample; (iii) fluorescence-detected LD (FDLD), which carries the same information for fluorescent dyes upon excitations with two orthogonally polarized light beams; (iv) anisotropy of the fluorescence emission (r), excited with non-polarized light, which is determined by the distribution of the emission transition dipole vectors in the sample and is analogous with LD and (v) the degree of polarization of the fluorescence emission (P), excited with polarized light, which depends on the depolarization of the emission e.g. due to the rotation of molecules during their excitation lifetimes. In fluorescence regimes, the DP images can be recorded in the confocal regime of the microscope, which thus warrants good spatial resolution and the possibility of mapping the anisotropy in three dimensions. In this paper, we outline the design and technical realization of our DP-LSM and give a few examples on DP imaging of different biological samples.

Confocal Fluorescence Detected Linear Dichroism Imaging of Isolated Human Amyloid Fibrils. Role of Supercoiling

Amyloids are highly organized insoluble protein aggregates that are associated with a large variety of degenerative diseases. In this work, we investigated the anisotropic architecture of isolated human amyloid samples stained with Congo Red. This was performed by fluorescence detected linear dichroism (FDLD) imaging in a laser scanning confocal microscope that was equipped with a differential polarization attachment using high frequency modulation of the polarization state of the laser beam and a demodulation circuit. Two- and three-dimensional FDLD images of amyloids provided information on the orientation of the electric transition dipoles of the intercalated Congo Red molecules with unprecedented precision and spatial resolution. We show that, in accordance with linear dichroism imaging (Jin et al. Proc Natl Acad Sci USA 100:15294, 2003), amyloids exhibit strong anisotropy with preferential orientation of the dye molecules along the fibrils; estimations on the orientation angle, of around 45°, are given using a model calculation which takes into account the helical organization of the filaments and fibrils. Our data also show that FDLD images display large inhomogeneities, high local values with alternating signs and, in some regions, well identifiable µm-sized periodicities. These features of the anisotropic architecture are accounted for by supercoiling of helically organized amyloid fibrils.

Cryo-imaging of photosystems and phycobilisomes in Anabaena sp. PCC 7120 cells

Primary photosynthetic reactions take place inside thylakoid membrane where light-to-chemical energy conversion is catalyzed by two pigment-protein complexes, photosystem I (PSI) and photosystem II (PSII). Light absorption in cyanobacteria is increased by pigment-protein supercomplexes--phycobilisomes (PBSs) situated on thylakoid membrane surfaces that transfer excitation energy into both photosystems. We have explored the localization of PSI, PSII and PBSs in thylakoid membrane of native cyanobacteria cell Anabaena sp. 7120 by means of cryogenic confocal microscopy. We have adapted a conventional temperature controlling stage to an Olympus FV1000 confocal microscope. The presence of red shifted emission of chlorophylls from PSI has been confirmed by spectral measurements. Confocal fluorescence images of PSI (in a spectral range 710-750 nm), PSII (in a spectral range 690-705 nm) and PBSs (in a spectral range 650-680 nm) were recorded at low temperature. Co-localization of images showed spatial heterogeneity of PSI, PSII and PBSs over the thylakoid membrane, and three dominant areas were identified: PSI-PSII-PBS supercomplex area, PSII-PBS supercomplex area and PSI area. The observed results were discussed with regard to light-harvesting regulation in cyanobacteria.

Three-Dimensional Architecture of the Granum-Stroma Thylakoid Membrane System Revealed by Electron Tomography

We Investigated The Three-Dimensional Architecture Of Isolated Granal Thylakoid Membranes By Using High Voltage (1,200 Kv) Electron Tomography On 250 Nm Thick Parts Of Isolated Intact Thylakoid Preparations Fixed And Stained With Conventional Electron Microscopy Techniques. High Resolution Reconstructed Tomographic Images Were Obtained Which Clearly Resolved The Membranes And The Lumenal Spaces, And Thus Also The Connections Between The Stacked And Non-Stacked Thylakoid Membranes. Based On Our Data, And Also Using The Electron Tomography Data Of Chloroplasts Within Cryo-Immobilized, Freeze Substituted Leaves (Shimoni Et Al. 2005), We Propose A Refined Model Of The Granum-Stroma Assembly. The Model Takes Into Account The Following Structural Factors And Membrane Properties: (I) Size Differences Between The Stroma Exposed Sides Of The Two Photosystems And Also The Protrusion Of The Atp Synthase In The Stroma; (Ii) The Lateral Segregation Of The Complexes Governed By The Self-Assembly Of Photosystem Ii (Psii) And Its Main Light Harvesting Complexes, The Lhcii, And The Formation Of Large Domains Enriched In Lhcii And Psii; (Iii) The Capability Of These Domains Of Stacking, A Step Initiating The Granum Formation And Stabilizing The Membrane Organization With The Lateral Heterogeneity; (Iv) The Fusion And Overlap Of Membranes During Their Growth. These Factors Can Bring About The Self-Assembly Of A Quasi-Helical Organization Of The Granumstroma Contiguous Thylakoid Membrane System That Encloses A Single Interior Aqueous Phase.

Differential polarization laser scanning microscopy: biological applications

With the aid of a differential polarization (DP) apparatus, developed in our laboratory and attached to our laser scanning confocal microscope, we can measure the magnitude and spatial distribution of 8 different DP quantities: linear and circular dichroism (LD&CD), linear and circular anisotropy of the emission (R and CPL, confocal), fluorescence detected dichroisms (FDLD&FDCD, confocal), linear birefringence (LB), and the degree of polarization of fluorescence emission (P, confocal). The attachment uses high frequency modulation and subsequent demodulation, via lock-in amplifier, of the detected intensity values, and records and displays pixel-by-pixel the measured DP quantity. These microscopic DP data carry important physical information on the molecular architecture of anisotropically organized samples. Microscopic DP measurements are thought to be of particular importance in biology. In most biological samples anisotropy is difficult to determine with conventional, macroscopic DP measurements and microscopic variations are of special significance. In this paper, we describe the method of LB imaging. Using magnetically oriented isolated chloroplasts trapped in polyacrylamide gel, we demonstrate that LB can be determined with high sensitivity and good spatial resolution. Granal thylakoid membranes in edge-aligned orientation exhibited strong LB, with large variations in its sign and magnitude. In face-aligned position LB was considerably weaker, and tended to vanish when averaged for the whole image. The strong local variations are attributed to the inherent heterogeneity of the membranes, i.e. to their internal differentiation into multilamellar, stacked membranes (grana), and single thylakoids (stroma membranes). Further details and applications of our DP-LSM will be published elsewhere.

Fluorescence-Detected Linear Dichroism of Wood Cell Walls in Juvenile Serbian Spruce: Estimation of Compression Wood Severity.

Fluorescence-detected linear dichroism (FDLD) microscopy provides observation of structural order in a microscopic sample and its expression in numerical terms, enabling both quantitative and qualitative comparison among different samples. We applied FDLD microscopy to compare the distribution and alignment of cellulose fibrils in cell walls of compression wood (CW) and normal wood (NW) on stem cross-sections of juvenile Picea omorika trees. Our data indicate a decrease in cellulose fibril order in CW compared with NW. Radial and tangential walls differ considerably in both NW and CW. In radial walls, cellulose fibril order shows a gradual decrease from NW to severe CW, in line with the increase in CW severity. This indicates that FDLD analysis of cellulose fibril order in radial cell walls is a valuable method for estimation of CW severity.