Katarina Logg

Resonance Raman spectroscopy of optically trapped functional erythrocytes

We introduce a novel setup combining a micro-Raman spectrometer with external optical tweezers, suitable for resonance Raman studies of single functional trapped cells. The system differs from earlier setups in that two separate laser beams used for trapping and Raman excitation are combined in a double-microscope configuration. This has the advantage that the wavelength and power of the trapping and probe beam can be adjusted individually to optimize the functionality of the setup and to enable the recording of resonance Raman profiles from a single trapped cell. Trapping is achieved by tightly focusing infrared (IR) diode laser radiation (830 nm) through an inverted oil-immersion objective, and resonance Raman scattering is excited by the lines of an argon:krypton ion laser. The functionality of the system is demonstrated by measurements of trapped single functional erythrocytes using different excitation lines (488.0, 514.5, and 568.2 nm) in resonance with the heme moiety and by studying spectral evolution during illumination. We found that great care has to be taken in order to avoid photodamage caused by the visible Raman excitation, whereas the IR trapping irradiation does not seem to harm the cells or alter the hemoglobin Raman spectra. Stronger photodamage is induced by Raman excitation using 488.0- and 514.5-nm irradiation, compared with excitation with the 568.2-nm line.

Image analysis algorithms for cell contour recognition in budding yeast

Quantification of protein abundance and subcellular localization dynamics from fluorescence microscopy images is of high contemporary interest in cell and molecular biology. For large-scale studies of cell populations and for time-lapse studies, such quantitative analysis can not be performed effectively without some kind of automated image analysis tool. Here, we present fast algorithms for automatic cell contour recognition in bright field images, optimized to the model organism budding yeast (Saccharomyces cerevisiae). The cell contours can be used to effectively quantify cell morphology parameters as well as protein abundance and subcellular localization from overlaid fluorescence data.

Continuous light exposure causes cumulative stress that affects the localization oscillation dynamics of the transcription factor Msn2p.

Light exposure is a potentially powerful stress factor during in vivo optical microscopy studies. In yeast, the general transcription factor Msn2p translocates from the cytoplasm to the nucleus in response to illumination. However, previous time-lapse fluorescence microscopy studies of Msn2p have utilized a variety of discrete exposure settings, which makes it difficult to correlate stress levels and illumination parameters. We here investigate how continuous illumination with blue light, corresponding to GFP excitation wavelengths, affects the localization pattern of Msn2p-GFP in budding yeast. The localization pattern was analyzed using a novel approach that combines wavelet decomposition and change point analysis. It was found that the Msn2p nucleocytoplasmic localization trajectories for individual cells exhibit up to three distinct and successive states; i) Msn2p localizes to the cytoplasm; ii) Msn2p rapidly shuttles between the cytoplasm and the nucleus; iii) Msn2p localizes to the nucleus. Many cells pass through all states consecutively at high light intensities, while at lower light intensities most cells only reach states i) or ii). This behaviour strongly indicates that continuous light exposure gradually increases the stress level over time, presumably through continuous accumulation of toxic photoproducts, thereby forcing the cell through a bistable region corresponding to nucleocytoplasmic oscillations. We also show that the localization patterns are dependent on protein kinase A (PKA) activity, i.e. yeast cells with constantly low PKA activity showed a stronger stress response. In particular, the nucleocytoplasmic oscillation frequency was found to be significantly higher for cells with low PKA activity for all light intensities.

The sodium pump Ena1p provides mechanistic insight into the salt sensitivity of vacuolar protein sorting mutants.

The vacuolar/endosomal network has an important but as yet undefined role in the cellular tolerance to salt stress. We hypothesized that the mechanistic basis for the importance of vacuolar protein sorting (vps) components in salt tolerance is the targeting of the crucial sodium exporter Ena1p to the plasma membrane. The link between Ena1p and the vps components was established by the observation that overexpression of Ena1p could suppress the salt sensitivity of the ESCRT knockouts vps20Delta, snf7/vps32Delta and snf8/vps22Delta. To further investigate this functional interaction, fluorescence microscopy was utilized to monitor localization of GFP-tagged Ena1p. For all analyzed vps mutants, Ena1p seemed properly localized to the plasma membrane, even during saline growth. However, quantitative differences in plasma membrane localized Ena1p were recorded; e.g. the highly salt sensitive pep12Delta mutant exhibited substantially enhanced Ena1p levels. In addition, the kinetics of Ena1p localization to the plasma membrane was severely delayed in several vps mutants, and this delay correlated to the salt specific growth defect. This paper discusses potential mechanistic hypotheses, like Ena1p transporter activity or localization kinetics, or ESCRT components influence on signaling, for linking endosomal sorting functions to cellular salt sensitivity.

Sulfate Assimilation Mediates Tellurite Reduction and Toxicity in Saccharomyces cerevisiae

ABSTRACT Despite a century of research and increasing environmental and human health concerns, the mechanistic basis of the toxicity of derivatives of the metalloid tellurium, Te, in particular the oxyanion tellurite, Te(IV), remains unsolved. Here, we provide an unbiased view of the mechanisms of tellurium metabolism in the yeast Saccharomyces cerevisiae by measuring deviations in Te-related traits of a complete collection of gene knockout mutants. Reduction of Te(IV) and intracellular accumulation as metallic tellurium strongly correlated with loss of cellular fitness, suggesting that Te(IV) reduction and toxicity are causally linked. The sulfate assimilation pathway upstream of Met17, in particular, the sulfite reductase and its cofactor siroheme, was shown to be central to tellurite toxicity and its reduction to elemental tellurium. Gene knockout mutants with altered Te(IV) tolerance also showed a similar deviation in tolerance to both selenite and, interestingly, selenomethionine, suggesting that the toxicity of these agents stems from a common mechanism. We also show that Te(IV) reduction and toxicity in yeast is partially mediated via a mitochondrial respiratory mechanism that does not encompass the generation of substantial oxidative stress. The results reported here represent a robust base from which to attack the mechanistic details of Te(IV) toxicity and reduction in a eukaryotic organism.

Computational high-throughput screening of fluid permeability in heterogeneous fiber materials

We explore computational high-throughput screening as a design strategy for heterogeneous, isotropic fiber materials. Fluid permeability, a key property in the design of soft porous materials, is systematically studied using a multi-scale lattice Boltzmann framework. After characterizing microscopic permeability as a function of solid volume fraction in the microstructure, we perform high-throughput computational screening of in excess of 35 000 macrostructures consisting of a continuous bulk interrupted by spherical/elliptical domains with either lower or higher microscopic permeability (hence with two distinct microscopic solid volume fractions and therefore two distinct microscopic permeabilities) to assess which parameters determine macroscopic permeability for a fixed average solid volume fraction. We conclude that the fractions of bulk and domains and the distribution of solid volume fraction between them are the primary determinants of macroscopic permeability, and that a substantial increase in permeability compared to the corresponding homogenous material is attainable.

Development of automatic image analysis algorithms for protein localization studies in budding yeast

Microscopy of fluorescently labeled proteins has become a standard technique for live cell imaging. However, it is still a challenge to systematically extract quantitative data from large sets of images in an unbiased fashion, which is particularly important in high-throughput or time-lapse studies. Here we describe the development of a software package aimed at automatic quantification of abundance and spatio-temporal dynamics of fluorescently tagged proteins in vivo in the budding yeast Saccharomyces cerevisiae, one of the most important model organisms in proteomics. The image analysis methodology is based on first identifying cell contours from bright field images, and then use this information to measure and statistically analyse protein abundance in specific cellular domains from the corresponding fluorescence images. The applicability of the procedure is exemplified for two nuclear localized GFP-tagged proteins, Mcm4p and Nrm1p.

Development of a novel Raman tweezers setup for single-cell studies

It has recently been shown that the combination of Raman spectroscopy and optical tweezers constitute a powerful tool for biological studies. Raman spectra of single cells immobilized in a sterile surrounding can then be recorded without the risk of surface-induced morphological cell changes. Further, the complete cellular environment can be changed while measuring dynamics in real time. We here introduce a novel Raman tweezers set-up ideal for resonance Raman studies of single cells. The system differs from earlier set-ups in that two separate laser beams, used for trapping and Raman excitation, are combined in a double-microscope configuration. This has the advantage that the wavelength and power of the trapping and probe beam can be adjusted individually, for example in order to optimize the functionality of the set-up or to record resonance Raman profiles from the same trapped cell. Further, the tweezers can be removed from the system without affecting the spectrometer configuration. Trapping is achieved by tightly focusing IR diode laser radiation (830 nm) through an inverted oil immersion objective with high numerical aperture (NA = 1.25), while Raman scattering is excited by the lines of an ArKr ion-laser. The backscattered Raman signal is collected by a single-grating spectrometer equipped with a microscope and a 60x water-immersion objective (NA = 0.9). The functionality of the system is demonstrated by measurements of trapped single functional erythrocytes using differen excitation lines (488, 514.5 568.2 nm) in resonance with the heme moiety and by studying the spectral evolution during illumination.