Max Sonnleitner

Single-Molecule Anisotropy Imaging

A novel method, single-molecule anisotropy imaging, has been employed to simultaneously study lateral and rotational diffusion of fluorescence-labeled lipids on supported phospholipid membranes. In a fluid membrane composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, in which the rotational diffusion time is on the order of the excited-state lifetime of the fluorophore rhodamine, a rotational diffusion constant, D(rot) = 7 x 10(7) rad(2)/s, was determined. The lateral diffusion constant, measured by direct analysis of single-molecule trajectories, was D(lat) = 3.5 x 10(-8) cm(2)/s. As predicted from the free-volume model for diffusion, the results exhibit a significantly enhanced mobility on the nanosecond time scale. For membranes of DPPC lipids in the L(beta) gel phase, the slow rotational mobility permitted the direct observation of the rotation of individual molecules characterized by D(rot) = 1.2 rad(2)/s. The latter data were evaluated by a mean square angular displacement analysis. The technique developed here should prove itself profitable for imaging of conformational motions of individual proteins on the time scale of milliseconds to seconds.

Single molecule microscopy of biomembranes (Review)

Recent advances in the development of new microscopy techniques with a sensitivity of a single molecule have gained access to essentially new types of information obtainable from imaging biomolecular samples. These methodologies are analysed here in terms of their applicability to the in vivo visualization of cellular processes on the molecular scale, in particular of processes in cell membranes. First examples of single molecule microscopy on cell membranes revealed new basic insight into the lateral organization of the plasma membrane, providing the captivating perspective of an ultrasensitive methodology as a general tool to study local processes and heterogeneities in living cells.

Organic photodiodes for biosensor miniaturization.

Biosensors have successfully demonstrated the capability to detect multiple pathogens simultaneously at very low levels. Miniaturization of biosensors is essential for use in the field or at the point of care. While microfluidic systems reduce the footprint for biochemical processing devices and electronic components are continually becoming smaller, optical components suitable for integration--such as LEDs and CMOS chips--are generally still too expensive for disposable components. This paper describes the integration of polymer diodes onto a biosensor chip to create a disposable device that includes both the detector and the sensing surface coated with immobilized capture antibody. We performed a chemiluminescence immunoassay on the OPD substrate and measured the results using a hand-held reader attached to a laptop computer. The miniaturized biosensor with the disposable slide including the organic photodiode detected Staphylococcal enterotoxin B at concentrations as low as 0.5 ng/mL.

Visualization of the Uptake of Individual HDL Particles in Living Cells Via the Scavenger Receptor Class B Type I

The scavenger receptor class B type I (SR-BI) plays an important role in mediating selective uptake of high-density lipoprotein (HDL)-derived cholesterol and cholesteryl ester in liver and steroidogenic tissues. The molecular mechanism by which this receptor mediates selective cholesteryl ester uptake remains still enigmatic. We applied ultrasensitive fluorescence microscopy to visualize the intracellular transport routes of HDL particles taken up via SR-BI in a Chinese hamster ovarian cell line. Although diffusion of the receptor bound particles on the cell surface is slow, internalization is accompanied by a dramatic increase in the mobility of the particles. HDL particles are endocytosed as clusters and actively transported to the perinuclear region of the cell. Costaining with organelle markers confirmed the involvement of an acidic compartment and the Golgi apparatus in the uptake process; finally, resecretion of the HDL particles was observed.

Imaging individual molecules by two-photon excitation

Abstract Wide-field epi-illumination of supported lipid membranes by a femtosecond near-infrared laser allowed us to identify and follow individual fluorescence labeled lipids in their motion within the membrane. The fluorophores were excited by two-photon absorption of the near-infrared beam as seen from a quadratic dependence of the fluorescence signal on the illumination intensity. The largely decreased background signal on two-photon excitation compared to that using one-photon excitation suggests that single-molecule imaging on native cell membranes will come into reach.

A sequence in the carboxy-terminus of the α1C subunit important for targeting, conductance and open probability of L-type Ca2+ channels

The role of the 80‐amino acid motif 1572–1651 in the C‐terminal tail of α1C Ca2+ channel subunits was studied by comparing properties of the conventional α1C,77 channel expressed in HEK‐tsA201 cells to three isoforms carrying alterations in this motif. Replacement of amino acids 1572–1651 in α1C,77 with 81 non‐identical residues leading to α1C,86 impaired membrane targeting and cluster formation of the channel. Similar to α1C,86, substitution of its 1572–1598 (α1C,77L) or 1595–1652 (α1C,77K) segments into the α1C,77 channel yielded single‐channel Ba2+ currents with increased inactivation, reduced open probability and unitary conductance, when compared to the α1C,77 channel. Thus, the C‐terminal sequence 1572–1651 of the α1C subunit is important for membrane targeting, permeation and open probability of L‐type Ca2+ channels.

Single Molecule Microscopy in Living Cells: Subtraction of Autofluorescence Based on Two Color Recording

A significant limitation of ultra-sensitive microscopy on living cells is set by background signal arising from cellular autofluorescence. Up to now, most strategies to circumvent this limitation were based on choosing long-wavelength dyes and selecting cell lines with reduced metabolism. In this article, we present a new strategy to identify and eliminate signal arising from autofluorescence. Two images are recorded simultaneously in distinct spectral channels. An algorithm, based on singular value decomposition, separates the contributions by the fluorophore of interest and autofluorescence. A first application of the method for imaging CD44-YFP in living cells is given.

High throughput FRET screening of the plasma membrane based on TIRFM

Monitoring protein function with high throughput at individual cell level is of high interest both for basic research and diagnostic applications. For this, following the changes in fluorescence resonance energy transfer (FRET) between a donor/acceptor pair, genetically encoded in the proteins of interest, is a frequently used tool. As proteins attached to or located in the plasma membrane represent a considerable fraction of total proteins, there is a need for high throughput imaging techniques suited for observation of proteins in the cell membrane only. A system is presented, which allows rapid imaging of large areas via total internal reflection fluorescence microscopy (TIRFM) conditions, using a focus‐hold system, multiwavelength excitation and dual color detection. The developed imaging system enables screening of large numbers of cells under TIRFM illumination combined with FRET imaging, thereby providing the means to record, e.g., FRET‐efficiency of a membrane‐associated protein labeled with a donor–acceptor pair. The capability of the system to perform live‐FRET scanning with TIRFM on stoichiometric FRET constructs, reaching throughputs of up to 1,000 cells/s at the optical resolution limit is demonstrated. A comparison with confocal microscopy shows that TIRFM offers a 4.2‐fold advantage in our conditions over confocal microscopy in detecting contributions from membrane‐localized proteins.

Single-molecule reader for proteomics and genomics.

Recent developments in ultrasensitive fluorescence microscopy enabled the detection and detailed characterization of individual biomolecules in their native environment. New types of information can be obtained from studying individual molecules, which is not accessible from ensemble measurements. Moreover, this methodological advance matches the need of bioscience to downscale the sample amount required for screening devices. It is envisioned that concentrations as low as approximately 1000 molecules contained in a sample of 1 nl can be detected in a chip-based assay. In this review, we overview state-of-the-art single molecule microscopy with respect to its applicability to ultrasensitive screening. Quantitative estimations will be given, based on a novel apparatus designed for large area screening at single molecule sensitivity.

Co-localization of CD3 and prion protein in Jurkat lymphocytes after hypothermal stimulation

While long‐term effects of temperature treatment in respect of, e.g., gene‐expression and cellular function have already been studied in some detail, nothing is known on the physiological responses of lymphocytes during short‐term hypothermal shifts. In this report, we characterized the effects of such a stimulation using the human lymphocyte cell line Jurkat E6.1 and present evidence that warming from 4 to 37 °C for only 2 min is sufficient to cause co‐localization of CD3, prion protein and the lipid‐raft ganglioside GM1 paralleling lymphocyte activation as observed by Ca2+ mobilization and mitogen‐activated protein kinase‐phosphorylation.