Graphene consisting of a monolayer of sp 2 -bonded carbon atoms is a relatively new member of the... more Graphene consisting of a monolayer of sp 2 -bonded carbon atoms is a relatively new member of the carbon family. But owing to its unique structure, exceptional electrical, optical and mechanical properties, graphene is a rapidly rising star on the horizon of materials science and condensed matter physics [1]. Since its discovery, optical microscopy, atomic force microscopy (AFM), scanning electron microscopy (SEM), transmission electron microscopy (TEM) and micro-Raman spectroscopy have been widely employed to investigate its optical properties, determine its thickness, resolve its atomic arrangement, and detect its film quality, respectively. We have applied several of those imaging and characterization techniques to investigate surface morphology and electronic properties of graphene, selected derivatives like graphene oxide (GO) or silanized graphene, graphene-metal nanoparticle composites as well as graphene analogues like hexagonal boron-nitride (h-BN) a) Imaging graphene with ...
Biomolecular interactions are responsible for the initiation, modulation and termination of DNA r... more Biomolecular interactions are responsible for the initiation, modulation and termination of DNA replication, RNA transcription, enzyme activity, infection, immune response, tissue generation, wound healing, cellular differentiation, programmed cell death and the activities of drugs, hormones or toxic substances. AFM (atomic force microscopy) allows scientists to visualize, probe and analyze the structures of biological molecules in their native environments with high resolution. Organic chemistry, surface chemistry, bioconjugaton chemistry and AFM can be combined and used to locate or quantify specific biomolecular interactions. For example, in molecular recognition force microscopy (MRFM), single molecule interactions are observed and quantified one by one as an AFM probe that has been modified with a ligand or an antibody approaches and then is subsequently withdrawn away from a surface that contains binding sites. Topography and recognition imaging (TREC) also relies on AFM probe...
Abstract. Colloidal suspensions of small silicon nanoparticles (diameter around 2 nm) with fast a... more Abstract. Colloidal suspensions of small silicon nanoparticles (diameter around 2 nm) with fast and efficient ultraviolet–blue photoluminescence (PL) band are fabricated by enhanced electrochemical etching of Si wafers. The detailed study of photoluminescence excitation spectra in a wide range of excitation photon energies (270–420 nm) reveals specific behavior of the Stokes shift of the fast PL band that agrees well with theoretical calculation of optical transitions in small silicon nanocrystals and is distinct from emission of silicon ...
Biotin-4-fluorescein (B4F) is a convenient molecular probe for (strept)avidin and for unlabeled b... more Biotin-4-fluorescein (B4F) is a convenient molecular probe for (strept)avidin and for unlabeled biotin in homogeneous fluorescence assays. The primary standard is a 16 μM working solution of d-biotin which is used to titrate an aliquot of a (strept)avidin stock solution while monitoring the tryptophane fluorescence of (strept)avidin. This serves to standardize the (strept)avidin stock solution, an aliquot of which is then titrated with a roughly 16 μM working solution of B4F while monitoring the fluorescence of B4F. Specific binding is accompanied by quenching, but after saturation of all binding sites, the appearance of free ligand causes a sharp rise of intense fluorescence, the beginning of which allows to calculate the effective concentration of B4F in the working solution. Measurement of avidin in a crude sample is exemplified by mixing 8 pmol of B4F with various amounts of diluted egg white in a volume of 1 mL. Hereby, the extent of fluorescence quenching linearly correlates with the concentration of functional avidin. Moreover, a sharp minimum of fluorescence is observed when exactly 2 pmol of avidin is present in the sample. The latter assay has been adapted to measure between 0.5 and 5 pmol of d-biotin in 1 mL of sample by adding 1.9 pmol of avidin and 8 pmol of B4F. This competitive assay correctly measures the small dose of d-biotin in multivitamin tablets (e.g., 150 μg in 5 g of solid) after subtracting the background fluorescence of the colored aqueous solution.
... running title: bioconjugation for single molecule microscopy Christian K. Riener, Gerald Kada... more ... running title: bioconjugation for single molecule microscopy Christian K. Riener, Gerald Kada, Cordula Borken, Ferry Kienberger, Peter Hinterdorfer, Hansgeorg Schindler, Gerhard J. Schütz, Thomas Schmidt, Christoph D. Hahn, and Hermann J. Gruber* ...
The lateral motion of single fluorescence labeled lipid molecules was imaged in native cell membr... more The lateral motion of single fluorescence labeled lipid molecules was imaged in native cell membranes on a millisecond time scale and with positional accuracy of 50 nm, using 'single dye tracing'. This first application of single molecule microscopy to living cells rendered possible the direct observation of lipid-specific membrane domains. These domains were sensed by a lipid probe with saturated acyl chains as small areas in a liquid-ordered phase: the probe showed confined but fast diffusion, with high partitioning (~100-fold) and long residence time (~13 s). The analogous probe with mono-unsaturated chains diffused predominantly unconfined within the membrane. With~15 saturated probes per domain, the locations, sizes, shapes and motions of individual domains became clearly visible. Domains had a size of 0.7 µm (0.2-2 µm), covering 13% of total membrane area. Both the liquid-ordered phase characteristics and the sizes of domains match properties of membrane fractions described as detergent-resistant membranes (DRMs), strongly suggesting that the domains seen are the in vivo correlate of DRMs and thus may be identified as lipid rafts. Keywords: fluorescence imaging/human coronary artery smooth muscle cell/lipid rafts/liquid-ordered lipid phase/ single molecule diffusion
Imaging diffusion of single fluorescence labeled lipids by Two Photon Excitation (TPE) has been a... more Imaging diffusion of single fluorescence labeled lipids by Two Photon Excitation (TPE) has been accomplished for an artificial lipid membrane [1] and on live cells . This opens the perspective to utilize the advantages of TPE, e.g. the reduced background, increased lateral and axial resolution and the possibility of synchronous multicolor experiments, to study dynamical rearrangements and interactions between biomolecules in living cells.
ABSTRACT A Two Photon Laser Scanning Microscope was used for detecting single Cy3-labeled lipids ... more ABSTRACT A Two Photon Laser Scanning Microscope was used for detecting single Cy3-labeled lipids on the plasma membrane of living human coronary artery smooth muscle cells. For Two-Photon-Excitation of Cy3 the fs-laser system was tuned to 700nm [1]. Figure A shows a fluorescence image of blank HASM cells taken with a mean power of 6mW at the sample and a dwell time of 50ms per pixel. The spots around the nucleus originate from autofluorescence of mitochondria. After taking this blank image the cells were incubated with a vesicle solution of POPC/DMPE-Cy3 at a molar ratio of 103.Lipid exchange between the vesicles and the cells resultedin a concentration of Cy3-labeled lipids of ∼1/50 μm2 on the cell membrane [2]. The vesicles were removed out of the measurement chamber by flushing with PBS buffer. For single molecule detection experiments a scanning region with almost no autofluorescence was chosen as indicated in Figure A. A sequence of images as shown Figure B was taken with a dwell time of 50ms per pixel. The fluorescence peaks represent single Cy3-labeled lipids diffusing on the cell membrane. Imaging the diffusion of single fluorescence labeled lipids by TPE has been accomplished for an artificial lipid membrane [3]. Here we show that single molecule detection with TPE is also possible on cells in vivo. This opens the perspective to utilize the advantages of Two Photon Excitation, e.g. the reduced background, increased lateral and axial resolution and the possibility of synchronous multicolor experiments, for single molecule imaging in living biological cells.
The lateral motion of single fluorescence labeled lipid molecules was imaged in native cell membr... more The lateral motion of single fluorescence labeled lipid molecules was imaged in native cell membranes on a millisecond time scale and with positional accuracy of approximately 50 nm, using 'single dye tracing'. This first application of single molecule microscopy to living cells rendered possible the direct observation of lipid-specific membrane domains. These domains were sensed by a lipid probe with saturated acyl chains as small areas in a liquid-ordered phase: the probe showed confined but fast diffusion, with high partitioning (approximately 100-fold) and long residence time (approximately 13 s). The analogous probe with mono-unsaturated chains diffused predominantly unconfined within the membrane. With approximately 15 saturated probes per domain, the locations, sizes, shapes and motions of individual domains became clearly visible. Domains had a size of 0.7 micrometer (0.2-2 micrometer), covering approximately 13% of total membrane area. Both the liquid-ordered phase characteristics and the sizes of domains match properties of membrane fractions described as detergent-resistant membranes (DRMs), strongly suggesting that the domains seen are the in vivo correlate of DRMs and thus may be identified as lipid rafts.
We present an easy-to-use combination of an atomic force microscope (AFM) and an epi-fluorescence... more We present an easy-to-use combination of an atomic force microscope (AFM) and an epi-fluorescence microscope, which allows live cell imaging under physiological conditions. High-resolution AFM images were acquired while simultaneously monitoring either the fluorescence image of labeled membrane components, or a high-contrast optical image (DIC, differential interference contrast). By applying two complementary techniques at the same time, additional information and correlations between structure and function of living organisms were obtained. The synergy effects between fluorescence imaging and AFM were further demonstrated by probing fluorescence-labeled receptor clusters in the cell membrane via force spectroscopy using antibody-functionalized tips. The binding probability on receptor-containing areas identified with fluorescence microscopy (''receptor-positive sites'') was significantly higher than that on sites lacking receptors. r
... running title: bioconjugation for single molecule microscopy Christian K. Riener, Gerald Kada... more ... running title: bioconjugation for single molecule microscopy Christian K. Riener, Gerald Kada, Cordula Borken, Ferry Kienberger, Peter Hinterdorfer, Hansgeorg Schindler, Gerhard J. Schütz, Thomas Schmidt, Christoph D. Hahn, and Hermann J. Gruber* ...
Plasmid DNA and viral RNA were imaged in a liquid environment by dynamic force microscopy (DFM) a... more Plasmid DNA and viral RNA were imaged in a liquid environment by dynamic force microscopy (DFM) and fine structures of DNA with heights of 1.8270.66 nm were obtained in topographical images. In simultaneously acquired phase images, DNA could be imaged with better contrast at lower imaging forces. By splitting the cantilever oscillation signal into lower and upper parts, the contribution of the adhesion between tip and sample to the topographical images was eliminated, resulting in better signal-to-noise ratio. DFM of the single stranded RNA genome of a human rhinovirus showed loops protruding from a condensed RNA core, 20-50 nm in height. The mechanical rigidity of the RNA was determined by single molecule pulling experiments. From fitting RNA stretching curves to the Worm-Like-Chain (WLC) model a persistence length of 1.070.17 nm was obtained. r
We demonstrate simultaneous transverse dynamic force microscopy and molecular recognition imaging... more We demonstrate simultaneous transverse dynamic force microscopy and molecular recognition imaging using tuning forks as piezoelectric sensors. Tapered aluminum-coated glass fibers were chemically functionalized with biotin and anti-lysozyme molecules and attached to one of the prongs of a 32 kHz tuning fork. The lateral oscillation amplitude of the tuning fork was used as feedback signal for topographical imaging of avidin aggregates and lysozyme molecules on mica substrate. The phase difference between the excitation and detection signals of the tuning fork provided molecular recognition between avidin/biotin or lysozyme/anti-lysozyme. Aggregates of avidin and lysozyme molecules appeared as features with heights of 1-4 nm in the topographic images, consistent with single molecule atomic force microscopy imaging. Recognition events between avidin/biotin or lysozyme/anti-lysozyme were detected in the phase image at high signal-to-noise ratio with phase shifts of 1-21. Because tapered glass fibers and shear-force microscopy based on tuning forks are commonly used for near-field scanning optical microscopy (NSOM), these results open the door to the exciting possibility of combining optical, topographic and biochemical recognition at the nanometer scale in a single measurement and in liquid conditions.
New image-processing methods were applied to atomic force microscopy images in order to visualize... more New image-processing methods were applied to atomic force microscopy images in order to visualize small details on the surface of virus particles and living cells. Polynomial line flattening and plane fitting of topographical images were performed as first step of the image processing. In a second step, a sliding window approach was used for low-pass filtering and data smoothing. The size of the filtering window was adjusted to the size of the small details of interest. Subtraction of the smoothed data from the original data resulted in images with enhanced contrast. Topographical features which are usually not visible can be easily discerned in the processed images. The method developed in this study rendered possible the detection of small patterns on viral particles as well as thin cytoskeleton fibers of living cells. It is shown that the sliding window approach gives better results than Fourier-filtering. Our method can be generally applied to increase the contrast of topographical images, especially when small features are to be highlighted on relatively high objects.
Fig. 5. Frequency spectra recorded at a cell nucleus of the live and fixed lung epithelial cell (... more Fig. 5. Frequency spectra recorded at a cell nucleus of the live and fixed lung epithelial cell (H441) in buffer. Curves are normalized to the main driving frequency. Inset: magnification of the second harmonic amplitude peaks.
The combination of fluorescence microscopy and atomic force microscopy has a great potential in s... more The combination of fluorescence microscopy and atomic force microscopy has a great potential in single-molecule-detection applications, overcoming many of the limitations coming from each individual technique. Here we present a new platform of combined fluorescence and simultaneous topography and recognition imaging (TREC) for improved localization of cellular receptors. Green fluorescent protein (GFP) labeled human sodium-glucose cotransporter (hSGLT1) expressed Chinese Hamster Ovary (CHO) cells and endothelial cells (MyEnd) from mouse myocardium stained with phalloidin-rhodamine were used as cell systems to study AFM topography and fluorescence microscopy on the same surface area. Topographical AFM images revealed membrane features such as lamellipodia, cytoskeleton fibers, F-actin filaments and small globular structures with heights ranging from 20 to 30 nm. Combined fluorescence and TREC imaging was applied to detect density, distribution and localization of YFP-labeled CD1d molecules on alpha-galactosylceramide (alphaGalCer)-loaded THP1 cells. While the expression level, distribution and localization of CD1d molecules on THP1 cells were detected with fluorescence microscopy, the nanoscale distribution of binding sites was investigated with molecular recognition imaging by using a chemically modified AFM tip. Using TREC on the inverted light microscope, the recognition sites of cell receptors were detected in recognition images with domain sizes ranging from approximately 25 to approximately 160 nm, with the smaller domains corresponding to a single CD1d molecule.
Optical lithography is a key technique in the development of semiconductor industry. However, dif... more Optical lithography is a key technique in the development of semiconductor industry. However, diffraction effects limit the minimal resolvable feature size to the Rayleigh diffraction limit of l/2, where l is the optical wavelength. Many technologies have been proposed in the past to replace optical lithography. Here, we present a new quantum optical method to do subwavelength lithography which is realizable by our current technology now. Using TEM, STEM, SEM and AFM measurements we show that 2 nm width lines could be written in novel materials such as fluorescent photosensitive glassceramics by a quantum multiphoton confinement effect. Exposure to the focus laser diode beam (l ¼650 nm) writes high-density lines with 4 nm pitch on the sample surface at room temperature, far beyond the diffraction limit, a fundamental barrier to the exploitation of optical lithography. 2 nm Quantum Optical Lithography is an important step to enable full-wafer-level nanofabrication at this resolution.
Biochimica et Biophysica Acta (BBA) - General Subjects, 1999
A new biotin^fluorescein conjugate with an ethylene diamine spacer was found to be the first fluo... more A new biotin^fluorescein conjugate with an ethylene diamine spacer was found to be the first fluorescent biotin derivative which truly mimicked D-biotin in terms of high affinity, fast association, and non-cooperative binding to avidin and streptavidin tetramers. These exceptional properties were attributed to the small size/length of the new ligand since all larger/ longer biotin derivatives are known for their mutual steric hindrance and anti-cooperative binding in 4:1 complexes with avidin and streptavidin tetramers. Specific binding of the new biotin^fluorescein conjugate towards avidin and streptavidin was accompanied by 84^88% quenching of ligand fluorescence. In the accompanying study this effect was used for rapid estimation of avidin and streptavidin in a new`single tube assay'. In the present study the strong quenching effect was utilized to accurately monitor stoichiometric titration of biotin-binding sites in samples with v 200 pM avidin or streptavidin. The concentration was calculated from the consumption of fluorescent ligand up to the distinct breakpoint in the fluorescence titration profile which was marked by the abrupt appearance of strongly fluorescent ligands which were in excess. Due to this protocol the assay was not perturbed by background fluorescence or coloration in the unknown samples. The new fluorescence titration assay is particularly suited for quick checks on short notice because getting started only means to thaw an aliquot of a standardized stock solution of fluorescent ligand. No calibration is required for the individual assay and the ligand stock solution needs to be restandardized once per week (or once per year) when stored at 325³C (or at 370³C, respectively). ß 0304-4165 / 99 / $^see front matter ß 1999 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 -4 1 6 5 ( 9 8 ) 0 0 1 7 8 -0
Graphene consisting of a monolayer of sp 2 -bonded carbon atoms is a relatively new member of the... more Graphene consisting of a monolayer of sp 2 -bonded carbon atoms is a relatively new member of the carbon family. But owing to its unique structure, exceptional electrical, optical and mechanical properties, graphene is a rapidly rising star on the horizon of materials science and condensed matter physics [1]. Since its discovery, optical microscopy, atomic force microscopy (AFM), scanning electron microscopy (SEM), transmission electron microscopy (TEM) and micro-Raman spectroscopy have been widely employed to investigate its optical properties, determine its thickness, resolve its atomic arrangement, and detect its film quality, respectively. We have applied several of those imaging and characterization techniques to investigate surface morphology and electronic properties of graphene, selected derivatives like graphene oxide (GO) or silanized graphene, graphene-metal nanoparticle composites as well as graphene analogues like hexagonal boron-nitride (h-BN) a) Imaging graphene with ...
Biomolecular interactions are responsible for the initiation, modulation and termination of DNA r... more Biomolecular interactions are responsible for the initiation, modulation and termination of DNA replication, RNA transcription, enzyme activity, infection, immune response, tissue generation, wound healing, cellular differentiation, programmed cell death and the activities of drugs, hormones or toxic substances. AFM (atomic force microscopy) allows scientists to visualize, probe and analyze the structures of biological molecules in their native environments with high resolution. Organic chemistry, surface chemistry, bioconjugaton chemistry and AFM can be combined and used to locate or quantify specific biomolecular interactions. For example, in molecular recognition force microscopy (MRFM), single molecule interactions are observed and quantified one by one as an AFM probe that has been modified with a ligand or an antibody approaches and then is subsequently withdrawn away from a surface that contains binding sites. Topography and recognition imaging (TREC) also relies on AFM probe...
Abstract. Colloidal suspensions of small silicon nanoparticles (diameter around 2 nm) with fast a... more Abstract. Colloidal suspensions of small silicon nanoparticles (diameter around 2 nm) with fast and efficient ultraviolet–blue photoluminescence (PL) band are fabricated by enhanced electrochemical etching of Si wafers. The detailed study of photoluminescence excitation spectra in a wide range of excitation photon energies (270–420 nm) reveals specific behavior of the Stokes shift of the fast PL band that agrees well with theoretical calculation of optical transitions in small silicon nanocrystals and is distinct from emission of silicon ...
Biotin-4-fluorescein (B4F) is a convenient molecular probe for (strept)avidin and for unlabeled b... more Biotin-4-fluorescein (B4F) is a convenient molecular probe for (strept)avidin and for unlabeled biotin in homogeneous fluorescence assays. The primary standard is a 16 μM working solution of d-biotin which is used to titrate an aliquot of a (strept)avidin stock solution while monitoring the tryptophane fluorescence of (strept)avidin. This serves to standardize the (strept)avidin stock solution, an aliquot of which is then titrated with a roughly 16 μM working solution of B4F while monitoring the fluorescence of B4F. Specific binding is accompanied by quenching, but after saturation of all binding sites, the appearance of free ligand causes a sharp rise of intense fluorescence, the beginning of which allows to calculate the effective concentration of B4F in the working solution. Measurement of avidin in a crude sample is exemplified by mixing 8 pmol of B4F with various amounts of diluted egg white in a volume of 1 mL. Hereby, the extent of fluorescence quenching linearly correlates with the concentration of functional avidin. Moreover, a sharp minimum of fluorescence is observed when exactly 2 pmol of avidin is present in the sample. The latter assay has been adapted to measure between 0.5 and 5 pmol of d-biotin in 1 mL of sample by adding 1.9 pmol of avidin and 8 pmol of B4F. This competitive assay correctly measures the small dose of d-biotin in multivitamin tablets (e.g., 150 μg in 5 g of solid) after subtracting the background fluorescence of the colored aqueous solution.
... running title: bioconjugation for single molecule microscopy Christian K. Riener, Gerald Kada... more ... running title: bioconjugation for single molecule microscopy Christian K. Riener, Gerald Kada, Cordula Borken, Ferry Kienberger, Peter Hinterdorfer, Hansgeorg Schindler, Gerhard J. Schütz, Thomas Schmidt, Christoph D. Hahn, and Hermann J. Gruber* ...
The lateral motion of single fluorescence labeled lipid molecules was imaged in native cell membr... more The lateral motion of single fluorescence labeled lipid molecules was imaged in native cell membranes on a millisecond time scale and with positional accuracy of 50 nm, using 'single dye tracing'. This first application of single molecule microscopy to living cells rendered possible the direct observation of lipid-specific membrane domains. These domains were sensed by a lipid probe with saturated acyl chains as small areas in a liquid-ordered phase: the probe showed confined but fast diffusion, with high partitioning (~100-fold) and long residence time (~13 s). The analogous probe with mono-unsaturated chains diffused predominantly unconfined within the membrane. With~15 saturated probes per domain, the locations, sizes, shapes and motions of individual domains became clearly visible. Domains had a size of 0.7 µm (0.2-2 µm), covering 13% of total membrane area. Both the liquid-ordered phase characteristics and the sizes of domains match properties of membrane fractions described as detergent-resistant membranes (DRMs), strongly suggesting that the domains seen are the in vivo correlate of DRMs and thus may be identified as lipid rafts. Keywords: fluorescence imaging/human coronary artery smooth muscle cell/lipid rafts/liquid-ordered lipid phase/ single molecule diffusion
Imaging diffusion of single fluorescence labeled lipids by Two Photon Excitation (TPE) has been a... more Imaging diffusion of single fluorescence labeled lipids by Two Photon Excitation (TPE) has been accomplished for an artificial lipid membrane [1] and on live cells . This opens the perspective to utilize the advantages of TPE, e.g. the reduced background, increased lateral and axial resolution and the possibility of synchronous multicolor experiments, to study dynamical rearrangements and interactions between biomolecules in living cells.
ABSTRACT A Two Photon Laser Scanning Microscope was used for detecting single Cy3-labeled lipids ... more ABSTRACT A Two Photon Laser Scanning Microscope was used for detecting single Cy3-labeled lipids on the plasma membrane of living human coronary artery smooth muscle cells. For Two-Photon-Excitation of Cy3 the fs-laser system was tuned to 700nm [1]. Figure A shows a fluorescence image of blank HASM cells taken with a mean power of 6mW at the sample and a dwell time of 50ms per pixel. The spots around the nucleus originate from autofluorescence of mitochondria. After taking this blank image the cells were incubated with a vesicle solution of POPC/DMPE-Cy3 at a molar ratio of 103.Lipid exchange between the vesicles and the cells resultedin a concentration of Cy3-labeled lipids of ∼1/50 μm2 on the cell membrane [2]. The vesicles were removed out of the measurement chamber by flushing with PBS buffer. For single molecule detection experiments a scanning region with almost no autofluorescence was chosen as indicated in Figure A. A sequence of images as shown Figure B was taken with a dwell time of 50ms per pixel. The fluorescence peaks represent single Cy3-labeled lipids diffusing on the cell membrane. Imaging the diffusion of single fluorescence labeled lipids by TPE has been accomplished for an artificial lipid membrane [3]. Here we show that single molecule detection with TPE is also possible on cells in vivo. This opens the perspective to utilize the advantages of Two Photon Excitation, e.g. the reduced background, increased lateral and axial resolution and the possibility of synchronous multicolor experiments, for single molecule imaging in living biological cells.
The lateral motion of single fluorescence labeled lipid molecules was imaged in native cell membr... more The lateral motion of single fluorescence labeled lipid molecules was imaged in native cell membranes on a millisecond time scale and with positional accuracy of approximately 50 nm, using 'single dye tracing'. This first application of single molecule microscopy to living cells rendered possible the direct observation of lipid-specific membrane domains. These domains were sensed by a lipid probe with saturated acyl chains as small areas in a liquid-ordered phase: the probe showed confined but fast diffusion, with high partitioning (approximately 100-fold) and long residence time (approximately 13 s). The analogous probe with mono-unsaturated chains diffused predominantly unconfined within the membrane. With approximately 15 saturated probes per domain, the locations, sizes, shapes and motions of individual domains became clearly visible. Domains had a size of 0.7 micrometer (0.2-2 micrometer), covering approximately 13% of total membrane area. Both the liquid-ordered phase characteristics and the sizes of domains match properties of membrane fractions described as detergent-resistant membranes (DRMs), strongly suggesting that the domains seen are the in vivo correlate of DRMs and thus may be identified as lipid rafts.
We present an easy-to-use combination of an atomic force microscope (AFM) and an epi-fluorescence... more We present an easy-to-use combination of an atomic force microscope (AFM) and an epi-fluorescence microscope, which allows live cell imaging under physiological conditions. High-resolution AFM images were acquired while simultaneously monitoring either the fluorescence image of labeled membrane components, or a high-contrast optical image (DIC, differential interference contrast). By applying two complementary techniques at the same time, additional information and correlations between structure and function of living organisms were obtained. The synergy effects between fluorescence imaging and AFM were further demonstrated by probing fluorescence-labeled receptor clusters in the cell membrane via force spectroscopy using antibody-functionalized tips. The binding probability on receptor-containing areas identified with fluorescence microscopy (''receptor-positive sites'') was significantly higher than that on sites lacking receptors. r
... running title: bioconjugation for single molecule microscopy Christian K. Riener, Gerald Kada... more ... running title: bioconjugation for single molecule microscopy Christian K. Riener, Gerald Kada, Cordula Borken, Ferry Kienberger, Peter Hinterdorfer, Hansgeorg Schindler, Gerhard J. Schütz, Thomas Schmidt, Christoph D. Hahn, and Hermann J. Gruber* ...
Plasmid DNA and viral RNA were imaged in a liquid environment by dynamic force microscopy (DFM) a... more Plasmid DNA and viral RNA were imaged in a liquid environment by dynamic force microscopy (DFM) and fine structures of DNA with heights of 1.8270.66 nm were obtained in topographical images. In simultaneously acquired phase images, DNA could be imaged with better contrast at lower imaging forces. By splitting the cantilever oscillation signal into lower and upper parts, the contribution of the adhesion between tip and sample to the topographical images was eliminated, resulting in better signal-to-noise ratio. DFM of the single stranded RNA genome of a human rhinovirus showed loops protruding from a condensed RNA core, 20-50 nm in height. The mechanical rigidity of the RNA was determined by single molecule pulling experiments. From fitting RNA stretching curves to the Worm-Like-Chain (WLC) model a persistence length of 1.070.17 nm was obtained. r
We demonstrate simultaneous transverse dynamic force microscopy and molecular recognition imaging... more We demonstrate simultaneous transverse dynamic force microscopy and molecular recognition imaging using tuning forks as piezoelectric sensors. Tapered aluminum-coated glass fibers were chemically functionalized with biotin and anti-lysozyme molecules and attached to one of the prongs of a 32 kHz tuning fork. The lateral oscillation amplitude of the tuning fork was used as feedback signal for topographical imaging of avidin aggregates and lysozyme molecules on mica substrate. The phase difference between the excitation and detection signals of the tuning fork provided molecular recognition between avidin/biotin or lysozyme/anti-lysozyme. Aggregates of avidin and lysozyme molecules appeared as features with heights of 1-4 nm in the topographic images, consistent with single molecule atomic force microscopy imaging. Recognition events between avidin/biotin or lysozyme/anti-lysozyme were detected in the phase image at high signal-to-noise ratio with phase shifts of 1-21. Because tapered glass fibers and shear-force microscopy based on tuning forks are commonly used for near-field scanning optical microscopy (NSOM), these results open the door to the exciting possibility of combining optical, topographic and biochemical recognition at the nanometer scale in a single measurement and in liquid conditions.
New image-processing methods were applied to atomic force microscopy images in order to visualize... more New image-processing methods were applied to atomic force microscopy images in order to visualize small details on the surface of virus particles and living cells. Polynomial line flattening and plane fitting of topographical images were performed as first step of the image processing. In a second step, a sliding window approach was used for low-pass filtering and data smoothing. The size of the filtering window was adjusted to the size of the small details of interest. Subtraction of the smoothed data from the original data resulted in images with enhanced contrast. Topographical features which are usually not visible can be easily discerned in the processed images. The method developed in this study rendered possible the detection of small patterns on viral particles as well as thin cytoskeleton fibers of living cells. It is shown that the sliding window approach gives better results than Fourier-filtering. Our method can be generally applied to increase the contrast of topographical images, especially when small features are to be highlighted on relatively high objects.
Fig. 5. Frequency spectra recorded at a cell nucleus of the live and fixed lung epithelial cell (... more Fig. 5. Frequency spectra recorded at a cell nucleus of the live and fixed lung epithelial cell (H441) in buffer. Curves are normalized to the main driving frequency. Inset: magnification of the second harmonic amplitude peaks.
The combination of fluorescence microscopy and atomic force microscopy has a great potential in s... more The combination of fluorescence microscopy and atomic force microscopy has a great potential in single-molecule-detection applications, overcoming many of the limitations coming from each individual technique. Here we present a new platform of combined fluorescence and simultaneous topography and recognition imaging (TREC) for improved localization of cellular receptors. Green fluorescent protein (GFP) labeled human sodium-glucose cotransporter (hSGLT1) expressed Chinese Hamster Ovary (CHO) cells and endothelial cells (MyEnd) from mouse myocardium stained with phalloidin-rhodamine were used as cell systems to study AFM topography and fluorescence microscopy on the same surface area. Topographical AFM images revealed membrane features such as lamellipodia, cytoskeleton fibers, F-actin filaments and small globular structures with heights ranging from 20 to 30 nm. Combined fluorescence and TREC imaging was applied to detect density, distribution and localization of YFP-labeled CD1d molecules on alpha-galactosylceramide (alphaGalCer)-loaded THP1 cells. While the expression level, distribution and localization of CD1d molecules on THP1 cells were detected with fluorescence microscopy, the nanoscale distribution of binding sites was investigated with molecular recognition imaging by using a chemically modified AFM tip. Using TREC on the inverted light microscope, the recognition sites of cell receptors were detected in recognition images with domain sizes ranging from approximately 25 to approximately 160 nm, with the smaller domains corresponding to a single CD1d molecule.
Optical lithography is a key technique in the development of semiconductor industry. However, dif... more Optical lithography is a key technique in the development of semiconductor industry. However, diffraction effects limit the minimal resolvable feature size to the Rayleigh diffraction limit of l/2, where l is the optical wavelength. Many technologies have been proposed in the past to replace optical lithography. Here, we present a new quantum optical method to do subwavelength lithography which is realizable by our current technology now. Using TEM, STEM, SEM and AFM measurements we show that 2 nm width lines could be written in novel materials such as fluorescent photosensitive glassceramics by a quantum multiphoton confinement effect. Exposure to the focus laser diode beam (l ¼650 nm) writes high-density lines with 4 nm pitch on the sample surface at room temperature, far beyond the diffraction limit, a fundamental barrier to the exploitation of optical lithography. 2 nm Quantum Optical Lithography is an important step to enable full-wafer-level nanofabrication at this resolution.
Biochimica et Biophysica Acta (BBA) - General Subjects, 1999
A new biotin^fluorescein conjugate with an ethylene diamine spacer was found to be the first fluo... more A new biotin^fluorescein conjugate with an ethylene diamine spacer was found to be the first fluorescent biotin derivative which truly mimicked D-biotin in terms of high affinity, fast association, and non-cooperative binding to avidin and streptavidin tetramers. These exceptional properties were attributed to the small size/length of the new ligand since all larger/ longer biotin derivatives are known for their mutual steric hindrance and anti-cooperative binding in 4:1 complexes with avidin and streptavidin tetramers. Specific binding of the new biotin^fluorescein conjugate towards avidin and streptavidin was accompanied by 84^88% quenching of ligand fluorescence. In the accompanying study this effect was used for rapid estimation of avidin and streptavidin in a new`single tube assay'. In the present study the strong quenching effect was utilized to accurately monitor stoichiometric titration of biotin-binding sites in samples with v 200 pM avidin or streptavidin. The concentration was calculated from the consumption of fluorescent ligand up to the distinct breakpoint in the fluorescence titration profile which was marked by the abrupt appearance of strongly fluorescent ligands which were in excess. Due to this protocol the assay was not perturbed by background fluorescence or coloration in the unknown samples. The new fluorescence titration assay is particularly suited for quick checks on short notice because getting started only means to thaw an aliquot of a standardized stock solution of fluorescent ligand. No calibration is required for the individual assay and the ligand stock solution needs to be restandardized once per week (or once per year) when stored at 325³C (or at 370³C, respectively). ß 0304-4165 / 99 / $^see front matter ß 1999 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 -4 1 6 5 ( 9 8 ) 0 0 1 7 8 -0
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Papers by Gerald Kada