The ability to detect a single analyte molecule represents the ultimate in sensitivity. Single molecule detection has emerged as a powerful tool to characterize heterogeneous systems, in which traditional bulk sampling methods provide a signal averaged over a large number of analytes. Traditionally, single molecule measurements have required highly controlled experimental conditions using ultrapure solvents to create a minimum level of interference. These constraints have primarily limited this technique to examination of systems in vitro. In this report we present the first instance of real-time single molecule detection in living cells. Our experimental approach allows dynamic monitoring of individual fluorophores in vivo, despite the highly complex cellular environment.

Single molecule fluorescence measurements are used to characterize the local dynamics in model lipid films of DPPC. Analysis of emission trajectory autocorrelations reveals a dependence in the single molecule emission fluctuations which is correlated with the surface pressure of the lipid monolayer. Comparison of DPPC monolayers transferred onto mica at (pi) equals 5 mN/m and (pi) equals 30 mN/m exhibit characteristic single molecule fluctuation times of 700 ms and 1.30 s, respectively. These fluctuation times are correlated with the order in the film. Comparison with recent near-field measurements of probe molecule orientation and AFM measurements of film topography, however, indicate that the lipid phase surrounding the probe molecule remains the same from (pi) equals 5 mN/m to 30 mN/m. The increase in the characteristic emission fluctuation times with increasing surface pressure, therefore, reflects a decrease in the freedom surrounding a single molecule. These fluctuations are consistent with long range concerted motions of the lipid tailgroups in the lipid film.

Single-molecule phosphorescence immunoassay microscopy was developed and applied for high-throughput screening of tumor markers at the single-molecule (SM) level with no need of separation processes. The screening of individual analyte in a mixture was based upon distinguished diffusion images of single molecules associated with its size and mass. As a working example, several molecular forms of serum prostate- specific antigens (PSA) were labeled with Ru(bpy)₃²⁺-NHS-ester and labeled PSA-free and PSA-complex were distinguished based upon their SM diffusion images using this SM microscopy. The bound and unbound PSA-free with its monoclonal antibody (MAB) were also detected using this SM microscopy. A novel solution-phase quantitative electro chemiluminescence (ECL) immunoassay was developed to measure affinity constants of PSA with its antibody and diffusion coefficients of labeled PSA. The ECL immunoassay was able to detect PSA at 1.7 pg/mL. Diffusion of labeled PSA-free and PSA-complex measured by ECL and SM microscopy was consistent demonstrating that distinguished SM diffusion images could be used to screen multiple analytes in a complex mixture. This also implied the possibility of real- time monitoring of kinetics of binding reactions using such SM microscopy.

We describe the use of scanning confocal fluorescence microscopy to probe the structure and temporal dynamics of unsupported planar lipid bilayer membranes. At high fluorescent label concentration, the shape and stability of the membranes are evaluated with submicron spatial resolution. At low label concentration, we observe individual molecules moving into and out of the confocal laser beam in real time. Lipid diffusion coefficients are measured via fluctuation correlation spectroscopy (FCS) and indicate nominal values of (0.1 +/- 0.2) X 10^-7 cm²/sec homogeneously distributed over the central portion of the bilayer. We also compare the real-time single-molecule fluorescence signatures to simulated photon emission patterns generated by a 2D random-walk model. Interestingly, analysis of the interval between fluorescence events suggests the presence of a diffusion bias that can be explained by a weak optical trapping mechanism. The trap causes labeled lipids to return to the confocal detection region more frequently than pure Einsteinian diffusion predicts.

Advances in ultra sensitive instrumentation have allowed the detection of single fluorescence molecules in solution, on dielectric surfaces, and in low-temperature solids. However, these studies are limited by the requirement of a high fluorescence quantum yield and do not often provide sufficient spectroscopic information for identification. These problems are addressed by surface-enhanced Raman scattering (SERS) which provides highly resolved vibrational information, does not suffer form rapid photo bleaching, and eliminates the need for fluorescent tagging of nonfluorescent species. Recent studies of single silver nanoparticles reveal intrinsic enhancement factors as large as 10¹⁵. These giant enhancement factors allow single- molecule SERS spectra to be obtained using visible or near- IR excitation. The detection efficiency of experiments using visible excitation has thus far ben relatively low. Approaches to detecting single labeled and unlabeled biomolecules and to increasing the overall detection efficiency of single-molecule SERS with visible excitation are presented.

Surface-enhanced Raman scattering (SERS) is a phenomenon resulting in strongly increased Raman signals form molecules which have been attached to nanometer sized metallic structures. The technique combines fingerprint capabilities of vibrational spectroscopy and ultra sensitive detection limits. Silver or gold colloidal clusters can provide total enhancement factors of about 14 orders of magnitude for non- resonant Raman scattering at near IR excitation. Since non- resonant near IR photons are used, photodecomposition of the probed molecule is avoided or, at least strongly reduced, and relatively high excitation intensities can be applied. In addition to the Stokes Raman signal, that linearly depends on excitation laser intensity, at excitation intensities higher than approximately 10⁵-10⁶ W/cm² and 10⁷ W/cm², 'pumped' anti-Stokes Raman scattering and surface enhanced hyper Raman scattering, respectively, can be observed. Both effects can provide a non-linear or two-photon Raman probe where the Raman scattering signal depends quadratically on the excitation laser intensity.

In disease diagnosis, DNA and proteins are distinguished based on charge and hydrodynamic radius. Many protein assays and DNA assays relevant to cancer diagnosis are based on electrophoresis. However, standard protocols are not only slow but also insensitive. We successfully demonstrated a high-throughput imaging approach that allows determination of the individual electrophoretic mobilities of many molecules at a time. Each measurement on ly requires a few ms to compete. This opens up the possibility of screening single copies of DNA or proteins within single biological cells for disease markers without performing polymerase chain reaction or other biological amplification.

Capillary electrophoresis with laser-induced fluorescence detection has the potential to become an important tool in analyzing the chemical contents of single cells. Singe-cell studies are important for understanding the molecular mechanisms of many biological processes, including serious disorders, such as concern and neurodegradative diseases. We developed a new technique, Metabolic Cytometry, that allows us to study complex metabolic pathways in single cells. The essence of the technique is the following. A fluorescently labeled metabolic probe is introduce by incubation into a cell where it is converted to a number of fluorescently labeled metabolites. After incubation the cell is injected into the capillary and lysed. The metabolites are then separated and detected using CE-LIF. If metabolic cytometry is combined with image cytometry, then metabolic activities can be correlated with cellular phenotype or genotype. We applied this powerful approach to correlate glycosylation with the cell's phase in the cell cycle.

Novel analytical tools for the analysis of intra- and extra- cellular events on a level of single cells have been developed. The toolbox comprises equipment for acoustical levitation and piezo-electric flow-through micro-dispersing for nano-volume sample handling and sample preparation.

We have focused on effects of local mechanical properties of the cell on cell motion. By using atomic force microscopy, we measured spatial distribution of local elastic modulus on mouse fibroblasts, which is living in a physiological condition. In order to examine validity of AFM elastic measurements, we measured local elastic modulus of gels as elastic reference materials. The results obtained with AFM were compared with values obtained by tensile creep method. It is verified that these values are proportional each other. The AFM experiments on living cells revealed that center area of cell surface is about 10 times softer than the surroundings and looks like a big softer hole in the elasticity image. We fixed the cell just after the AFM measurements and carried out immunofluorescence observation for cytoskeletal filaments of actin filaments, microtubules and intermediate filaments. A comparison between distribution of local elasticity and cytoskeletones indicates that harder area on the cell results mainly from concentration of actin filaments. However, we found that some areas like the big softer hole do not correspond to distribution of actin filaments.

Conformation change of DNA under binding a DNA binding protein was studied using an AFM. An originally designed AFM mounted on an inverse microscope was used for imaging DNA. High mobility group (HMG) protein and pUC118 DNA were used as DNA binding protein and DNA for investigation in this study. The pUC118 DNA and its mixture with HMG2BJ were imaged by the AFM. In the AFM image of pUC118, DNA strands showed open structure with a few helix sites. The height of the helix part is 1.5 times higher than the ordinary DNA strand. Centrally AFM images of the mixture showed HMG-like particles on DNA strands. The height of the particle is 2-3 times higher than the DNA strands which showed the particles were not aggregating parts but HMG. The HMG bound on a crossing part of DNA which make a loop.

The temporal behavior of bursts from single molecules reveals whether adsorbates are diffusing at chemical interfaces or are specifically adsorbed. The chemical interface of water/C₁₈-monolayer on silica is studied. Burst data for single molecules of DiI show that regions of the surface having more nanoindentations are associated with a greater number of specific adsorption events. Lysozyme is shown to adsorb irreversibly, and an oligonucleotide is shown to adsorb reversibly but more strongly than DiI to nanointendations at this interface. For each adsorbate, the specific adsorption at the nanoindentations is attributed to hydrogen bonding to exposed silanols.

A new fluorescent method was developed for single molecule studies. The fluorophores were excited by the evanescent wave field produced either on the core surface of the optical fiber probe or on the flat surface of a quartz prism. The first configuration was used for single molecule detection. Single rhodamine 6G and fluorescein molecules have ben detected. The number of rhodamine 6G molecules imaged by the optical fiber probe showed an excellent linear relationship with the concentrations of the fluorophores. It represents a simpler fluorescent method for the detection of single molecules in solution and at an interface. The second configuration was used to monitor single molecule reaction. Direct observation of single molecule generation from a chemical reaction was achieved at a solid-liquid interface. The reaction between fluorescamine and immobilized N'-(3- trimethoxysilylpropyl)diethylenetriamine was studied at the single molecule level. Time-lapse fluorescence images of single molecule products were recorded to follow the chemical reaction to its completion. Analysis of the photoelectron intensity of the flourescent product and its distribution shows that the reaction kinetics goes through a transition from zeroth-order as the reaction proceeds. This approach offered a novel means to study single molecule reactions at the solid-liquid interface.

In recent years, laser induced fluorescence detection and spectroscopy of single molecules (SMD) has seen a tremendous development. Besides fundamental research on individual molecular systems, practical applications of this sensitive detection technique for analytical and diagnostic purposes are becoming more and more important. For a wider applicability of the SMD technique it is desirable to detect not only the presence or absence of a molecule within a given detection volume,, but also t be able to quantify this fluorescence absolutely. Modified flow cytometry system for SMD that are ensuring such a quantifiable fluorescence detection of single molecules are already successfully applied for e.g. DNA fragment sizing. In our talk, we present a modified confocal detection set-up for performing SMD on surfaces that aims at a quantified detection of single molecule fluorescence. First experimental results presented and emerging problems are discussed.

In recent years, the fluorescence detection of single molecules (SMD) in liquids and on surfaces under ambient conditions has seen a tremendous development. One of the difficulties of SMD is the efficient suppression of background signals, mostly Rayleigh and Raman scattering. Diverse methods were developed to minimize the scattering signals, e.g. usage of highly efficient emissions filters; minimization of the detection volume by diffraction limited focusing of the exciting laser beam and confocal imaging of the fluorescence, or using near-IR dyes employing the fact that the scattering intensity decreases with the fourth power of the wavelength. However, this strong wavelength dependency of the scattering signal made it, until now, hard to detect single fluorophores in the UV-region. One may use two- or three-photon excitation, but the necessary excitation intensities are often so high that they excite also non- linear scattering processes. Here, a new scheme is proposed for efficient fluorescence excitation of surface immobilized molecules via frequency doubling of the exciting laser light within a thin layer of an optically non-linear material, which may be much more efficient then the usual direct two- photon excitation.

A novel optical biosensor matrix has been developed to exploit the native fluorescence of certain proteins. This matrix uses a gold colloid monolayer attached to an end of a fiber as a substrate for protein attachment. The effect of the gold monolayer size has been investigated through the techniques of fluorescence, scanning electron microscopy, and transmission electron microscopy. It has been shown that the size of the gold colloid does produce a marked difference in the fluorescence intensity measured. It is surmised through the use of microscopy techniques that the intensity changes seen in the fluorescence emission are not a result of surface coverage, or availability of sites for protein adsorption, but instead of quenching or enhancement by the gold itself.

This paper describes the synthesis and characterization of submicron phospholipid coated polystyrene particles, named lipobeads, with pH sensing capability. The phospholipids used to coat the particles are labeled with fluorescein and tetramethylrhodamine, which serves as a referencing fluorophore for increased accuracy of the pH measurements. The synthesis of the pH sensing lipobeads is realized by the covalent attachment of the fluorescent phospholipids to the surface of styrene-divinylbenzene micron or submicron sized particles. The pH dynamic range of the sensing particles is between pH 5.5 and 7 with a sensitivity of 0.1 pH units and they are photostable under the experimental conditions used in these studies. The fluorescent lipobeads are used to monitor pH changes in volume limited samples and to measure the pH of single macrophages. The lipobeads are ingested by the macrophages and directed to lysosomes, which are the cellular organelles involved in the phagocytosis process. Despite the high lyososomal levels of digestive enzymes and acidity, the absorbed particles remain stable for 6 hours in the cells when the cells are stored in a PBS buffer solution at pH 7.4.

A method is described for immobilizing double-stranded DNAs to a planar gold surface with high density and uniform spacing. This is accomplished by adsorbing biotinylated DNAs onto a nearly close-packed monolayer of the protein streptavidin. This streptavidin monolayer, which offers approximately 5 X 10¹² biotin sites per cm², is prepared first by adsorbing it onto a mixed self-assembled monolayer on gold which contains biotin-terminated and oligo-terminated alkylthiolates in a 3/7 ratio. This DNA- functionalized surface resists non-specific protein adsorption and is useful for probing the kinetics and equilibrium binding of proteins to DNA with surface plasmon resonance. This is demonstrated with the Mnt protein, which is found to bind in 3.8:1 ratio to its immobilized DNA operator sequence. This is consistent with its behavior in homogeneous solution, where it binds as a tetramer to its DNA. A sequence with a single base-pair mutation shows nearly as much Mnt binding, but a completely random DNA sequence shows only 5 percent of this binding. This proves that DNA-binding proteins bind sequence-specifically to double-stranded DNAs which are immobilized to gold with this streptavidin linker layer.

Molecular Radar represents techniques capable of probing in real time biological processes in living samples. Compared to biochemistry techniques, MR focuses on spatial and temporal features of the process. It also integrates different types of measurements in order to interrogate a complicated biological system from different perspectives and to reveal correlation among the measured parameters. Based on a two-photon fluorescence microscope, we are developing a MR with fluorescence imaging and fluorescence correlation spectroscopy functions. Using the system, we obtained free calcium ion response in a transmembrane signal transduction study. In a different effort, the system is being used to obtain autofluorescence of elastin in rat aorta.

The utilization of supercritical fluids as a solvent medium in analysis has been on the rise over the past decades. The study of analytically relevant supercritical fluid systems through standard spectroscopic techniques presents unique challenges due to the extreme conditions that must be realized. This is also true of the study of biological macromolecules under extreme conditions of temperature and pressure. This presentation will focus on the spectroscopic approaches that we have developed and utilized to study these systems ranging form microvolume techniques to standard high-pressure vessels.

In this paper we discuss the mechanisms of image formation in the mid-IR of a transmission mode near-field microscope are studied. It is found that the amount of light propagating from a sub-wavelength aperture through a flat substrate strongly increases the tip nears the same. This effect tends to generate topographic artifacts in near-field images that can be eliminated through the use of flat sample preparation techniques. The transmitted power is strongly influenced by the refractive index of the sample, leading to a substantial difference between a near-field and a far- field spectrum. A phenomenological model, which makes predictions in good agreement with experiment, describing tunneling of light through a sub-wavelength aperture into a substrate is developed. The model predicts spectral sensitivity enhancement with decreasing aperture size.

A SNOAM system is capable of obtaining simultaneous topographic and optical images with a resolution beyond than the diffraction limit of far field optical imaging. Fluorescence tagging combined with optical resolutions of better than 100nm allow us to detect structures not possible with conventional microscopes. Also in contrast with electron microscopy SNOAM has the ability to look at biological structures in the liquid medium. Presently there is much interest in understanding the processes that lead to LTP in neuron synapses. LTP is widely associated with memory function in neurons. Hence, better understanding will lead to advances in medicine, as well as neuron-based memory and processing devices. Better understanding is also crucial to the development of neuron-electronic interfaces. In this research, neuron networks are grown on a patterned polylysine substrate. Polylysine is patterned using micro lithographic techniques. Neurons are extracted from the hippocampus of chick embryos, and are then grown on this pattern under standard sterile incubating conditions. The neurons are stimulated to release the neurotransmitter glutamate. The glutamate is then fluorescently imaged with Amplex-red SNOAM.

Single molecule imaging with optical methods has become an important tool in biophysical studies. However, when imaging molecules at room temperature using far field optics, one can only resolve molecules that are separated by a distance greater than the diffraction limit of the microscope, about 220 nanometers. Near field techniques have allowed researchers to image with resolutions on the order of 30-50 nanometers. However, there are numerous reasons to try to push the resolution limit further. One that particular concerns our group is the \notion to try to image information in DNA in order to measure sequence information. To that end, we have developed a new type of near field microscope, the fluorescence apertureless near field microscope.

Near-field scanning optical microscopy (NSOM) is utilized to study the 3D orientation of fluorescent probe molecules doped into thin polymer films and lipid monolayers. A simplified model of the fields near the NSOM tip aperture is used to simulate the single molecule near-field fluorescence features and extract the orientation of the probe molecules in the films. These measurements are particular useful for semi-ordered systems such as lipid monolayers that are often utilized as models of biological membranes. These single molecule measurements can reveal new features and provide details previously hidden in bulk methods that average over large ensembles of molecules.

A new technique of focused ion beam (FIB) microscopy/nanomachining is proposed. Ultrahigh resolution 3D tomography can be performed on whole cell, without preparation. Very fast imaging times allow quasi real time microscopy. FIB is a source of ions which can be precisely oriented and focused on the sample. The resolution can be as low as 15 nm. Higher currents produce 'cutting' and 'attaching' operations of very high precision where at the same time the resulting secondary ions and electrons again provide follow up sample imaging.

Nan-imaging of Entamoeba histolytica was carried out by using Atomic Force Microscope (AFM). The structure of the nucleus, endoplasm and ectoplasm were studied separately. The diameter of the nucleus in living E. histolytica was found to be of the order of 10 micrometers which is slightly higher than the earlier reported value. The presence of karysome was detected in the nucleus. Well-organized patterns of chromatoid bodies located within the endoplasm, were detected and their repetitive patterns were examined. The organized structure was also extended within the ectoplasm. The dimensions and form of the organization suggest that chromatic bodies are constituted with ribosomes ordered in the form of folded sheet. Such structures were found to be absent in non-living E. histolytica. AFM images were also captured just in the act when ameba was extending its pseudopods. Alteration in the ultrastructure caused during the process of extension was viewed. Well marked canals of width 694.05 nm. And height 211.05 nm are clearly perceptible towards the direction of the pseudopods. 3D images are presented to appreciate the height variation, which can not be achieved by conventional well-established techniques such as electron microscopy.

We have prepared DNA catenanes and studied the topological structures of DNA catenanes by atomic force microscopy (AFM) and electrophoresis. Nicked DNA was synthesized by the addition of DNASE I to a solution of plasmid pBR322. Catenated DNA molecules were prepared by the reaction of topoisomerase I with nicked DNA. Catenation reactions were monitored by the agarose gel electrophoresis. A droplet of solution, containing DNA catenanes extracted from the band of 1.0 percent agarose gel electrophoresis by a centrifugal filter device, was applied to a freshly cleaved mica surface. After drying the specimens, AFM measurements were carried out by using a silicon cantilever. The single molecule images of DNA catenanes were clearly observed for the first time by AFM using a tapping mode at room temperature and in an ambient atmosphere.

Detection of in situ hybridization to human metaphase chromosomes provides important information about gene mappings and about analysis of chromosomal disorders. We applied atomic force microscopy (AFM) to the detection of in situ hybridization to get better resolution as compared to light microscopy. Chromosomes were spread over a glass substrate and hybridized with DNA probes labeled with biotin or digoxigenin. The hybridized probes were reacted with streptavidin or anti-digoxigenin antibody, both of which were conjugated with 5-nm gold colloidal particles. We missed direct detection of the conjugated gold colloidal particles by micro-meter scale AFM scanning , but obtained clear topographic difference between the site of hybridization and the chromosome arm with the help of silver enhancement. We thus clearly detected the in situ hybridization using chromosome painting probes, alpha satellite probes, and locus specific gene probes by AFM. The in situ hybridization to DNA fiber was also detected by AFM. The detection of in situ hybridization by AFM has advantages over fluorescence in situ hybridization: no reduction of signal intensity under light irradiation. Application of AFM to the detection of in situ hybridization will be a useful method to analyze chromosomes.

We have developed a thermal lens microscope which indices and detects photothermal effect at sub-single molecules level in/on liquids and condensed media. The thermal lens microscope can determine non-fluorescent molecules without receiving serious effects of light scattering in/on various condensed phase substances, and it can be applied to imaging of the distribution of non-fluorescent molecules by scanning on the sample. These characteristics of the thermal lens microscope are suitable for ultra sensitive analysis and imaging of biomedical substance in/on a single cell, separation media, and microfabricated chemical devices. We applied the thermal lens microscope to determine an ultratrace chemical species in various media.