Tri-mode optical biopsy probe with fluorescence endomicroscopy,Raman spectroscopy,and time-resolved fluorescence spectroscopy

We present an endoscopic probe that combines three distinct optical fibre technologies including: A high-resolution imaging fibre for optical endomicroscopy, a multimode fibre for time-resolved fluorescence spectroscopy, and a hollow-core fibre with multimode signal collection cores for Raman spectroscopy. The three fibers are all enclosed within a 1.2 mm diameter clinical grade catheter with a 1.4 mm end cap. To demonstrate the probe's flexibility we provide data acquired with it in loops of radii down to 2 cm. We then use the probe in an anatomically accurate model of adult human airways, showing that it can be navigated to any part of the distal lung using a commercial bronchoscope. Finally, we present data acquired from fresh ex vivo human lung tissue. Our experiments show that this minimally invasive probe can deliver real-time optical biopsies from within the distal lung - simultaneously acquiring co-located high-resolution endomicroscopy and biochemical spectra.     

Arrangement of chromatin in the nucleus

Summary The factors responsible for producing some degree of order to the arrangement of chromatin in the nucleus are reviewed. They are the following: 1. Chromosomes are attached to the nuclear membrane, nucleolus and intranuclear matrix. As a result they have a relatively fixed position in the nucleus. 2. In some species somatic pairing results in alignment of homologs. This is rare in mammals. 3. The association of ribosomal DNA and 5S DNA with the nucleolous results in the close approximation of the chromosomes carrying these DNA sequences. In man and other animals the most obvious consequence is satellite association. 4. Heterochromatin is condensed onto the inner nuclear membrane and periphery of the nucleolous while genetically active chromatin occupies the more central portion of the nucleus. The results is a peripheral location of late replicating DNA and a central location of early relicating DNA. 5. The DNA replication points tend to be associated with the nuclear matrix. Autoradiography of briefly labelled cells shows a high frequency of grains associated with nuclear matrix material. 6. Heterochromatin association results in chromocenters and ectopic pairing. 7. In addition to all these is the Rabl orientation or alignment of centromeres with centromeres and telomeres with telomeres. This polarization of the chromosomes results from the traction on the centromeres by the spindle fibers. There is no firm evidence for any higher degrees of order that might bring specific functioning genes into close proximity.Supported by NIH Grant GM 15886     

Nanosecond time-resolved emission spectroscopy of a fluorescence probe adsorbed to L-alpha-egg lecithin vesicles.

Nanosecond time-resolved emission spectra (TRES) are fluorescence emission spectra obtained at discrete times during the fluorescence decay. The complete data-set obtainable is a surface representing the intensity at all wavelengths and times during the emission decay time. When 2-p-toluidinonaphthalene-6-sulfonate (2,6 p-TNS) is adsorbed to egg lecithin vesicles, an excited-state reaction associated with energetic changes of the emitting species occurs on the nanosecond time scale. Convolution of the fluorescence decay with the excitation response introduces an artifact in the time-dependent spectra. A precedure is described by which this artifact can be eliminated. The data for the generation of time-resolved emission spectra are obtained with a computer-interfaced instrument based on the single-photon counting method.     

Nanosecond time-resolved emission spectroscopy of a fluorescence probe bound to L- -egg lecithin vesicles

The monophoton counting technique was used to measure nanosecond time-resolved fluorescence emission spectra of 2-p-toluidino-naphthalene-6-sulfonate (2,6 p-TNS) adsorbed to lipid bilayer vesicles. A time-dependent red shift in the emission maximum was observed and the rate of this red shift was shown to be temperature dependent. Analysis of fluorescence decay curves obtained at different emission wavelengths indicates that the time-dependent spectral shifts are due to an excited-state reaction such as solvent relaxation or an excited-state interaction between the chromophore and a polar residue of the phospholipid.     

Fluorescence correlation spectroscopy diffusion laws to probe the submicron cell membrane organization

To probe the complexity of the cell membrane organization and dynamics, it is important to obtain simple physical observables from experiments on live cells. Here we show that fluorescence correlation spectroscopy (FCS) measurements at different spatial scales enable distinguishing between different submicron confinement models. By plotting the diffusion time versus the transverse area of the confocal volume, we introduce the so-called FCS diffusion law, which is the key concept throughout this article. First, we report experimental FCS diffusion laws for two membrane constituents, which are respectively a putative raft marker and a cytoskeleton-hindered transmembrane protein. We find that these two constituents exhibit very distinct behaviors. To understand these results, we propose different models, which account for the diffusion of molecules either in a membrane comprising isolated microdomains or in a meshwork. By simulating FCS experiments for these two types of organization, we obtain FCS diffusion laws in agreement with our experimental observations. We also demonstrate that simple observables derived from these FCS diffusion laws are strongly related to confinement parameters such as the partition of molecules in microdomains and the average confinement time of molecules in a microdomain or a single mesh of a meshwork.     

The use of site-directed mutagenesis and time-resolved fluorescence spectroscopy to assign the fluorescence contributions of individual tryptophan residues in Bacillus stearothermophilus lactate dehydrogenase

Site-directed mutagenesis has been used to generate two mutant Bacillus stearothermophilus lactate dehydrogenases: in one, Trp-150 has been replaced with a tyrosine residue and, in the other, both Trp-150 and -80 are replaced with tyrosines. Both enzymes are fully catalytically active and their affinities for substrates and coenzymes, and thermal stabilities are very similar to those of the native enzyme. Time-resolved fluorescence measurements using a synchrotron source have shown that all three tryptophans in the native enzyme fluoresce. By comparing the mutant and native enzymes it was possible, for the first time, to assign, unambiguously, lifetimes to the individual tryptophans: Trp-203 (7.4 ns), Trp-80 (2.35 ns) and Trp-150 (less than 0.3 ns). Trp-203 is responsible for 75-80% of the steady-state fluorescence emission, Trp-80 for 20%, and Trp-150 for less than 2%.     

Single cell fluorescence imaging using metal plasmon-coupled probe

This work constitutes the first fluorescent imaging of cells using metal plasmon-coupled probes (PCPs) at single cell resolution. N-(2-Mercapto-propionyl)glycine-coated silver nanoparticles were synthesized by reduction of silver nitrate using sodium borohyride and then succinimidylated via ligand exchange. Alexa Fluor 647-labeled concanavalin A (con A) was chemically bound to the silver particles to make the fluorescent metal plasmon-coupled probes. The fluorescence images were collected using a scanning confocal microscopy. The fluorescence intensity was observed to enhance 7-fold when binding the labeled con A on a single silver particle. PCPs were conjugated on HEK 293 A cells. Imaging results demonstrate that cells labeled by PCPs were 20-fold brighter than those by free labeled con A.     

Detection of motional heterogeneities in lipid bilayer membranes by dual probe fluorescence correlation spectroscopy

We report the detection of heterogeneities in the diffusion of lipid molecules for the three-component mixture dipalmitoyl-PC/dilauroyl-PC/cholesterol, a chemically simple lipid model for the mammalian plasma membrane outer leaflet. Two-color fluorescence correlation spectroscopy (FCS) was performed on giant unilamellar vesicles (GUVs) using fluorescent probes that have differential lipid phase partition behavior—DiO-C18:2 favors disordered fluid lipid phases, whereas DiI-C20:0 prefers spatially ordered lipid phases. Simultaneously-obtained fluorescence autocorrelation functions from the same excitation volume for each dye showed that, depending on the lipid composition of this ternary mixture, the two dyes exhibited different lateral mobilities in regions of the phase diagram with previously proposed submicroscopic two-phase coexistence. In one-phase regions, both dyes reported identical diffusion coefficients. Two-color FCS thus may be detecting local membrane heterogeneities at size scales below the optical resolution limit, either due to short-range order in a single phase or due to submicroscopic phase separation.     

Detection of motional heterogeneities in lipid bilayer membranes by dual probe fluorescence correlation spectroscopy

We report the detection of heterogeneities in the diffusion of lipid molecules for the three-component mixture dipalmitoyl-PC/dilauroyl-PC/cholesterol, a chemically simple lipid model for the mammalian plasma membrane outer leaflet. Two-color fluorescence correlation spectroscopy (FCS) was performed on giant unilamellar vesicles (GUVs) using fluorescent probes that have differential lipid phase partition behavior--DiO-C18:2 favors disordered fluid lipid phases, whereas DiI-C20:0 prefers spatially ordered lipid phases. Simultaneously-obtained fluorescence autocorrelation functions from the same excitation volume for each dye showed that, depending on the lipid composition of this ternary mixture, the two dyes exhibited different lateral mobilities in regions of the phase diagram with previously proposed submicroscopic two-phase coexistence. In one-phase regions, both dyes reported identical diffusion coefficients. Two-color FCS thus may be detecting local membrane heterogeneities at size scales below the optical resolution limit, either due to short-range order in a single phase or due to submicroscopic phase separation.     

Measuring diffusion in cell membranes by fluorescence correlation spectroscopy

Fluorescence Correlation Spectroscopy (FCS) can measure diffusion on the cell surface with unparalleled sensitivity. In appropriate situations, this can be the most sensitive and accurate method for measuring receptor interaction and oligomerization. Here we attempt to describe FCS in sufficient detail so that the reader is able to judge when there is a compelling reason to choose this technique, understand the basic theory behind it, construct a FCS spectrometer in the laboratory, and analyze the data to obtain a meaningful estimate of the physical parameters.     

Sub millimetre flexible fibre probe for background and fluorescence free Raman spectroscopy

Using the shifted-excitation Raman difference spectroscopy technique and an optical fibre featuring a negative curvature excitation core and a coaxial ring of high numerical aperture collection cores, we have developed a portable, background and fluorescence free, endoscopic Raman probe. The probe consists of a single fibre with a diameter of less than 0.25 mm packaged in a sub-millimetre tubing, making it compatible with standard bronchoscopes. The Raman excitation light in the fibre is guided in air and therefore interacts little with silica, enabling an almost background free transmission of the excitation light. In addition, we used the shifted-excitation Raman difference spectroscopy technique and a tunable 785 nm laser to separate the fluorescence and the Raman spectrum from highly fluorescent samples, demonstrating the suitability of the probe for biomedical applications. Using this probe we also acquired fluorescence free human lung tissue data.     

The fluorescence properties of a DNA probe

Steady-state and dynamic fluorescence measurements have been performed on DAPI in solution and in complexes formed with a number of synthetic and natural polydeoxynucleotides. The decay of DAPI in buffer at pH 7 was decomposed using two exponentials having lifetime values of approximately 2.8 ns and 0.2 ns. The double exponential character of the decay was maintained over a large pH range from 3 to 9. At pH 1 the short component dominated, whereas at pH 12, only the long component was detectable. Two distinct spectra were associated with the two lifetime components; the short component was shifted to the red. The short lifetime component occurs in the presence of water. In water the excitation spectra depended on the emission wavelength and there was no viscosity dependence of the two forms. To explain these results we propose that there is a ground state conformer in which preferential solvation of the indole ring allows proton transfer in the excited state. DAPI complexed with polydeoxynucleotides retained most of the features of the decay of DAPI in solution. However, the complexes with fuly AT-containing polymers stabilized the longer lifetime form of DAPI because the stronger binding enhanced solvent shielding. A gradual increase of the short lifetime component, which monitors dye solvent exposure, was obtained as the AT content was decreased. For polyd(GC) the decay was similar to that of free DAPI.Abbreviations DAPI 4-6-diamidino-2-phenylindole - POPOP 1,4-bis(5-phenyl-2-oxazolyl)-benzene; 2,2-p-phenylene-bis(5-phenyloxazole) Financial support for this work was provided by a M.P.I. grant 1984, Roma, Italy for M.L.B. and NSF-PCM 84-03107 and PHS-IP41RR03155 for E.G.