Pin-Hole Array Correlation Imaging: Highly Parallel Fluorescence Correlation Spectroscopy

In this work, we describe pin-hole array correlation imaging, a multipoint version of fluorescence correlation spectroscopy, based upon a stationary Nipkow disk and a high-speed electron multiplying charged coupled detector. We characterize the system and test its performance on a variety of samples, including 40 nm colloids, a fluorescent protein complex, a membrane dye, and a fluorescence fusion protein. Our results demonstrate that pin-hole array correlation imaging is capable of simultaneously performing tens or hundreds of fluorescence correlation spectroscopy-style measurements in cells, with sufficient sensitivity and temporal resolution to study the behaviors of membrane-bound and soluble molecules labeled with conventional chemical dyes or fluorescent proteins.     

Learning how planarization can affect dichroic patterns in polyfluorenes

The circular dichroism spectra of a single chain of polyfluorene was predicted for a p-twisted helix conformation and local planarized polymer sections in the presence and in the absence of thermal vibrations. Under thermal vibrations at 300 K, the planarized section of polyfluorene affords a red-shifted positive dichroic band between 446 and 456 nm, preserving a degree of chirality. The S1 ← S0 excitation energy decreases from 3.29 eV, for the p-twisted helix to 2.77 or 2.71 eV, for planarized sections with one or two coplanar twists, respectively. Thermal vibrations and intramolecular rotations eventually affect the circular dichroism spectrum patterns, where planarized bent conformers induce a positive band towards 450 nm.     

Reassessment of the size of the supermolecular state of Dishevelled-3

Amendment of the interpretation of recently published size‐exclusion chromatography data for Dishevelled‐3 on Superdex 200 and Sephacryl S‐400 has led to an increase in the estimated size of the supermolecular state from 2000 to 35 000 kDa, a value that essentially duplicates the redetermined and reported estimates obtained by fluorescence correlation spectroscopy on live cells. The earlier discrepancy between the sizes of the extensively aggregated form of this scaffold protein in vivo and in vitro is thereby eliminated. Copyright © 2011 John Wiley & Sons, Ltd.     

DNA methylation of cord blood cell types: Applications for mixed cell birth studies

Epigenome-wide association studies of disease widely use DNA methylation measured in blood as a surrogate tissue. Cell proportions can vary between people and confound associations of exposure or outcome. An adequate reference panel for estimating cell proportions from adult whole blood for DNA methylation studies is available, but an analogous cord blood cell reference panel is not yet available. Cord blood has unique cell types and the epigenetic signatures of standard cell types may not be consistent throughout the life course. Using magnetic bead sorting, we isolated cord blood cell types (nucleated red blood cells, granulocytes, monocytes, natural killer cells, B cells, CD4⁺T cells, and CD8⁺T cells) from 17 live births at Johns Hopkins Hospital. We confirmed enrichment of the cell types using fluorescence assisted cell sorting and ran DNA from the separated cell types on the Illumina Infinium HumanMethylation450 BeadChip array. After filtering, the final analysis was on 104 samples at 429,794 probes. We compared cell type specific signatures in cord to each other and methylation at 49.2% of CpG sites on the array differed by cell type (F-test P < 10^?8). Differences between nucleated red blood cells and the remainder of the cell types were most pronounced (36.9% of CpG sites at P < 10^?8) and 99.5% of these sites were hypomethylated relative to the other cell types. We also compared the mean-centered sorted cord profiles to the available adult reference panel and observed high correlation between the overlapping cell types for granulocytes and monocytes (both r=0.74), and poor correlation for CD8⁺T cells and NK cells (both r=0.08). We further provide an algorithm for estimating cell proportions in cord blood using the newly developed cord reference panel, which estimates biologically plausible cell proportions in whole cord blood samples.     

Fluorescence correlation spectroscopy of intact nuclear pore complexes

No methods proposed thus far have the sensitivity to measure the transport of single molecules through single nuclear pore complexes (NPCs) in intact cells. Here we demonstrate that fluorescence correlation spectroscopy (FCS) combined with real-time tracking of the center of mass of single NPCs in live, unperturbed cells allows us to detect the transport of single molecules in a reference system of a pore with high temporal (millisecond) and spatial (limited by diffraction) resolution. We find that the transport of the classical receptor karyopherin-β1 (Kapβ1) is regulated so as to produce a peculiar distribution of characteristic times at the NPC. This regulation, which is spatially restricted to the pore, depends on the properties and metabolic energy of Kapβ1. As such, this method provides a powerful tool for studying nucleocytoplasmic shuttling at the nanometer scale under physiological conditions.     

Single cell analysis of ligand binding and complex formation of interleukin-4 receptor subunits

Interleukin-4 (IL-4) is an important class I cytokine involved in adaptive immunity. IL-4 binds with high affinity to the single-pass transmembrane receptor IL-4Rα. Subsequently, IL-4Rα/IL-4 is believed to engage a second receptor chain, either IL-2Rγ or IL-13Rα1, to form type I or II receptor complexes, respectively. This ternary complex formation then triggers downstream signaling via intracellular Janus kinases bound to the cytoplasmic receptor tails. Here, we study the successive steps of complex formation at the single cell level with confocal fluorescence imaging and correlation spectroscopy. We characterize binding and signaling of fluorescently labeled IL-4 by flow cytometry of IL-4-dependent BaF3 cells. The affinity to ectopically expressed IL-4Rα was then measured by single-color fluorescence correlation spectroscopy in adherent HEK293T cells that express the components of the type II IL-4R but not type I. Finally, IL-4-induced complex formation was tested by dual-color fluorescence cross-correlation spectroscopy. The data provide evidence for codiffusion of IL-4-A647 bound IL-4Rα and the type II subunit IL-13Rα1 fused to enhanced green fluorescent protein, whereas type I complexes containing IL-2Rγ and JAK3 were not detected at the cell surface. This behavior may reflect hitherto undefined differences in the mode of receptor activation between type I (lymphoid) and type II (epithelial) receptor expressing cells.     

Quantification of bacterial polyhydroxyalkanoic acids by Nile red staining

The fluorescence properties of one chemically and seven biologically produced polyhydroxyalkanoic acids were investigated as film castings and in living cells respectively after staining with Nile red. All these polyesters show a similar fluorescence behaviour, revealing a clear fluorescence maximum at an excitation wavelength between 540 nm and 560 nm and an emission wavelength between 570 nm and 605 nm. This could be shown by the use of two-dimensional fluorescence spectroscopy and flow cytometry. The examination of native poly(3-hydroxybutyric acid), poly(3HB), granules isolated from cells of Ralstonia eutropha H16 showed that the addition of 6.0 μg Nile red is necessary for total staining of 1.0 mg granules. The fluorescence intensity at an excitation wavelength of 550 nm and an emission wavelength of 600 nm showed high correlation to the poly(3HB) concentration of grana suspensions at different grana concentrations. These results and the staining of cell suspensions during cultivation experiments revealed that Nile red has a high potential for the quantitative determination of hydrophobic bacterial polyhydroxyalkanoic acids. Received: 13 November 1998 / Received revision: 4 February 1999 / Accepted: 12 February 1999     

Effects of external potassium supply on stomatal closure induced by abscisic acid

Abstract Epidermal strips of Commelina communis with ‘isolated’ stomata were incubated on Trizma-maleate buffer containing 0-500 mM KCL, with or without 10^?4 M ABA, for 2.5 h. The resulting stomatal apertures indicate that there is no absolute requirement for live epidermal and subsidiary cells for ABA-mediated closure. This implies that ABA has a direct effect on influx or efflux of K⁺ into or out of the guard cells rather than on uptake of K⁺ by the subsidiary cells. The possible in vivo role of subsidiary cells in stomatal closure is discussed.     

Antenna organization and evidence for the function of a new antenna pigment species in the green photosynthetic bacterium Chloroflexus aurantiacus

Whole cells and isolated chlorosomes (antenna complex) of the green photosynthetic bacterium Chloroflexus aurantiacus have been studied by absorption spectroscopy (77 K and room temperature), fluorescence spectroscopy, circular dichroism, linear dichroism and electron spin resonance spectroscopy. The chlorosome absorption spectrum has maxima at 450 (contributed by carotenoids and bacteriochlorophyll (BChl) a Soret), 742 (BChl c) and 792 nm (BChl a) with intensity ratios of 20:25. The fluorescence emission spectrum has peaks at 748 and 802 nm when excitation is into either the 742 or 450 nm absorption bands, respectively. Whole cells have fluorescence peaks identical to those in chlorosomes with the addition of a major peak observed at 867 nm. The CD spectrum of isolated chlorosomes has an asymmetric-derivative-shaped CD centered at 739 nm suggestive of exciton interaction at least on the level of dimers. Linear dichroism of oriented chlorosomes shows preferential absorption at 742 nm of light polarized parallel to the long axis of the chlorosome. This implies that the transition dipoles are also oriented more or less parallel to the long axis of the chlorosome. Treatment with ferricyanide results in the appearance of a 2.3 G wide ESR spectrum at g 2.002. Whole cells grown under different light conditions exhibit different fluorescence behavior when absorption is normalized at 742 nm. Cells grown under low light conditions have higher fluorescence intensity at 748 nm and lower intensity at 802 nm than cells grown under high light conditions. These results indicate that the BChl c in chlorosomes is highly organized, and transfers energy from BChl c (742 nm) to a connector of baseplate BChl B792 (BChl a) presumably located in the chlorosome baseplate adjacent to the cytoplasmic membrane.     

Dynamic molecular confinement in the plasma membrane by microdomains and the cytoskeleton meshwork

It is by now widely recognized that cell membranes show complex patterns of lateral organization. Two mechanisms involving either a lipid-dependent (microdomain model) or cytoskeleton-based (meshwork model) process are thought to be responsible for these plasma membrane organizations. In the present study, fluorescence correlation spectroscopy measurements on various spatial scales were performed in order to directly identify and characterize these two processes in live cells with a high temporal resolution, without any loss of spatial information. Putative raft markers were found to be dynamically compartmented within tens of milliseconds into small microdomains (? <120 nm) that are sensitive to the cholesterol and sphingomyelin levels, whereas actin-based cytoskeleton barriers are responsible for the confinement of the transferrin receptor protein. A free-like diffusion was observed when both the lipid-dependent and cytoskeleton-based organizations were disrupted, which suggests that these are two main compartmentalizing forces at work in the plasma membrane.     

Nanoaperture Fluorescence Enhancement in the Ultraviolet

We study fluorescence excitation and emission enhancement in the ultraviolet regime for molecules confined within sub-wavelength metal apertures. Calculations are performed across a range of excitation wavelengths for individual apertures constructed in gold, silver, and aluminum. As expected, enhancement in the ultraviolet is greatest with aluminum. Using excitation and emission wavelengths appropriate for tryptophan, we find that more than 10× net increase in fluorescence count rate should be obtainable for aluminum apertures of ~75 nm diameter. These results suggest that many applications utilizing native protein fluorescence could become practical, even to the single molecule level.     

Two-photon fluorescence correlation spectroscopy: method and application to the intracellular environment.

We report on the application of two photon molecular excitation to fluorescence correlation spectroscopy. We demonstrate the first fluorescence correlation spectroscopy measurements of translational mobility in the cytoplasm of living cells. Two-photon excitation inherently excites small sample volumes in three dimensions, providing depth discrimination similar to confocal microscopy, without emission pinholes. We demonstrated accurate measurements of the diffusion constant, D, for particles of several different known sizes, in bulk solutions of different viscosity. We then showed measurements of translational diffusion for 7- and 15-nm radius latex beads in the cytoplasm of mouse fibroblast cells. We measured time-dependent diffusion coefficients. When first injected in the cells, the spheres moved from two to five times slower than in water, with average rates of 18 x 10(-8) cm2/s for the 7 nm and 5 x 10(-8) cm2/s for the 15 nm radius spheres. After a few hours, spheres stick to the cells, and the motion slows down 10 to 100 times.     

Dark states in monomeric red fluorescent proteins studied by fluorescence correlation and single molecule spectroscopy

Monomeric red fluorescent proteins (mRFPs) have become indispensable tools for studying protein dynamics, interactions and functions in the cellular environment. Their emission spectrum can be well separated from other fluorescent proteins, and their monomeric structure preserves the natural function of fusion proteins. However, previous photophysical studies of some RFPs have shown the presence of light-induced dark states that can complicate the interpretation of cellular experiments. In this article, we extend these studies to mRFP1, mCherry, and mStrawberry by means of fluorescence correlation spectroscopy and prove that this light-driven intensity flickering also occurs in these proteins. Furthermore, we show that the flickering in these proteins is pH-dependent. Single molecule spectroscopy revealed reversible transitions from a bright to a dark state in several timescales, even up to seconds. Time-resolved fluorescence spectroscopy showed multiexponential decays, consistent with a “loose” conformation. We offer a structural basis for the fluorescence flickering using known crystal structures and point out that the environment of Glu-215 is critical for the pH dependence of the flickering in RFPs. We apply dual-color fluorescence correlation spectroscopy inside live cells to prove that this flickering can seriously hamper cellular measurements if the timescales of the flickering and diffusion are not well separated.     

Differentiation of live, dead and treated cells of Escherichia coli O157:H7 using FT-IR spectroscopy

Aims: To apply specific collection techniques and spectroscopy to differentiate between live and dead Escherichia coli O157:H7 cells, as well as cells subjected to various inactivation treatments, including heat, salt, UV, antibiotics and alcohol. Methods and Results: Fourier transform‐infrared (FT‐IR) spectroscopy was used to analyse E. coli O157:H7 cells, after filtration or immunomagnetic collection. Partial least squares analysis of the spectra quantified live E. coli O157:H7 in the presence of dead cells with an R² > 0·996. Canonical variate analysis (CVA) not only differentiated between spectra of 100% dead and 100% live cells but also between 1% live : 99% dead and 100% dead. CVA using principal components also differentiated between the spectra of the differentially treated cells at a 95% confidence level, and Cooman plots showed clear separation between clusters of spectra of bacteria exposed to the different inactivation treatments. Mahalanobis distances (MD) corroborated the results of CVA. Conclusions: These results demonstrated the effectiveness of rapid cell collection and FT‐IR spectroscopy techniques to differentiate between live and dead E. coli O157:H7 cells. Significance and Impact of the Study: This technique has potential applications for use with foods subjected to various inactivation treatments.     

Changes in intrinsic fluorescence during the production of viable but nonculturable Escherichia coli

The potential of intrinsic fluorescence spectroscopy to detect and differentiate viable but nonculturable bacteria in the presence of culturable bacteria was explored. Escherichia coli cells, starved for 210 days in nutrient-free normal saline, show new fluorescence emissions near 400 and 440 nm, and reduced emission near 340 nm. Received 7 July 1997/ Accepted in revised form 26 November 1997     

SINGLE-CELL PIGMENTATION OF PORPHYRA LINEARIS ANALYZED BY FOURIER TRANSFORM MULTI-PIXEL SPECTROSCOPY AND IMAGE ANALYSIS1

The novel method of Fourier transform multi-pixel spectroscopy was used for the nondestructive analysis of and comparison of pigmentation in different regions of live thalli of the red alga Porphyra linearis. Because the thallus in this alga consists of a monolayer of nonoverlapping cells, we were able to analyze the pigmentation of single cells by combining light absorbance with natural fluorescence data. From the image of each cell in the vegetative male and female reproductive and holdfast regions, more than 4 ± 10⁴ fluorescence and absorbance spectra were obtained. Specific pigments in the different regions were localized by the use of a software program of similarity mapping followed by image construction. The reconstructed images revealed subcellular localization of each pigment according to specific spectroscopic fingerprints. The results showed that the vegetative and female reproductive cell types had a significantly higher content of phycoerythrin than of phycocyanin, and quite similar chlorophyll a levels. Most of the holdfast cells were poorly pigmented, but had more chlorophyll a than phycoerythrin or phycocyanin. The male reproductive cells contained only traces of pigments. Thus, by using Fourier transform multipixel spectroscopy, we were able to characterize the pigmentation of different regions of the thallus and follow the distribution patterns of the different pigments on the subcellular level along the differentiation gradient of the alga.     

Nanoscopy in a living multicellular organism expressing GFP

We report superresolution fluorescence microscopy in an intact living organism, namely Caenorhabditis elegans nematodes expressing green fluorescent protein (GFP)-fusion proteins. We also superresolve, by stimulated emission depletion (STED) microscopy, living cultured cells, demonstrating that STED microscopy with GFP can be widely applied. STED with GFP can be performed with both pulsed and continuous-wave lasers spanning a wide wavelength range from at least 556–592 nm. Acquiring subdiffraction resolution images within seconds enables the recording of movies revealing structural dynamics. These results demonstrate that numerous microscopy studies of live samples employing GFP as the marker can be performed at subdiffraction resolution.     

Neither total culture fluorescence nor intracellular fluorescence are indicative of NAD(P)H levels in Escherichia coli MG 1655

Summary The blue fluorescence emitted by microbial cells irradiated with UV light at 360 nm is usually supposed to provide a good estimate of the cell NAD(P)H content. Here we present an example of a microbial fermentation in which culture fluorescence, both in the cells and in the medium, was almost exclusively due to the presence of a fluorophore that displayed an emission spectrum very similar to that of NAD(P)H but that we show by biochemical studies to be a different compound. Our results demonstrate that studies on the redox state of cells should be based on on-line fluorescence data only after appropriate control experiments to establish a definitive correlation between fluorescence and NAD(P)H levels. Offprint requests to: J. E. Bailey     

Detecting Amyloid-β Aggregation with Fiber-Based Fluorescence Correlation Spectroscopy

Soluble aggregates critically influence the chemical and biological aspects of amyloid protein aggregation, but their population is difficult to measure, especially in vivo. We take an optical fiber-based fluorescence correlation spectroscopy (FCS) approach to characterize a solution of aggregating amyloid-β molecules. We find that this technique can easily resolve aggregate particles of size 100 nm or greater in vitro, and the size distribution of these particles agrees well with that obtained by conventional FCS techniques. We propose fiber FCS as a tool for studying aggregation in vivo.