Article Information

• PDF (85 kb)
• Full Text with Large Figures
• Supporting Material
• Cited by in Scopus (2)

PubMed

• Articles by Kanchan Garai
• Articles by S. Maiti

Related Articles

• Correlation Spectroscopy of Minor Fluorescent Species: Signa...

  Correlation Spectroscopy of Minor Fluorescent Species: Signal Purification and Distribution Analysis Biophysical Journal, Volume 92, Issue 6, 15 March 2007, Pages 2184-2198 Ted A. Laurence, Youngeun Kwon, Eric Yin, Christopher W. Hollars, Julio A. Camarero and Daniel Barsky Abstract We are performing experiments that use fluorescence resonance energy transfer (FRET) and fluorescence correlation spectroscopy (FCS) to monitor the movement of an individual donor-labeled sliding clamp protein molecule along acceptor-labeled DNA. In addition to the FRET signal sought from the sliding clamp-DNA complexes, the detection channel for FRET contains undesirable signal from free sliding clamp and free DNA. When multiple fluorescent species contribute to a correlation signal, it is difficult or impossible to distinguish between contributions from individual species. As a remedy, we introduce “purified FCS”, which uses single molecule burst analysis to select a species of interest and extract the correlation signal for further analysis. We show that by expanding the correlation region around a burst, the correlated signal is retained and the functional forms of FCS fitting equations remain valid. We demonstrate the use of purified FCS in experiments with DNA sliding clamps. We also introduce “single-molecule FCS”, which obtains diffusion time estimates for each burst using expanded correlation regions. By monitoring the detachment of weakly-bound 30-mer DNA oligomers from a single-stranded DNA plasmid, we show that single-molecule FCS can distinguish between bursts from species that differ by a factor of 5 in diffusion constant. Abstract | Full Text | PDF (1052 kb)

• Image Correlation Spectroscopy. II. Optimization for Ultrase...

  Image Correlation Spectroscopy. II. Optimization for Ultrasensitive Detection of Preexisting Platelet-Derived Growth Factor-β Receptor Oligomers on Intact Cells Biophysical Journal, Volume 76, Issue 2, 1 February 1999, Pages 963-977 Paul W. Wiseman and Nils O. Petersen Abstract Previously we introduced image correlation spectroscopy (ICS) as an imaging analog of fluorescence correlation spectroscopy (FCS). Implementation of ICS with image collection via a standard fluorescence confocal microscope and computer-based autocorrelation analysis was shown to facilitate measurements of absolute number densities and determination of changes in aggregation state for fluorescently labeled macromolecules. In the present work we illustrate how to use ICS to quantify the aggregation state of immunolabeled plasma membrane receptors in an intact cellular milieu, taking into account background fluorescence. We introduce methods that enable us to completely remove white noise contributions from autocorrelation measurements for individual images and illustrate how to perform background corrections for autofluorescence and nonspecific fluorescence on cell population means obtained via ICS. The utilization of photon counting confocal imaging with ICS analysis in combination with the background correction techniques outlined enabled us to achieve very low detection limits with standard immunolabeling methods on normal, nontransformed human fibroblasts (AG1523) expressing relatively low numbers of platelet-derived growth factor- (PDGF-) receptors. Specifically, we determined that the PDGF- receptors were preaggregated as tetramers on average with a mean surface density of 2.3 clusters m after immunolabeling at 4°C. These measurements, which show preclustering of PDGF- receptors on the surface of normal human fibroblasts, contradict a fundamental assumption of the ligand-induced dimerization model for signal transduction and provide support for an alternative model that posits signal transduction from within preexisting receptor aggregates. Abstract | Full Text | PDF (413 kb)

• Characterization of the Photoconversion on Reaction of the F...

  Characterization of the Photoconversion on Reaction of the Fluorescent Protein Kaede on the Single-Molecule Level Biophysical Journal, Volume 89, Issue 5, 1 November 2005, Pages 3446-3455 P.S. Dittrich, S.P. Schäfer and P. Schwille Abstract Fluorescent proteins are now widely used in fluorescence microscopy as genetic tags to any protein of interest. Recently, a new fluorescent protein, Kaede, was introduced, which exhibits an irreversible color shift from green to red fluorescence after photoactivation with =350–410nm and, thus, allows for specific cellular tracking of proteins before and after exposure to the illumination light. In this work, the dynamics of this photoconversion reaction of Kaede are studied by fluorescence techniques based on single-molecule spectroscopy. By fluorescence correlation spectroscopy, fast flickering dynamics of the chromophore group were revealed. Although these dynamics on a submillisecond timescale were found to be dependent on pH for the green fluorescent Kaede chromophore, the flickering timescale of the photoconverted red chromophore was constant over a large pH range but varied with intensity of the 488-nm excitation light. These findings suggest a comprehensive reorganization of the chromophore and its close environment caused by the photoconversion reaction. To study the photoconversion in more detail, we introduced a novel experimental arrangement to perform continuous flow experiments on a single-molecule scale in a microfluidic channel. Here, the reaction in the flowing sample was induced by the focused light of a diode laser (=405nm). Original and photoconverted Kaede protein were differentiated by subsequent excitation at =488nm. By variation of flow rate and intensity of the initiating laser we found a reaction rate of 38.6s for the complete photoconversion, which is much slower than the internal dynamics of the chromophores. No fluorescent intermediate states could be revealed. Abstract | Full Text | PDF (194 kb)

• …more

Copyright © 2007 The Biophysical Society. All rights reserved.
Biophysical Journal, Volume 92, Issue 7, L55-L57, 1 April 2007

doi:10.1529/biophysj.106.101485

Biophysical Letters

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

Kanchan Garai^*, Ruchi Sureka^† and S. Maiti^*, ,  

^* Tata Institute of Fundamental Research, Colaba, Mumbai, India
^† Manipal Academy of Higher Education, Manipal, Karnataka, India

Address reprint requests and inquiries to Sudipta Maiti, Tel.: 091-222-278-2716; Fax: 091-222-280-4610.

Recent studies suggest that the soluble aggregates of amyloid-β peptide (Aβ), rather than the amyloid deposits, are the main pathogenic species in Alzheimer’s disease (AD) 1,2,3,4. Similar species are also thought to be responsible for many other aggregation-related diseases, such as Huntington’s and Parkinson’s 2,5. Hence, many of the current clinical strategies target these aggregates to prevent neuronal loss in AD 6,7. Unfortunately, presymptomatic diagnosis of the disease has achieved limited success 8. An effective strategy for diagnosis would be to detect the soluble aggregates, which appear early and are potently toxic, in vivo. Monitoring the effectiveness of a candidate drug or a treatment routine, say in a model animal, also requires monitoring of these aggregates inside the brain over time.

In vitro, such aggregates can be detected using fluorescence correlation spectroscopy (FCS) 9,10,11. FCS measurements have helped uncover chemical agents that can lower the population of these aggregates 11. Unfortunately, FCS uses bulky optics, and at present it is not possible to use it inside the brain. Here we explore the possibility of measuring protein aggregation in a minimally invasive manner, extending a recently demonstrated scheme that uses a single mode fiber for obtaining FCS data from fluorescent beads 12. Comparatively lower sensitivity, higher background fluorescence, and higher photobleaching make this technique inappropriate for detecting single fluorescent molecules. However, an aggregated protein particle, where a fraction of the monomers is labeled with a fluorophore, can present a target much brighter than a single molecule, and may possibly be amenable to the fiber technique. Here we monitor an aggregating Aβ solution in vitro with fiber-based FCS, and compare the results with conventional FCS.

We first calibrate the fiber FCS instrument with a solution of fluorescent beads of 13nm radius. The fiber optic arrangement is similar to that reported earlier 12. In this setup, a single mode optical fiber (mode field radius=1.7μm) is used both for delivering the excitation light (543nm) and also for collecting the fluorescence from the sample. This setup is automatically confocal and can be used for FCS measurements 12,13. We fit the FCS data using the MEMFCS routine 14, which analyzes the data in terms of a quasicontinuous distribution of diffusion constants. Such analysis is required for characterizing highly heterogeneous aggregating protein solutions 10,11. The size distribution obtained from the bead solution using the fiber FCS setup is shown in Fig. 1 (solid line). The single-peaked distribution is centered around 30ms. The size distribution obtained from the same specimen using a conventional FCS instrument 15 of the same specimen is shown as a dotted line in Fig. 1. We see that the two size distributions are similar, except that the fiber FCS curve is peaked at a time point that is 21 times higher than that of the conventional FCS. This is due to the larger probe volume of the fiber FCS instrument, and this provides a size calibration for any unknown particle measured with this technique.

Display large version of this figure

Figure 1

Calibration of the fiber FCS instrument. The size distributions obtained using fiber FCS (solid line) and conventional FCS (dotted line) from fluorescent beads of radius 13nm.

We note that the probe volume can be theoretically calculated from the geometry of the fiber and that of the optical system, and agrees well with the experimentally observed probe volume 12.

We then examine our ability to characterize the aggregation state of an Aβ solution by examining it in vitro, first with fiber FCS and then with conventional FCS. We prepare a 10-μM Aβ solution mixed with 100 nM rhodamine-labeled Aβ (RAβ)(both purchased from rPeptide, Athens, GA) at pH 7.4 in HEPES buffer and incubate it for 6h at room temperature.

Figure 2A shows the autocorrelation data (solid squares) obtained from a fiber FCS measurement of this sample. The data are fit (solid line) with MEMFCS. The size distribution obtained from this data is shown in Figure 2C (solid line). The same solution then is examined by conventional FCS. The autocorrelation data and the fit are shown in Figure 2B. The error bars in the autocorrelation traces in all the figures are calculated from 10 identical measurements for 3min each. The size distribution is plotted (as a dotted line) in Figure 2C. The abscissa denotes the hydrodynamic size in nanometers. This axis has been calibrated using the ratio of the size and the diffusion time of the beads obtained from Fig. 1. The fiber FCS data show the existence of a clear peak centered around 200nm, and extending from ∼130 to ∼270nm. The conventional FCS data on the other hand show a similar peak at 210nm, but in addition, they also show a peak at 2.1nm. The heights of the largest peaks are normalized to unity. The similarity of the second peak observed in conventional and that observed with fiber FCS establishes that the larger aggregates can be effectively seen with fiber FCS. On the other hand, it is clear that the peak at the smaller size range, which contains the monomer and the small oligomers (the calculated value of the hydrodynamic radius for the monomer is 1.7nm), is resolved only by conventional FCS.

Display large version of this figure

Figure 2

Measurement from aggregating Aβ solution. Autocorrelation data (solid squares) with fit (solid line) obtained from 10μM Aβ+100 nM RAβ after 6h of preparation of the solution using (A) fiber FCS and (B) conventional FCS instrument. (C) Size distributions obtained using fiber FCS (solid line) and conventional FCS (dotted line). The size axis has been calibrated using the data from Fig. 1.

We note here that due to the relatively long residence time of the particles in the fiber FCS confocal volume, photobleaching may be a concern. We estimate the probability of bleaching from the molar extinction coefficient for rhodamine-B in water at 543nm (∼50,000 l/mol cm), and the average intensity of the excitation light in the FCS observation volume (∼3×10²⁵ photons/m²). The number of photons absorbed by a rhodamine label during the residence time of an aggregate (∼0.5s) is <1.2×10⁵. The average number of photons absorbed by a bioconjugated rhodamine-6G molecule before photobleaching is ∼3×10⁵16. We thus expect a large fraction of the oligomers to remain unbleached. Because the oligomers are multiply labeled, photobleaching is unlikely to have a major effect on the FCS data.

We also examine a control Aβ solution that has been aged for 2 weeks, and that does not have appreciable amounts of the soluble aggregates left in it. The fiber FCS instrument does not show any appreciable correlation from this specimen (Figure 3A), whereas the conventional FCS experiment shows a short time correlation extending to ∼2ms (Figure 3B). The analysis of the conventional FCS data (Figure 3C, dotted line) shows that the specimen only contains monomers and small oligomers, and no appreciable amount of higher order aggregates. This establishes the reliability of the fiber FCS technique to faithfully report the presence or absence of the large soluble aggregates in an aggregating amyloid-β solution.

Display large version of this figure

Figure 3

Measurement from an equilibrium Aβ solution. Autocorrelation data (solid squares) with fit (solid line) obtained from 10μM Aβ+100 nM RAβ after 2 weeks of preparation of the solution using (A) fiber FCS and (B) conventional FCS instrument. (C) Size distribution from conventional FCS (dotted line). The size axis has been calibrated using the data from Fig. 1.

The ability to resolve particles directly depends on the signal/noise of the instruments. The results show that particles in the size range of 1.7–5.0nm, which are most likely labeled with a single fluorescent label, do not have adequate brightness to be observed by the fiber FCS technique. However, many reports have shown that the larger soluble aggregates, or protofibrils, are potently toxic to the neuronal cells 1. These species are detected well by the fiber FCS technique. Optical fibers of diameters larger than what has been used here have been introduced into living rodents and their brains have been optically monitored over long periods 17. If such fibers are used for FCS, with some amount of fluorescently labeled amyloid-β introduced into the cerebrospinal fluid, it should be possible to monitor the population of large soluble aggregates in vivo, e.g., in the brain of an Alzheimer model mouse 18. This study thus highlights a possible route toward quantitative monitoring of the aggregation of physiological molecules in vivo.