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Project IV - Technology Development For Studies of Cellular Regulation
Aim 2: Studies of protein aggregation and folding

Measuring α-synuclein aggregation and HSP-70 effects using combined FCS and fluorescent burst analysis.   Using the instrumentation and software outlined in Aim 1, we have begun experiments to measure the progression of intracellular aggregation of α-synuclein-GFP and the effect of co-expression of HSP-70 in the distribution of oligomers.   Figure 1 shows results from a typical experiment on cell lysates at 16 hours and 24 hours after transfection.   Cells were frozen as various time points in LN and then thawed and immediately diluted for the assay to avoid further aggregation of α-synuclein-GFP.   To better sample the solutions we slowly flow the sample through a micro-channel.  With our current transfection protocol, expression levels are too high to use FCS techniques in vivo, although we are able to see individual bursts as larger oligomers diffused through the focal volume.   These pilot experiments show that we can quantitatively follow the progression of oligomer formation using our hardware and software.   Using FCS alone on the same data streams without first separating out the bright bursts showed no consistent differences between times points since the apparent dwell time of each curve is dictated by a few of the largest photon bursts. The method shown in Figure 1 provides a quantitative analysis of large aggregates and a semi-quantitative measurement of the smaller oligomers through the correlations of the photon stream in regions where there are no distinguishable large particle bursts.    We refer to this method of locating large bursts and autocorrelating regions in between as “segmental FCS”.

To further quantify the size distribution of the smallest and most biomedically relevant oligomers we have begun to employ single molecule channel detection and extremely high sensitivity detection.  We have previously demonstrated single molecule fluorescence measurements in nanofluidic devices (Foquet et al, 2002; Foquet et al, 2004) using heavily labeled fragments of DNA.   In this work we were able to determine DNA fragment sizes based on the number of photons collected as the fragments pass though the excitation volume.   The DNA fragments were stained with a DNA binding dye and the number of photons collected per fluorescence burst generated by passage through the focal volume was linearly proportional to the fragment size allowing for the fragment size distribution to be determined.  Using software shown in Aim 1 developed for the work shown in (Foquet et al, 2002), analysis of the photon bursts arising from the passage of a single molecule are carried out on the collected photon traces.  Histograms of the number of photons per burst were calculated to determine the distribution of the particle sizes.   In this early work we were able to show that we could accurately size a DNA fragment sample in 6 minutes using only 10-6 of the volume of sample and examining ~9000 fragments.  However, the ability of these systems to detect individual molecules and to discriminate between the numbers of fluorophores with high sensitivity has yet to be fully determined. In our previous work we were detecting thousands of fluorophores per molecule (DNA fragment) and differences in molecular size were easily determined. 

Our requirement here is to accurately count low integer numbers of fluorophores that correspond one-to-one with the number of Αβ monomers. To achieve this goal a dramatic increase in system collection efficiency is required; however, the experimental strategy is the same.  One can calculate the number of photons that need to be collected from each particle passing through the focal volume to be able to discriminate between integer numbers of fluorophores per particle.   Based on a Poisson distribution and its standard deviation, ~50 photons must be collected per molecular passage through the focal volume to statistically tell the difference between one and two fluorophores (i.e. between a monomer or a dimmer of aβ;).  This defines the maximum sensitively we require.  Assuming a fluorescence lifetime in the 2.5 ns range (typical for most fluorophores), a single fluorophore will produce 400 photons per ms.  Using electrokinetic flow we can achieve flow speeds of 0.5 to 1 mm/ms and therefore can imagine a maximum collection (assuming 10% collection efficiency) of about 20-40 photons per focal volume transit using a high NA objective (spot size is ~ 1 micron due to underfilling). In previous nanochannel single molecule detection work using this same channel design in which single quantum dots were detected (Stavis et al. 2005) we were only able to collect between a 2 to 3 photons per transit at maximum, but this was using APDs with 100 ns dead time.  The detector and acquisition advances shown in Project I will eliminate the detector dead time effects and provide at least a 10 fold increase in detection.  To further improve our collection we propose to use a 4pi collection scheme that will double the collection and provide the sensitivity needed to be able to fully discriminate between oligomers at the desired level of resolution

Using photolithography, we have fabricated 500 nm wide square fluidic channels in optical grade fused silica wafers. In our device, a dilute sample of fluorescently labeled particles is driven by voltage from the inlet to the outlet passing through the focal volume of the excitation beam one molecule at a time.  The confinement region of the channels is 500 nm wide, 500 nm deep and 10 microns long as shown in Figure 2. These are fabricated in arrays of typically 20 channels on each wafer.

Figure 2. A. Single molecule detection in a nano-channel.



Cantuti-Castelvetri, I., J. Klucken, et al. (2005). "Alpha-synuclein and chaperones in dementia with Lewy bodies." J Neuropathol Exp Neurol64(12): 1058-66.

Foquet, M., J. Korlach, et al. (2002). "DNA fragment sizing by single molecule detection in submicrometer-sized closed fluidic channels." Anal Chem 74(6): 1415-22.

Foquet, M., J. Korlach, W. R. Zipfel, W. W. Webb and H. G. Craighead, (2004) Focal Volume Confinement by Submicrometer-Sized Fluidic Channels Analytical Chemistry 76(6), 1618-1626.

Stavis, S. M., J. B. Edel, et al. (2005). "Single molecule studies of quantum dot conjugates in a submicrometer fluidic channel." Lab Chip 5(3): 337-43.




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