Development of System and Methods for the Sub-Pixel Resolution Characterization of Nanoscale Constructs Containing Multiple Fluorophores

An ongoing challenge in the development of nanoelectronics and nanophotonics is the nondestructive high resolution localization in space of single molecules and multi-molecular assemblies. The apparent barrier to the use of optical microscopy at the sub-100 nm scale is the well known Abbe Limit, the diffraction limit to resolution. This laboratory is adapting a technique first developed by Spudich called Single-molecule High-Resolution Co-localization (SHREC). By using two chromatically different fluorescent molecules or two chromatically similar, but time multiplexed molecules as probes one can measure distances less than 100nm optically. We are developing methods for utilizing SHREC for the determination of the separation of two fluorophores in single DNA molecules or DNA origami constructs.Two channel fluorescence imaging has been implemented using an Optosplit equipped Nikon microscope. The construction and characterization of the two types of DNA based test objects will be presented. Because a pre-requisite for single molecule microscopy is the production and maintenance of coverslips and solutions with essentially no fluorescent contamination, we have constructed highly effective systems to remove adventitious fluorescent contamination from substrates and from buffer solutions. We developed a vector based protocol for image series analysis to discriminate between single fluorophores and linear assemblies of fluorophores. The application of this process to a quantum dot sample will be presented.

ABSTRACT

Anuradha Rajulapati, David Neff, Wanqiu Shen,Ph.D. Hong Zhong. Ph.D. , Rusty Parrett, Micheal Norton, Ph.D. :Department of Chemistry, Marshall University.

Acknowledgements

Dr. M.L. Norton for maintaining the MBIC imaging facilities.

1.L. Stirling Churchman, Ronald S. Rock, John F. Dawson, and James A. Spudich, et.al. Single molecule high-resolution colocalization of Cy3and Cy5 attached to macromolecules measures intramolecular distances through timePNAS February 1, 2005 vol. 102 no. 5 1419–1423 . 2. Seong Ho Kang . Yun-Jeong Kim .Edwards S.Yeung_Detection of Single-molecule DNA hybridization by using dual-color total internal reflection fluorescence microscopy Anal Bioanal Chem (2007) 387:2663–2671 3. Extracellular DNA and Type IV pili mediate surface attachment by Acidovorax temperans Bjorn, D. Heijstra Franz B. Pichler,Quanfeng Liang, Razel G. Blaza, Susan J. Turner Antonie van Leeuwenhoek (2009) 95:343–349. 4. I. F. Sbalzarini and P. Koumoutsakos. Feature point tracking and trajectory analysis for video imaging in cell biology. J. Struct. Biol., 151(2): 182–195, 2005. 5. video spot tracker; http://cismm.cs.unc.edu/about/ 6. image J thanks to NIH.gov

REFERENCES

Removal of fluorescent contaminants from solutions and glass substrates

When imaging large samples such as cultured cells, researchers have generally not been concerned with contamination of cover-slips and solutions by fluorescent molecules, this type of sample typically contains many molecules that have been concentrated on discrete structures. That is, the sample is much brighter than the background. However when imaging single molecules as in the present work, the detector (camera) must be very sensitive. Imaging with a detector of this type allows us to see the fluorescent material that has contaminated the coverglass surface (see fig. 2A). With background contamination this severe we cannot be sure if we are imaging sample molecules or contamination molecules. For that reason, we treat all coverslips and solutions as described below.

Proceedure for treatment of water used in rinses and solutions:House water from MU BBSC D.I. tap (this is water deionized by reverse osmosis) is further purified by distillator seen in figure 1A. This water still shows fluorescent contamination (fig. 2D) so we followed distillation with UV light exposure using a high intensity UV chamber (Minipure by Atlantic Ultraviolet Corporation, dosage ~ 120mW/cm2) to photobleach these contaminants coverslip after applying and drying pure water is seen in figure 2D. Components of this setup are seen in (figure 1A).

Proceedure for removing fluorescent contamination from coverslips: We place Fisher brand premium coverslips (#1.5~170um) in teflon coverslip holder and place the holder in a beaker of Acetone (HPLC grade, Sigma Aldrich). Coverslips are sonicated for 30 minutes, rinsed with water (H2O prepared as described above), and dried with nitrogen gas.To eliminate remaining fluorescent particles, we exposure coverslips to a low pressure UV lamp (SEN LIGHT CORP., Japan, UVL20US-60) for 10min (apparatus shown in figure 1B). Distance from lamp to coverglass was ~2cm for a dosage power of 125mW/cm2 (this actually accounts for the absorption area of particles within the water) Examples of coverslips imaged after each of the preceding steps can be seen in figure 2 A-E).

condensor for distillator

UV water treatment chamber

Sonicatior (left) and UV coverglass treatment chamber (right). UV bulb is at top of chamber, coverslips are raised to within 2cm of source on platform within.

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Results of solution and coverslip preparation: In figure2A, is an untreated coverslip right from the box showing fluorescent contamination in both red and green channels.

B shows a coverslip after 30min sonication with acetone showing few fluorescent contaminants .

In C, a coverslip fully treated as explained above shows no fluorescent contamination in either red or green channels.

After full coverslip preparation and subsequent application and evaporation of distilled water (ours or HPLC double distilled from Fisher), rinse, and blown dry with nitrogen gas, we still see in D some fluorescent contamination.

In E we see a fully prepared coverslip that was treated with water as the surface in D. However, in this case, we used our water that was treated as described in proceedures section above (including UV exposure) . We see fluorescent contamination neither in red nor green channels.

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Although the fluorescent objects we are currently studying are much smaller than a single pixel (each pixel here is ~260nm for 60x objective or ~155 nm for 100x objective), we use fitting equations provided in software, ImageJ (IJ by NIH) and Video Spot Tracker (VST by CISMM) to find the ‘centers’ of these objects and achieve sub-pixel resolution. The Image J particle tracker uses ‘Moment Scaling Spectra’ (Ref. 4) while VST uses Gaussian fitting similar to what is shown below in figure 7.

Single molecule localization and tracking

How does a simplified 2D Gaussian fit work?The original image (figure 7 left) is a dispersion of qdots on a glass cover-slip. A sub-region was extracted from the image for clarity (center). Pixels have integer coordinates, here the brightest pixel in the sub-region is at (5,5). After fitting an equation to the intensity distribution, we get a sub-pixel coordinate (4.9,4.6) as the location of the object. In terms of calibrated space (nm not pixels), this is a positional ‘correction’ of ~25nm in x and ~100nm in y.

profile of vertical line with fit

profile of horizontal line with fit

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Intensity profiles measured along the yellow lines above are fit to a Gaussian curve (right). The original data is seen at right as yellow tracing, the fit is the white curve. The center of the qdot has been re-defined not as 5,5 (brightest pixel) but as 4.9,4.6 (Gaussian center)

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Locating single molecules and qdots with sub-pixel accuracyFig 8

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At right in figure 8D is an example of how a single ‘stationary’ qdot (from figure 8A) can be localized with sub-pixel accuracy but appears to move within that pixel. The 3x3 pixel regions at left, labeled time 1-7, are actual pixels sampled from movie in 8A. The larger white boxes in the 3x3 array at right represent these pixels. The tracing at center (blue line) is the ‘trajectory’ of the qdot as calculated by VST. Notice that the time points on the trajectory correspond to the image

In fig 8A-C are images from our 2 channel optical system as seen in figure 4. A is the 525 qdots spread on a coverslip we show the green channel only, we used a movie of this sample for analysis in the fig 8D. Fig. 8B is the R-DNA spread on a clean glass coverslip (red channel only shown). Fig. 8C is the annelaed DNA sample on the glass cover slip. We used images from 8A-C for analysis of X-Y scattering that are shown in fig 9.

regions at left. This is the type of localization data that is plotted on x/y coordinates below in figure 9. All plots in figure 9 are in nm units, the 2 plots for each point are a comparison of the 2 different math treatments provided by either VST or IJ. The tightest grouping of locations are seen in the R-DNA plots where the spread is ~ +/- 30nm. These types of plots will be used in future work to establish the stability of our system and to help us recognize fluorescent constructs that are measurably asymetrical. For example, a 2 fluorophore construct , depending on its length, should show a distribution that is skewed along the axis of orientation of the construct.

DNA/fluorophore constructs

Time resolved behaviour of fluorophores in our system blinking and bleaching: Organic molecular fluorophores such as rhodamine are known to photobleach, that is to absorb photon energy and enter a state that is reactive with other species (often oxygen). Once a molecule bleaches, it cannot recover. Rhodamine bleaching is seen fig. 6C. Inorganic fluorophores (CdSe qdots) generally do not photobleach. Bleaching in our system is quantified in figure 6A, we believe the 7.5% (not 0% as expected) bleaching for qdots to be a result of final frame blinking. Blinking, is a phenomenon in which a fluorophore enters a metastable state in which it temporarily cannot fluoresce. Fluorophores do recover from blinking as we see in 6B. Blinking of quantum dots and other fluorophores can cause difficulty in quantifying emissions over time (imagine a qdot blinks out for 50% of an image exposure, it will appear half as bright in the final image). The standard deviation bars in 6A are a measure of eveness of distribution of particles on the surface.

Duplex DNA Preparation: We are developing a control object or ‘ruler’ to test/calibrate our system, we chose the dsDNA molecule below with fluorophores at each end, spaced at ~15nm. To make the 50bp duplex DNA molecules, two nucleotides with the sequence and modifications shown below (Integrated DNA Technologies,US) were hybridized. The oligonucleotide 5’-AAG GGG CTT TCT TGC TCT TAT TAT ATT GCT ATT TCA TTG TAT GA CCG AAA-3’ was labelled with carboxy fluorescein at its 5’ end (F-DNA). The complimentory oligonucleotide was labelled with rhodamine red at it’s 3’ end (R-DNA).These molecules were mixed at 1:1 stoichiometry (checked by OD 260nm). The fluorophores affect the absorbance reading so that we must account for them in absorbance/mass calculations.

Proceedure for gel analysis of dna/fluorophore constructs: 8% polyacrylamide native gel: Lane 1&2- annealed DNA with Fluorescein and Rhodamine at ends. Lane 3-DNA with rhodamine only at 5’ end. Lane 4-DNA with fluorescein only at 5’ end. Figure 3 A shows a PAGE image of 50mer DNA with fluorophores and its components. See in lanes 1 and 2 that our 1:1 ratio of the two strands is not perfect, we do see some extra unannealed R-DNA material in these lanes (arrows). Figure 3B-D shows these DNA moleclues as imaged with our 2 channel optical microscope (fig. 4). In short, left side of camera chip ‘sees’ green emissions and right side red emissions. Notice that our fluorescein labeled molecules do not appear in these images. We believe that this is a result of insufficient excitation energy for fluorescein (see below).

Proceedure for taking 2 channel (optosplit) images of dna/fluorophore constructs: Clean the coverslips and water for solutions as described in panel 1. We then pre-treat the coverslip with 100mM MgCl2 to facilitatie adsorption of the charged DNA molecules to glass (ref.3). We put the DNA sample on coverslip and incubate for 5min, wash with treated water and dry with N2 gas. We observe our sample with a fluorescence microscope (Nikon te200, Japan) having Optosplit II (Cairn, see fig. 4A) with an EM(electron multiplier) CCD camara (e2v chip, Qimaging camera package). Ideally, with this setup we can see the two wavelength ranges in perfect registration on the same camera chip (fig. 5 and 3B-D).

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Annealed product50 mer DNA with Rhodamine at 5’ end50 mer DNA with Fluorescein at 3’ end

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So why does our 2 channel setup not work for fluorescein ?According to our optical microscopy system, the DNA fluorophore constructs are supposed to show up in the both channels of optosplit microscope, but it’s not. So, we matched the output of our Hg lamp (green in figure 4B) with our excitation filters (black in 4B). We also took the excitation spectrum as it came from the microscope (using a spec-trometer). This is seen in figure 4C as the blue tracing. It shows an excitation peak at ~550nm and a much lower, even intensity at shorter λ. Actually it will sufficiently excite only rhodamine (abs. red arrow) not fluorescein (abs. green arrow). So we conclude that this is the reason fluorescein is not showing up in my experimental results (see figure 3B, left side ‘green’ channel ).

So, we have designed a new strand to compliment our R-DNA. This strand has attachment chemistry for a 5’ quantum dot (qdot 525, excitation range shown in figure 4C, blue arrow). We have established that qdot 525nm are clearly visible in our ‘green channel ‘ (fig.5A left and 5B merged channels) and have begun to study their behaviour in relation to the organic fluorophore rhodamine.

Fig 4

HgXe lamp; peaks at 405, 436, 546, 577nm

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Cairn optosplit 2 image splitter

1-excitation filter (see B below)2- dichroic mirror3-dichroic mirror4- long λ ‘red’ emission filter (595/50)5-short λ‘green’emission(515/30) )

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http://www.marshall.edu/mbic/

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