Development of System and Methods for the Sub-Pixel Resolution Characterization of Nanoscale Constructs
Containing Multiple Fluorophores
Anuradha Rajulapati, David Neff, Wanqiu Shen,Ph.D. Hong Zhong. Ph.D. , Rusty Parrett, Micheal Norton, Ph.D. :Department of Chemistry, Marshall University.
ABSTRACT
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.
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.
DNA/fluorophore constructs
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.
5’-/56-FAM/AAG GGG CTT TCT TGC TCT TAT TAT ATT GCT ATT TCA TTG TAT GA CCG AAA-3’
B
C
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.
FIG 1
A
C
B
D
B
B
E
Fig 2
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.
B
1
2
4
3
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.
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 subregion 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.
data
fit
4.9805
profile of horizontal line with fit
Intensity profiles
4.6293
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
profile of vertical line with fit
center of the qdot has
been re-defined not
as 5,5 (brightest pixel)
but as 4.9,4.6
(Gaussian center)
Fig 7
C
Locating single molecules and qdots with sub-pixel accuracy
D
this gel is not stained
Annealed product
50 mer DNA with Rhodamine at 5’ end
50 mer DNA with Fluorescein at 3’ end
Fig 3
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 3BD).
Cairn Optosplit 2 image splitter
So why does our 2 channel setup
1-excitation filter (see B below)
Sample
HgXe lamp;
2- dichroic mirror
not work for fluorescein ?
peaks at 405,
3-dichroic mirror
4- long λ ‘red’ emission filter (595/50)
436,
546,
According to our optical microscopy
5-short λ‘green’emission(515/30) )
577nm
system, the DNA fluorophore
camera chip
Cairn optosplit
constructs are supposed to show up
2 image splitter fixed mirrors
in the both channels of optosplit
microscope, but it’s not. So, we
fixed mirror
matched the output of our Hg lamp
(green in figure 4B) with our excitation
A
fixed mirror
filters (black in 4B). We also took the
Excitation intensities resulting from Hg lamp output
excitation spectrum as it came from
and optosplit filtering
range of qdot
the microscope (using a specabsorption
trometer). This is seen in figure 4C as
range of rhodamine
absorption
the blue tracing. It shows an
range of fluorescein
absorption
excitation peak at ~550nm and a
much lower, even intensity at shorter
wave length, nm
Wavelength, nm
C
λ. Actually it will sufficiently excite
B
only rhodamine (abs. red arrow) not
Fig 4
fluorescein (abs. green arrow). So
we conclude that this is the reason
B
A
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.
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.
Fig 8
In fig 8A-C are images from our 2
qdot
R-DNA
Annealed R-DNA/F-DNA
channel optical system as seen in
A
B
C
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
D
used images from 8A-C for analysis of
X-Y scattering that are shown in fig 9.
time 1
At right in figure 8D is an example of
how a single ‘stationary’ qdot (from
2
figure 8A) can be localized with subpixel accuracy but appears to move
3
within that pixel. The 3x3 pixel regions
4
at left, labeled time 1-7, are actual
pixels sampled from movie in 8A.
5
The larger white boxes in the 3x3 array
at right represent these pixels. The
6
tracing at center (blue line) is the
‘trajectory’ of the qdot as calculated by
7
260nm
VST. Notice that the time points on the
trajectory correspond to the image
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.
Fig 9
5
6
qdot point 1
100
100
-100
50
50
0
0
-50
0
-50
-100
100
50
50
50
100
0
100
50
50
0
0
100 -100
-100
-50
-50
-100
-100
-100
100
50
50
100
-50
-100
-100
100
0
0
0
VST
100
-100
-50
-100
100
50
0
0
100
-100
0
-50
-50
-100
-100
Image J
100
Image J
annealed point 2
0
0
-50
50
-100
100-100
VST
100
-100
0
0
50
100
Image J
100
-100
Image J
50
100
-100
VST
50
-50
0
-50
0
-50
0
0
100
-100
R-DNA point 2
100
0
100
-50
R-DNA point 1
-100
0
0
Image J
VST
100
50
-100
-100
-100
qdot point 2
100
-50
0
-50
annealed point 1
bars are
SD
6A
6A
blink off
blink on
6B
4
2
7
VST
Fig 5
3
1
VST
100
Image J
62% decrease
UV water treatment
chamber
A
7.5% decrease
condensor for
distillator
Proceedure for gel analysis of
dna/fluorophore constructs: 8%
polyacrylamide native gel: Lane 1&2annealed 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).
Intensity ,a.u.
A
~15nm
% T for filters, a.u. for Hg output
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).
5’-/5RhoR/TTT CGG TCA TAC AAT GAA ATA GCA ATA TAA TAA GAG CAA GAA AGC CCC TT- 3’
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).
Single molecule localization and tracking
REFERENCES
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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
bleach off
Acknowledgements
Dr. M.L. Norton for maintaining the MBIC imaging facilities.
6C
http://www.marshall.edu/mbic/