Bioimaging 6 (1998) 43–53. Printed in the UK
PII: S0966-9051(98)90833-4
Near-field optical microscopy for DNA
studies at the single molecular level
M F Garcia-Parajo†, J-A Veerman, S J T van Noort,
B G de Grooth, J Greve and N F van Hulst
Applied Optics Group, Department of Applied Physics and MESA Research Institute,
University of Twente, PO Box 217, 7500 AE Enschede, The Netherlands
Submitted 2 December 1997, accepted 16 January 1998
Abstract. An aperture-type near-field optical microscope (NSOM) with two
polarization detection channels has been used to image fluorescently labelled DNA with
high spatial resolution and single molecule fluorescence sensitivity. The sample has
been engineered such that there is only one rhodamine dye per DNA strand. Lateral
and vertical DNA dimensions in the shear-force image are 14 ± 2 nm and
1.4 ± 0.2 nm, respectively. No sample deformation was observed under our imaging
conditions. Near-field fluorescence imaging of individual fluorophores shows an optical
resolution of 70 nm at full-width at half-maximum. Large intensity differences between
individual rhodamine molecules attached to DNA are observed from the NSOM
images. Statistics on rhodamine dyes in different environments (attached to glass,
embedded in a polymer layer and attached to DNA) show bleaching rates of 10−5 .
Total intensity line profiles together with in-plane angle orientation are used to
characterize individual dyes. Rhodamine dyes show strong intensity fluctuations
independent of the particular environment. These results are in contrast with the more
stable photophysical behaviour as observed for carbocyanine molecules embedded in
polymer matrices. The mobility of rhodamine—both lateral and rotational—is clearly
influenced by its immediate surrounding and attachment to the surface.
Keywords: NSOM, near-field fluorescent imaging, shear-force imaging, single
molecule detection, DNA–dye interactions, photodynamics
1. Introduction
Detection and spectroscopy of single molecules at ambient
and biologically relevant conditions, combined with
great advances in genetic engineering, has opened new
opportunities of research in DNA analysis, diagnosis and
fundamental studies. A particular challenge—within the
context of the Human Genome project [1]—is the detection
of biomolecules containing a single fluorophore as the
molecule transits a focused laser beam [2]. Clearly, the final
goal is fast DNA sequencing. Another challenging area of
research is the understanding of molecular events involved
in DNA transcription and gene regulation. In many of these
events, the monitoring of structural and conformational
changes of single biomacromolecules (proteins and/or
the DNA itself) is of vital importance. Already the
study of enzymatic reactions at a single molecular level
[3–5] has started to offer detailed information regarding
molecular interaction and dynamics information that would
† E-mail address: [email protected]
c 1998 IOP Publishing Ltd
0966-9051/98/010043+11$19.50 be virtually impossible to obtain from ensemble-averaged
investigations.
While scanning force microscopy (SFM) is becoming
an increasingly used research tool to further the understanding of biomolecular processes by imaging the
dynamics of the macromolecule structure and assembly
in real time [6–8], chemical specificity is lost with
SFM. Near-field scanning optical microscopy (NSOM)
offers a powerful tool to combine the high spatial
resolution of SFM with the spectroscopic information
offered by optical methods.
In 1993, imaging of
individual dye molecules was achieved using an NSOM
configuration [9].
Since then, a wealth of studies
at the single molecular level, with superspatial and/or
temporal resolution, has been performed [10–14]. Further
advances in the field show that single molecule experiments
do provide a sensitive tool for studying the local
environment of a single molecule at ambient conditions
[15]. Moreover, slow dynamic processes have been
studied in the near field at the single molecular
43
M F Garcia-Parajo et al
level, including translational and rotational diffusion
[13, 16, 17].
In NSOM, an aperture probe of sub-wavelength
dimensions is scanned in close proximity (within the near
field, i.e. less than 10 nm) to the specimen under study.
Using the probe as a near-field excitation source, the
interaction with the sample surface induces changes in the
far-field radiation, which is collected by conventional optics
and directed to a highly sensitive detector to provide an
optical image [18, 19]. An independent mechanism is used
to control the distance separation between the probe and
the sample within 1 to 10 nm, and generate simultaneously
a topographic image [20]. In this way, a singular feature
pertaining to NSOM is produced: correlative optical and
topographical imaging with a spatial resolution mainly
determined by the probe configuration. Another unique
characteristic of the near-field excitation is given by the
finite size of the probe itself: decreasing the area of
illumination obviously reduces the interaction volume and
background scatter, which is of major importance to
minimize photodamage of the sample and to enhance the
sensitivity for single molecule detection.
The challenge of monitoring biochemical reactions
using a NSOM imposes special demands on both sample
preparation and data analysis. NSOM, like SFM, is a
surface technique and therefore the biochemical reactions
have to proceed on the surface, which might in turn
negatively influence the process. Undesirable effects
of the near-field probe on the process should also be
minimized and taken into account when analysing the
data. Individual dynamic events of a biomolecule will
be observable only when one fluorophore is attached
to the biomolecule.
Additionally, fluorescence of a
single dye displays rich dynamics associated with the
molecular properties of the dye itself and its immediate
surrounding. Therefore, in the study of biomolecule
dynamics it is crucial to understand first the photophysical
properties and dynamics of individual fluorophores under
relevant conditions. This knowledge will then be used for
distinguishing fluorophore dynamics from the biomolecule
dynamics. In this paper we show that NSOM is a powerful
technique for the identification of specific tagged sites
on the DNA macromolecule. Individual DNA molecules
tagged with only one fluorophore at one end of the
DNA fragment are visualized with unprecedented optical
and topographical lateral and vertical resolution, together
with single molecule fluorescence sensitivity. Next, we
present statistical data on the photodynamics of two
biologically relevant dyes as a function of their different
surroundings. Photobleaching rates, total emitted intensity
and in-plane orientation per molecule are presented for
both dyes, on the millisecond and second timescales. We
find that rhodamine molecules exhibit, in general, much
faster dynamic behaviour, independent of the surrounding
(adsorbed on glass, imbedded in a thin polymer layer
or attached to the DNA macromolecule) as compared to
carbocyanine molecules.
44
2. Materials and methods
2.1. Sample preparation
As a model system to demonstrate ultimate sensitivity
imaging we chose double stranded (ds) DNA, 1 kb in
length (∼340 nm) labelled at one end with rhodamine, i.e.
one fluorophore per DNA fragment. The 1 kbp dsDNA
were manufactured by polymerase chain reaction (PCR)
and already labelled with rhodamine 6G succinimidyl ester,
R6G-SE (Eurogentec, SA, Belgium). The 50 -phosphate
end of the synthesized dsDNA fragment was chemically
modified using standard aminophosphoramidite modifiers
to introduce a NH2 reactive group. A 6 methylene (CH2 )
linker was chosen between the terminal phosphate and the
amino moiety. R6G-SE was then allowed to react with
the NH2 group to form a carboxamide bond. Because
of the selectivity of this reaction, only one fluorophore
per DNA fragment is produced. Fluorescence strands
have been purified by enhanced high-performance liquid
chromatography (trade mark of Eurogentec) to remove
unlabelled fragments. Labelling efficiency is better than
90%, as guaranteed by the manufacturer. Freshly cleaved
mica disks (Ted Pella, CA, USA) (thickness ∼150 µm)
were used as substrates for immobilizing the DNA. dsDNA
fragments were diluted in a buffer containing 4 mM
HEPES, 1 mM NaCl and 1 mM MgCl2 , pH 6.5 to a final
concentration of 3.2 ng µl−1 for deposition. The buffers
were made of MilliQ filtered deionized water. A 5 µl drop
was deposited onto the freshly cleaved mica. After the
sample settled for a couple of minutes, it was washed with
water (0.5 ml of doubly distilled water from a squirt bottle)
and then briefly dried in a stream of compressed air. SFM
in the tapping mode was used to initially characterize the
samples. In practice, we found between 30 and 65 DNA
molecules µm−2 , in rough agreement with the calculated
areal density of 80 molecules µm−2 , with the discrepancy
probably attributable to the well known unevenness of
the DNA binding to the mica surface [8]. After SFM
inspection, samples were rigidly mounted onto a metallic
support and fixed to the microscope scanning table with
three small magnets.
To gain understanding of the effect of DNA over
the fluorophore photophysical properties and dynamics,
we prepared samples in which the same fluorophore
R6G-SE (Molecular Probes, C-6127) was spread on a
previously acetone-cleaned silica glass cover slip at a
concentration of 10 nM, and also embedded in a thin
polymethylmethacrylate (PMMA) layer. Similarly, as a
reference calibration sample, carbocyanine dye molecules
(DiI-C18 from Molecular Probes, D-282) were also
investigated. The samples were prepared by embedding the
dyes in PMMA as described elsewhere [21]. In all cases,
the surface coverage was typically a few dye molecules per
square micrometre.
Near-field optical microscopy for DNA studies at the single molecular level
to separate the fluorescence signal into two perpendicular
polarization components. Both signals are then sent to
photocounting avalanche photodiodes (APD, SPCM-100
from EG&G Optoelectronics, Canada). Excitation powers
varied from 5 to 80 nW at the end of the near-field
probe (depending on aperture diameter and throughput
efficiency). Counts rates between 103 and 104 counts s−1
per detector from a single molecule were obtained with
background signals of 102 –103 counts s−1 per detector.
The single molecule detection sensitivity of the NSOM
configuration was complemented with molecular resolution
topographic imaging using shear-force detection. The
shear-force feedback scheme is based on tuning fork piezoelectric elements [20] and on the detection of the phase
difference between the driving excitation and the tuning
fork signal. Improvements in tuning fork time response
and sensitivity have been discussed elsewhere [17, 22].
Further sensitivity for DNA imaging has been achieved by
increasing the quality factor Q of the tuning fork–probe
configuration (between 800 and 1200), and by extending the
bandwidth of the feedback and phase detection electronics
(currently limited by the piezo-electric scanner resonance,
at ∼2 kHz). A custom-designed metallic holder was
used for sample mounting, providing high mechanical
rigidity while leaving a 6 mm diameter opening for optical
measurements. Mechanical vibrations have been kept
below 0.3 Hz by suspending the entire microscope with
elastic cords. Additionally, the microscope head is enclosed
during measurements to reduce airflow. All measurements
were performed at ambient conditions. Typical relative
humidity is ∼65% during experiments.
Figure 1. Schematic set-up of the aperture-type NSOM used in
our experiments. The scanning stage is mounted on top of an
inverted optical microscope. Shear-force feedback is used to
control the tip-to-sample separation. Fluorescence is collected in
transmission using a 1.3 NA immersion oil objective, split in
two polarization directions and detected with two photon
counting APDs.
2.2. The instrument
The NSOM configuration used in these experiments has
been described in detail elsewhere [16]. A schematic is
shown in figure 1. Briefly, the scanning sample stage is
mounted on a Zeiss, Axiovert 135 TV inverted optical
microscope. Local excitation is achieved by coupling
the 514 nm line of an Ar/Kr laser into the aluminium
coated optical fibre probe (typical aperture diameter <
100 nm). A tuning fork based shear-force feedback scheme
has been implemented to control the tip–sample distance
separation and generate at the same time a topographic
image. The optical detection path is similar to that found
in any conventional far-field ultrasensitive fluorescence
microscope, except from a broadband polarizing beamsplitter cube (Newport, 400–700 nm) which has been added
3. Results
3.1. Shear-force and near field fluorescence images of
DNA/dye
As an example of the high lateral and vertical resolution
that can be obtained using the shear-force modality of the
NSOM, figure 2 shows a 1.5 × 1.5 µm2 scan area image
(200 × 200 pixels) of plasmid DNA deposited as described
above. The image was obtained using an aluminium coated
NSOM probe. The resonance frequency of the tuning fork–
probe ensemble was f0 = 33 958 Hz with a quality factor,
Q = 1200. The tuning fork was driven externally with
dither amplitude of 0.1 pm peak-to-peak resulting in a
lateral tip displacement of approximately 6 pm†. Phase
feedback was used to control the tip–sample separation and
to generate the topographic image [22]. The phase set point
was chosen close to the out-of-contact value. The scan
speed was 1.5 µm s−1 . The line trace in figure 2 shows
a full-width at half-maximum (FWHM) of 14 nm, and a
† From macroscopic measurements we have determined the tip amplitude
as a function of the dither piezo-amplitude. We have observed that the
tip amplitude does not follow directly the amplification given by the fork
Q factor, depending on the mechanical mounting of the tip onto the fork.
45
M F Garcia-Parajo et al
Figure 2. A 1.5 × 1.5 µm2 (200 × 200 pixels) shear-force image
of plasmid DNA as deposited on mica. The time per pixel is
5 ms. The width of the DNA is 14 ± 2 nm at FWHM, with a
height of 1.4 ± 0.2 nm as seen from the line trace on the figure.
height of 1.4 nm. These values are comparable to those
obtained using conventional SFM techniques [23, 24]. Due
to the metal grain structure at the tip end region which
differs from tip to tip, it is difficult to obtain images with
reproducible good quality. However, once a ‘good’ tip
has been selected, the quality of imaging reproduces nearly
100% from image to image.
The non-disturbing character of shear force microscopy
has been pointed out by several workers [21, 25] despite
the lack of understanding of the nature of the shearforce interaction. An estimation of the friction force
transferred to the sample can be easily obtained using
the harmonic oscillator model for the tuning fork–probe
configuration [26]. With Q = 1200 and a static spring
constant kstat = 15 kN m−1 (for a 32 kHz tuning fork), an
effective spring constant within the resonance of kdyn =
1.25 N m−1 is obtained. The effective force is then
7.5 pN, close to the thermal noise of the drag force (the
noise limited drag
√ force detected by the fork is [26] Fn =
[kstat kB Te ]1/2 / 3Q where kB is the Boltzmann constant
and Te is the temperature of the fork. At Te = 300 K,
with Q = 1200, Fn ∼ 4 pN). It has been established that
deformation of proteins and many biological systems in
aqueous solution using SFM techniques occurs at forces
higher than 100 pN [27]. It is important to note that
46
the sensitivity of the technique is ultimately determined
by the effective spring constant of the fork, therefore the
importance of having large Q values, even if speed is
sacrificed.
Figure 3 shows a combined topographic (a) and
fluorescence (b) image of 1 kbp dsDNA fragments labelled
with a single R6G dye molecule at the 50 end of
each fragment. The scan area is 1.5 × 1.5 µm2 , with
200 × 200 pixels per image and an integration time of
10 ms/pixel. Because of technical difficulties, the images
were not acquired simultaneously, and therefore they do not
correspond exactly to the same surface area. In figure 3(a),
DNA fragments of 340 nm length (as expected) are clearly
resolved with a filament height of 1.4 nm. In figure 3(b),
the near-field fluorescence image of individual R6G dye
molecules attached to one end of the DNA strands is shown.
The diameter of the individual intensity peaks is 70 nm at
FWHM. The averaged background is 40 counts/pixel, with
a maximum R6G molecule signal of 240 counts/pixel. The
measured number of counts per pixel is consistent with the
emission expected from a single R6G molecule under our
excitation conditions (with an excitation power of ∼10 nW
at the end of the NSOM probe as measured in the far-field,
an absorption cross section of 2 × 10−16 cm2 for R6G, an
assumed emission quantum efficiency of 0.8 and estimated
collection efficiency of the optimized optical path of 0.1,
we expect to detect ∼104 counts s−1 , i.e. ∼100 counts/pixel
per molecule).
An interesting feature on the fluorescent image of
figure 3(b) is the large difference in intensity emitted
by individual R6G molecules. While some molecules
emitted a maximum of 240 counts/pixel, some others can
barely be distinguished from the background. Moreover, in
correlation with the number of DNA molecules observed
on the topographic image and with a fluorescence labelling
efficiency of 90%, we expected to detect many more R6G
dye molecules than the actual number detected. Both
findings as well as ‘noisiness’ on the majority of the
molecules were observed in most of the images. The next
section presents and discusses some statistical data over
these findings.
3.2. Dynamic behaviour of the fluorescent molecules
To gain understanding over the markedly different
behaviour of R6G on DNA as compared to more stable
dyes like carbocyanine (DiI), we have imaged many
molecules and extracted statistical information. Figure 4
shows the signal decay due to photobleaching for DiI
molecules embedded in PMMA (a), R6G in PMMA (b),
and R6G adsorbed on glass (c). The experiments were
conducted using the same NSOM probe and the same
excitation conditions (well below molecule saturation).
The data were taken from consecutive 3 × 3 µm2
(200 × 200 pixels) fluorescence images over the same
sample surface and counting the number of molecules
Near-field optical microscopy for DNA studies at the single molecular level
Figure 3. Shear-force image (a) and near-field fluorescence image (b) of 1 kbp (340 nm length) DNA fragments labelled with R6G.
The scan size is 1.5 × 1.5 µm2 , with 200 × 200 pixels and an integration time of 10 ms/pixel. The sample consists of one fluorophore
per strand. The shear-force image shows well resolved DNA strands with a resolution comparable to SFM techniques. The near-field
fluorescence image shows the intensity of individual R6G dyes. The optical resolution (FWHM) is 70 nm. Note the variation of
intensity for different dyes. The maximum signal is 240 counts/pixel. The background is 40 counts/pixel.
present on each image. Taking 10 × 10 pixels for
the typical emission spot of each molecule (tip diameter
∼150 nm), and an excitation time of 10 ms per pixel,
each molecule is excited for one second in each image.
Thus, we plot the number of molecules present in
each image as a function of the illumination time per
molecule.
Figure 4(a) shows slow and gradual bleaching for
DiI molecules embedded in PMMA. In fact, after 4 s
of illumination only nine out of 59 molecules have been
bleached. This is in contrast to what is observed for R6G
dyes in the same environment, i.e. PMMA. As depicted
in figure 4(b), 44% of the molecules are bleached after
1 s of illumination. More gradual bleaching is observed
in seconds 2 and 3. Similar behaviour is observed for
the same molecule R6G, but now adsorbed on glass, as
shown in figure 4(c). In fact, during the first second of
illumination approximately 30% of the molecules display
discrete photobleaching and emit fluorescence during the
first three or four line scans only.
After 1 s of
illumination approximately 50% of the molecules have
been bleached. A slower and more gradual bleaching
rate is observed for seconds 2 to 4. We have not been
able to collect enough statistical data over the R6G-DNA
samples, mainly because instrumentation adjustments are
necessary during the first few scannings. Nevertheless,
correlating the topographic images of DNA and taking
into account 90% fluorescence labelling efficiency, we
normally observed only half of the molecules that are
expected to be present on the surface, consistent with
the results found in PMMA and glass.
Similarly,
subsequent imaging can be performed with slow bleaching
rates.
A rough estimation of the number of photons emitted
by a molecule during 1 s of illumination can be obtained
by adding all the counts in each pixel. An average
value is obtained by adding the counts of all molecules
and dividing by the total number of molecules in one
image. Taking into account a collection efficiency of
0.1, we obtain an emission of ∼3 × 105 photons per
molecule per second in the case of R6G in glass.
Based on the observation that approximately 50% of the
R6G molecules photobleach after 1 s of illumination,
we then estimate a photobleaching rate of ∼10−5 . In
the case of DiI molecules we have measured a similar
number of emitted photons per second of illumination,
i.e. of ∼3 × 105 , but contrary to the R6G dyes, DiI
molecules in PMMA live many more photocycles before
photodissociation (emission > 106 photons, as estimated
from our images).
The distinct behaviour of DiI and R6G molecules is
clearly displayed in figure 5. Both images are 200 × 200
pixels, with an integration time of 10 ms/pixel. Figure 5(a)
is a 3 × 3 µm2 scan area of DiI molecules embedded in
PMMA. The aperture diameter is approximately 120 nm.
The orientation of the excitation polarization is along 45◦ ,
with polarization characteristics of the near-field probe
greater than 1:13 for all directions when viewed in the farfield. Figure 5(b) is a 1.5 × 1.5 µm2 scan area of R6G
molecules attached to DNA as deposited on mica. The
image has been generated with a 70 nm aperture probe.
The orientation excitation polarization is not exactly known
due to the birefringence effect of mica. Nevertheless,
we have adjusted the incoming polarization such that an
equal amount of signal is obtained in transmission on both
APD detectors. The diameters of the fluorescent spots
47
M F Garcia-Parajo et al
Figure 4. Signal decay due to photobleaching for (a) DiI
molecules embedded in PMMA, (b) R6G in PMMA and (c) R6G
adsorbed on glass. Measurements were performed with the same
NSOM probe and the same excitation conditions (well below
molecule saturation). Plots have been generated by counting the
number of molecules present in each 3 × 3 µm2 image. Each
molecule is excited for 1 s in each image. Approximately 50%
of the R6G molecules photobleach after 1 s of illumination. The
photobleaching rate is ∼10−5 for R6G. DiI molecules in PMMA
show more gradual and slow photobleaching behaviour.
48
correspond to the convolution of the molecule fluorescent
intensity with the near-field probe profile. Each spot
is approximately 10 × 10 pixels. Each image consists
of two data sets, one for each fluorescence polarization
direction. The data have been colour coded, red for the
0◦ detector and green for the 90◦ detector, and added
up to create one image. As a result, the colour of each
image reflects the relative contribution of each polarization
component (i.e. equal amounts of red and green give
yellow) while the brightness reflects the total fluorescence
intensity.
Two main observations can be drawn from these
images. First, the different but defined in-plane orientation
of individual DiI molecules when compared to R6G
molecules. Figure 5(a) shows individual DiI molecules
with significantly different in-plane orientations. As an
example, three molecules have been outlined in the image:
one with an orientation close to 0◦ (red molecule), one
with an orientation close to 90◦ (green molecule) and
finally one with an in-plane orientation close to 45◦ . Also,
stationary dipoles are observed for most of the molecules,
evidenced by the uniform colour within one fluorescent
spot. In contrast, most of the molecules in figure 5(b)
display an overall yellow scale colour. Individual green
and red pixels are recognized within one fluorescent spot
of 70 nm FWHM. A second observation is the quite
stable and constant intensity emission of DiI molecules
compared to the noisy and stripy behaviour of R6G dyes. In
figure 5(b) we have outlined four different noisy molecules.
Close inspection of the image reveals, however, that many
molecules exhibit this behaviour.
We next try to obtain some insight into the dynamics of
DiI and R6G molecules immobilized on different surfaces.
Figure 6 shows the total number of counts collected
for four different molecules ((a), (c), (e), (g)) and their
corresponding in-plane orientations ((b), (d), (f), (h))
as a function of sampling time. Figures 6(a) and (b)
correspond to a DiI molecule embedded in PMMA, while
figures 6(c)–(h) correspond to R6G molecules embedded
in PMMA ((c) and (d)), adsorbed on glass ((e) and (f))
and attached to DNA ((g) and (h)). The first three
molecules have been imaged with the same NSOM probe
(tip aperture diameter ∼150 nm). The last molecule
has been imaged with a ∼70 nm aperture NSOM probe.
The graphs have been constructed taking a vertical line
profile (perpendicular to the line scan) along the centre
of the fluorescent spot of the molecule. We have taken
between 14 and 22 pixels to describe a molecule. In
figures 6(a)–(f) the pixel size is 15 nm, and in (g) and
(h) the pixel size is 7.5 nm. The pixel time is 10 ms
in (a)–(f) and 100 ms in (g) and (h). The time interval
between pixels is 4 s. Despite heterogeneity of molecules
in different environments, the data shown in figure 6 are
quite representative of the behaviour exhibited by most
of the molecules analysed (a total of 49 DiI molecules
embedded in PMMA, 10 R6G molecules in PMMA, 73
Near-field optical microscopy for DNA studies at the single molecular level
Figure 5. Near-field fluorescence images of individual dye molecules. Each image is 200 × 200 pixels, and the integration time is
10 ms/pixel. (a) A 3 × 3 µm2 scan area of DiI molecules in PMMA; (b) a 1.5 × 1.5 µm2 scan area of R6G molecules attached to DNA
through a 6C linker. The data have been colour coded: red, 0◦ orientation (parallel to the x scan direction); green, 90◦ orientation;
yellow, equal fluorescent signal in both polarization channels. Different but defined in-plane orientation is observed for individual DiI
molecules as compared to R6G molecules. R6G molecules display more ‘stripy’ and noisy behaviour. Note that the FWHM of the
fluorescent spot in (b) is 70 nm.
R6G molecules on glass and 32 R6G molecules attached to
DNA).
Several observations can be drawn from these data.
Stable spatial positioning of DiI molecules is evident
from figure 6(a), where the molecule nicely maps the
intensity profile of the NSOM probe, with a diameter
of 150 nm at FWHM. The R6G molecules embedded
in PMMA and attached to DNA are also mapping the
Gaussian profile of the NSOM probe, as shown in plots
(c) and (g), respectively. This is certainly not the case
for the R6G molecule adsorbed in glass (plot (e)). There
a much ‘flatter’ and abrupt profile is obtained. Different
behaviour is also observed when looking at the individual
intensity contributions per pixel. While (a) shows intensity
fluctuations within the NSOM probe profile of less than
10%, much larger intensity fluctuations are observed in
plots (c), (e) and (g). The total intensity per pixel seems to
vary up to three times below or above the value given by
the convolution of the NSOM probe profile.
Plots on the in-plane angle also show some differences
between molecules and their surroundings. Together with
stable lateral positioning and stable intensity emission, the
DiI molecule in plot (b) also has a stable angle orientation
of 25.5◦ ± 3.5◦ . Despite intensity fluctuations of R6G
in PMMA, its in-plane angle orientation (plot (d)) also
seems rather stable with a value of 61.5◦ ± 6.5◦ . Plots
(f) and (h) give values close to 45◦ . It is important to note
that while DiI and R6G molecules embedded in PMMA
show a clear defined angle which is away from 45◦ , all
of the investigated R6G molecules adsorbed in glass give
values around 45◦ , i.e. equal number of counts in both
polarization channels. In the case of R6G attached to DNA
a similar behaviour occurs, although occasionally a slight
preference in one channel was observed. An additional
effect is induced by the well known retarding effect of the
mica substrate. This retardation influences the degree of
polarization of the emitted light. As a result, it is hard
to draw conclusions on the in-plane orientation and any
fluctuation based on the present data.
4. Discussion
4.1. Shear-force and near-field fluorescence imaging of
DNA
We have shown in figures 2 and 3 that DNA imaging can be
readily performed using the shear-force modality of NSOM,
together with single molecule fluorescence sensitivity. As
mentioned in the introduction, in order to consider NSOM
as a useful instrument for fundamental studies at the
biomolecular level, the influence of the tip over the sample
should be minimized. The NSOM probe might influence
the sample in two possible ways: by inducing sample
deformation due to interaction forces during shear force
imaging, and by inducing sample photodamage during
NSOM imaging. We have calculated the interaction forces
during imaging and compared with literature. We conclude
that values below 10 pN will hardly affect the sample
characteristics. However, interaction forces will grow
inversely proportional to the tuning fork quality factor
Q. Additionally, for small interaction forces the tip
displacement should be kept as small as possible. It is
therefore important to keep the Q factor of the fork as high
as possible, and to optimize the pre- and post-amplification
49
M F Garcia-Parajo et al
Figure 6. Total emitted intensity ((a), (c), (e), (g)) and in-plane angle orientation ((b), (d), (f), (h)) of four different dye molecules:
(a), (b) a DiI molecule in PMMA; (c), (d) R6G in PMMA; (e), (f) R6G on glass; (g), (h) R6G on DNA as a function of sampling time.
Graphs have been obtained taking a vertical line profile (perpendicular to the line scan) along the centre of the fluorescent spot. The
integration time per data point is 10 ms (100 ms in (g) and (h)). The time interval between points is 4 s (see text for a discussion of the
different plots).
stages of the tuning fork signal for maximum signal-tonoise ratio. In our case we have shown that the detection
limit of our electronics is close to the thermal noise of the
fork. Other workers have also imaged DNA [28] and liquid
crystal films at the liquid–vapour interface [25] using shearforce microscopy. In the latter case, even the movement
of the liquid-crystal monolayers could be followed in the
topography as a function of time. It is clear that shearforce microscopy is an extremely useful technique in its
own right for the study of soft materials and/or samples
that are loosely bound to the surface. The latter is actually
a requirement for the study of DNA–protein interactions
where steric hindrance by the surface might affect the
biochemical reactions [8].
The fluorescence image shown in figure 3(b) shows a
high optical resolution of 70 nm at FWHM, well beyond
the optical diffraction limit. To our knowledge, this is
the smallest single molecule fluorescence imaging spot
reported. Most near-field single molecule fluorescence
images have been generated with 100 nm apertures or
larger. Because of the small excitation volume, the surface
sample density can be much larger which is of advantage
in the study of real biological systems. Moreover, the
50
fluorophore can be localized with an accuracy of a few
nanometres. The differences in intensity obtained for
individual fluorophores reflect the heterogeneity of the
dyes and the interaction with their immediate surroundings
[10, 14]. Different in- and out-plane orientations also lead
to different excitation efficiencies and therefore different
emission rates per molecule [16, 29].
4.2. Photodynamics of individual molecules
We have followed the behaviour of a large number of
R6G dyes and DiI dyes at different conditions. Statistics
over our data show that R6G and DiI molecules emit
a similar number of photons per second, under equal
excitation conditions. However, approximately 50% of
R6G molecules photodissociate after emission of ∼105
photons, while DiI molecules exhibit a much longer
lifetime.
Furthermore, the results indicate that the
photobleaching behaviour of R6G does not depend on the
surface to which the molecules have been immobilized, but
it is rather a photophysical property of the dye itself. While
we do not have enough data to propose a specific model
for the behaviour of R6G, the bi-exponential bleaching
Near-field optical microscopy for DNA studies at the single molecular level
Figure 6. (Continued)
rate behaviour of R6G (and in general of most rhodamine
dyes) is rather well known [30] and observed on molecules
immobilized on different substrates. Our results agree with
these observations.
Results obtained on DiI molecules show the slow
dynamics of these dyes when embedded in polymer
matrices. Within the timescale of our measurements we
only observed two out of 49 molecules that exhibit some
rotational activity. We have recently shown that the dipole
orientation of DiI molecules can be determined within a
few degrees of accuracy with our NSOM configuration, and
that stationary dipoles are obtained for periods of up to 1 h
[16]. The results on R6G are more difficult to interpret. Our
data suggest that lateral position and in-plane orientation are
reasonably stable when R6G molecules are embedded in a
polymer matrix, like PMMA. On the other hand, the ‘stripy’
aspect in most of our R6G–glass images, combined with the
total intensity and angular orientation data presented here,
indicate that the molecules do not sit fixed on the surface
but move much faster than the timescale of our experiment.
Because the adsorption of R6G on glass is through OH
groups, it is reasonable to expect a large dependence of
R6G adsorption/desorption upon humidity conditions. This
fact might explain why some workers have reported fixed
absorption polarization and fixed spatial positioning for
rhodamines adsorbed on glass [10, 11] while some others
observed that R6G molecules adsorbed on glass might have
diffusion coefficients similar to the liquid situation [31]. If
lateral diffusion occurs faster than our observation times,
the same is expected with rotational diffusion. Within
10 ms of integration time a random orientation will be
obtained, resulting in unpolarized emission. At present
we are conducting experiments where sample preparation
(hydrophilicity of the glass surface) and relative humidity
conditions are being carefully controlled. As expected,
R6G attached to DNA shows a stable lateral position
within a few nanometres. The dye shows strong intensity
fluctuations, comparable to the situation in the polymer and
on glass. So far, we do not have enough data for definite
conclusions about the behaviour of R6G when attached to
DNA and the possible influence of the linker on the dye
dynamics.
5. Conclusions
We have demonstrated the usefulness of NSOM for
dedicated studies where high spatial resolution combined
with ultra high sensitivity are required. Lateral and vertical
resolution is comparable to standard SFM techniques while
51
M F Garcia-Parajo et al
negligible damage is produced on the sample. Near-field
fluorescence imaging of individual fluorophores shows an
optical resolution of 70 nm at FWHM. Taking into account
current biochemical techniques, specific sequences and/or
genes can be easily tagged with one or more fluorophores.
Co-localization studies are readily possible where, in
principle, single base pair resolution can be obtained.
Simultaneous topography and fluorescence mapping at the
molecular scale uniquely afforded by this technique will
lead to new diagnosis and analytical methods.
In a first approach to analyse the dynamics of a
single fluorophore linked to DNA we have presented some
statistical information on R6G dye molecules in different
surroundings and compared to DiI molecules embedded
in a thin polymer film. A direct conclusion from our
data is the different photophysical properties of R6G
molecules as compared to DiI: bleaching rates and intensity
fluctuations are much larger in R6G than in DiI. These
are mainly properties of the dye itself and do not depend
on the particular environment. The environment clearly
influences the mobility of the dye, both lateral and rotational
diffusion. Systematic investigation of the (co)-localization
and photodynamics of the DNA–dye system is the subject
of current research.
Acknowledgments
The authors would like to thank K Karrai for fruitful
discussions on shear-force detection and interactions forces,
and T Ruiter, K van der Werf, F Segerink, W Rensen,
O Willemsen and L Kuipers for their assistance and useful
suggestions. MFG-P is partly financed by the European
HCM network on ‘Near-field Optics and Nanoscale Science
and Technology’. J-AV and SJTvN are supported by the
Dutch Foundation for Fundamental Research (FOM).
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