Application Note
Visualisation, Sizing and Counting of Fluorescent and
Fluorescently-Labelled Nanoparticles
Introduction
Fluorescent molecules have long been used to specifically
label particular structures and features in complex mixtures
or matrices to allow their presence and spatial distribution to
be determined. Historically based on organic molecules, a
very wide range of such fluorophores have been developed,
the selection of which is determined by the application
required.
Background
Fluorescent molecules, depending on their structure and
properties, exhibit specific excitation and emission spectra,
have different solubilities and stabilities in different chemical/
solvent environments and possess varying quantum
efficiencies and resistance to photobleaching. Their ability to
be discriminated from non-labelled background through the
use of selective optical filters allows the user to identify and
quantify almost any type of structure. This is the case
whether mediated by antibody, nucleic acid fragment or
other structures with a specificity for, and an ability to bind to
a target analyte, or if used as a direct fluorescent ‘stain’ with
a direct affinity for lipids, sugars, proteins, etc. Recently, a
new class of luminescent semiconductor nanocrystal, called a
quantum dot, which exhibits significantly enhanced
luminescence, chemical stability and resistance to photobleaching has become available. Fluorophores are thus found
to be used throughout the bioanalytical sciences.
In the example, a mixture of 100nm fluorescent
(Fluoresbrite™, PolySciences Inc.) and 400nm non-fluorescent
calibration polystyrene particles was measured under
scattered light (Figure 1A) and through an optical fluorescence
filter (Figure 1B). Under scattered light, both fluorescent and
non-fluorescent particles were observed, sized and counted,
while under the fluorescence filter only the 100nm fluorescent
particles could be visualised.
Note that it was also possible to retain concentration
information on the fluorescently labelled nanoparticles for
comparative labelling efficiency purposes.
A
NanoSight’s Fluorescent Options
NanoSight’s fluorescence versions of the instrument range
allow fluorescent nanoparticles to be individually tracked in
real-time from which labelled particle size and concentration
can be determined. Under light scatter mode, the total
number of particles can be measured and subsequently
compared to the concentration of labelled particles when
measuring in fluorescence mode.
B
NanoSight Example 1 — Polystyrene beads,
size discrimination
The 405nm laser can be used to excite fluorescently loaded
polystyrene beads (which excite at 441nm and emit at
486nm).
Figure 1: Particle Size Distribution profiles (yellow graph) of a
mixture of 100nm fluorescent and 400nm non-fluorescent
polystyrene particles analysed under A) scatter mode and B)
fluorescent (optically filtered) mode.
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Application Note
NanoSight Example 2 — Polystyrene beads,
fluorescence discrimination
NanoSight’s fluorescent versions of their instrument range
allow not only fluorescent nanoparticles to be individually sized
in real-time but also for them to be counted at the same time.
Particle Concentration
In the following example an approx. 50:50 mixture of
fluorescently labelled (Fluoresbrite) 100nm and unlabelled
100nm polystyrene beads were analysed under light scatter
mode (red line and top image) and when fluorescently filtered
(white line and bottom image).
NanoSight Example 3— Detection of
Individual Quantum Dots (QDots®)
Semiconductor nanocrystals have recently emerged as a
powerful and attractive alternative to conventional
fluorescent labels due to their great chemical and optical
stability and ease of use. Now commercially available as
pre-functionalised kits with a choice of emission
wavelengths, these interesting materials are rapidly
gaining
in popularity in the biosciences. While
conventionally restricted to being imaged when
immobilised (i.e. visualised by long exposure microscopy
or when used to multiply label larger structures (e.g.
Cellular structures)), NanoSight’s fluorescent versions of
their LM Series instrument allow, for the first time,
quantum dots to be visualised, sized and counted when
unbound and moving freely under Brownian motion in
liquids.
The following example is an analysis of a suspension of
Invitrogen’s
non-functionalised
QD655
QDot®
nanocrystals in an aqueous buffer. Excited by NanoSight’s
405nm (blue) laser and detected through a suitable filter,
these 655nm emitting QDot® structures are visualized,
sized and counted on an individual basis in less than 60
seconds.
Particle Size
Figure 2. 50:50 mixture of 100nm unlabelled polystyrene beads and
fluorescently labelled 100nm beads shown in scatter mode (red
line) and fluorescent mode (white line).
Mixture
Mixture of 100 nm fluorescence
and 100 nm non-fluorescence
polystyrene nanoparticles
Mode
Under scatter
light
Under optical
filter
(fluorescence)
Particle size [nm]
100
98
Concentration
[*108particles/ml]
8.88
3.44
10-12nm uncoated QDot®
Biomolecule
Polymer
Figure 3. Table showing difference in concentration measured for a
50:50 mixture of 100nm unlabelled polystyrene beads and 100nm fluorescently labelled beads when analysed under light scatter mode and
fluorescence mode. The sizes remain the same, but the number of
particles seen when only the fluorescently- labelled part of the population is observed through the filter, fall as expected.
Shell
Core
15-20nm biomolecule-coated QDot®
Figure 4. Analysis of non-functionalised QD655 QDot®
nanocrystals in fluorescence mode.
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Application Note
Particle Concentration
Note also there is slight evidence of the presence of nonfunctionalised QDots® at 14nm. The veracity of this peak
would need to be confirmed with further analysis.
Particle Size
Figure 5. Analysis of functionalised QDot® sample.
The differences seen can be explained by the presence of
lipid vesicles in the plasma which are of a similar size to
the cellular vesicles but are not labelled with the cell
tracker peptide. This gives a higher concentration measurement under light scatter mode because all of the particles can be seen, including those that have not been
labelled. Once the fluorescence filter is inserted only
those that have been labelled with the QDOT conjugated
cell tracker can be visualised and analysed, giving a much
lower concentration measurement in fluorescence mode
than scatter mode. After ultracentrifugation, the nonlabelled lipid vesicles are not pelleted with the cellular
vesicles and are removed from the sample. This results in
similar concentration measurements being obtained in
both scatter and fluorescence mode.
Vesicles x 108 /mL
The following plot is of a functionalised QDot® sample in which
initial particle interaction with binding ligands in the sample is
evident. Note the mode of the smaller peak is compatible with
the dimensions of a protein coated QDot® but aggregates
(multimers) are also appearing.
NanoSight Example 4— Fluorescently
Labelled Sub-Micron Biological Structures
This is an example of a plasma sample, containing cellular
vesicles labelled with a cell tracker peptide which is conjugated
to quantum dots. In both A and B of figure 6, the blue line
shows the sample as analysed under light scatter mode and
the red line shows the sample as analysed under fluorescence
mode. Part A shows the sample prior to ultracentrifugation.
Note that the concentration of labelled particles seen under
fluorescence mode is much lower than that measured under
light scatter mode. In part B, the same sample after
ultracentrifugation, a very similar concentration is seen in both
light scatter and fluorescence mode.
Figure 6. Image taken from [1]. Fluorescence analysis of cellular
vesicles in plasma. A) Plasma labelled with cell tracker peptide and
analysed in light scatter (Blue line) and fluorescence (red line)
modes. B) Same sample after ultracentrifugation under light scatter (blue line) and fluorescence (red line) modes.
Size nm
Figure 7. Image taken from [1]. Fluorescently labelled cellular
vesicles in light scatter (blue line) mode and fluorescence mode
with correct antibody (red line) and isotype control antibody
(green line).
Figure 7 shows a sample of clinically significant cellular
vesicles that were labelled with a quantum dot conjugated antibody raised against a molecular antigen on the
microvesicle of interest. The plot shows the quantum
dot labelled vesicles under light scatter (blue line) and
fluorescence (red line) modes. The green line shows the
same sample labelled with an isotype control antibody
(one that has no affinity for the molecular antigen of interest) and analysed in fluorescence mode. The very low
concentration of particles seen when the isotype control
antibody is used shows the labelling specificity of the
antibody.
[1] Rebecca A. Dragovic, Christopher Gardiner, Alexandra S. Brooks,
Dionne S. Tannetta, David J.P. Ferguson, Patrick Hole, Bob Carr, Christopher W.G. Redman, Adrian L. Harris, Peter J. Dobson, Paul Harrison, Ian
L. Sargent 2011. Sizing and phenotyping of cellular vesicles using
Nanoparticle Tracking Analysis. Nanomedicine: Nanotechnology, Biology
and Medicine, 7(6), pp. Pages 780-788.
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Application Note
Addressing Photobleaching
Photobleaching occurs when a fluorophore permanently loses
the ability to fluoresce due to photon-induced chemical
damage or covalent modification. Photobleaching is not well
characterised, but can result in a dramatic loss in the
fluorescence emission intensity and subsequently reduces the
ability to perform accurate sample analysis with the NanoSight
system.
NanoSight have developed two solutions to overcome the
problem of photobleaching during sample analysis and further
increase the accuracy of particle counting and sizing when
operating in fluorescence mode.
Solution 1 - Synchronisation Cable
A synchronisation cable is used to pulse the laser in time with
the camera shutter, thus limiting the exposure time of the
fluorophore to the illumination source and slowing down the
process of photobleaching. Figure 8 shows how use of the
synchronisation cable can improve fluorescence concentration
measurements when capturing for longer time periods,
improving the accuracy of these measurements.
A
When no synchronisation cable is used, there is a
substantial reduction in measured concentration of the
same sample at 60 seconds compared to 30 seconds, and
this is even further reduced for a 90 second capture. When
comparing this to results obtained with the synchronisation
cable attached, the concentration measurement remains
constant from a 30—60 second capture time. Although this
is reduced for a 90 second capture, the percentage of
fluorescence signal obtained (compared to scatter as 100%)
is still higher with the synchronisation cable than without.
Solution 2 - Syringe Pump
To further improve the accuracy of fluorescence
measurements a syringe pump can be integrated into all
systems of the NanoSight instrument range to enable the
user to continuously introduce a constant flow of fresh
sample into the viewing area. The flow speed is user
controlled and can be adjusted to suit the bleach rate of a
particular fluorophore. For example, when viewing a
sample labelled with a fluorophore that bleaches very
quickly, the flow rate can be increased to minimise the
amount of time that the labelled particles are exposed to
the beam. This enables longer capture durations to be used
whilst retaining the same level of fluorescence signal
detection.
Particles X 108/mL
Figure 9. The syringe pump
connected to the NanoSight
LM14.
B
Figure 8. Comparison of Scatter and fluorescence concentration
measurements. A) Shows an average concentration count in
scatter and fluorescence mode, with and without the synchronisation cable for 30, 60 and 90 second capture durations. B)
Shows the percentage of fluorescence signal detected when
compared to scatter mode as 100%.
Figure 10. Shows a screen shot of particles under flow being
tracked by NanoSight NTA software. The automatic drift correction allows particles to be tracked and sized correctly
whilst moving at a steady flow.
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Application Note
NanoSight Example 5— Fluorescently
Labelled Cellular Vesicles Using Syringe Pump
430nm Longpass
Filter- Cut-on: 430+/-3nm
Trans: >/=90% avg
80%min 440-750nm
Block: >/=OD4 @ 420nm
Size: 22 +0/-0.25mm
Thick: </=3.5mm
In this example, a sample of sub micron cellular vesicles
labelled with a fluorescent protein were analysed. The
fluorescent signal bleached within 3-5 seconds which did not
allow a long enough capture time to calculate a statistically
accurate particle size distribution with the NTA software. In
this example it was necessary to use the syringe pump to flow
the sample through the chamber. This supplied a constant
supply of fresh, non-bleached sample into the chamber,
allowing a 90 second capture time to be used so that a more
accurate analysis of this sample could be carried out.
500nm Longpass
Filter- Cut-on: 500 +/-3nm
Trans: >/=90% avg
80%min 504 -750nm
Block: >/=OD4 @ 490nm
Size: 22 +0/-0.25mm
Thick: </=3.5mm
Figure 11 shows the sample under scatter (red line) and
fluorescence (purple line) modes. The image in the top right
hand corner is a screen shot of the sample under flow in
fluorescence mode during analysis. Use of the syringe pump
allowed analysis of a sample that would have otherwise
bleached too quickly to capture a long enough video to build
up an accurate particle size distribution. The slight difference
in concentration seen in scatter mode and fluorescence mode
is probably due to contaminants present in the sample which
were not labelled with the fluorescent protein and were
therefore not included in the analysis of the sample in
fluorescence mode.
565nm Longpass
Filter- Cut-on: 565 +/-3nm
Trans: >/=90% avg
80%min 570 -750nm
Block: >/=OD4 @ 550nm
Size: 22 +0/-0.25mm
Thick: </=3.5mm
650nm Longpass
Filter- Cut-on: 650 +/-3nm
Trans: >/=90% avg
80%min 654 -750nm
Block: >/=OD4 @ 640nm
Size: 22 +0/-0.25mm
Thick: </=3.5mm
Scatter mode
Fluorescence mode
*Contact NanoSight for alternative filter sets.
Contact Details
For further information, contact NanoSight or your local
distributor, listed at www.nanosight.com
Key Features Summary
Figure 11. Plot of scatter (red line) versus fluorescence (purple line)
mode. Image in top right shows a screen shot of the same sample under
flow using the syringe pump during analysis.
NanoSight Options—Lasers and Filters
The NanoSight system uses either a 405nm (violet), 488nm
(blue), 532nm (green) or 638nm (red) laser source to excite
suitable fluorophores whose fluorescence can then be
determined using matched 430nm, 500nm, 565nm or 650nm
long-pass filters respectively.

Particles can be measured, sized and counted under
two modes: scattered light and optical filter
(fluorescence)

Small sample volume required

Low cost of instrument

Visualisation of individual fluorescent particles

Addition of synchronisation cable and syringe pump
improve analysis of fluorescently labelled particles
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