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. 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. 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 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. Page 1
Particle Concentration 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. 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 Mode Mixture of 100 nm fluorescence and 100 nm non-fluorescence polystyrene nanoparticles Under scatter light Under optical filter (fluorescence) Particle size [nm] 100 98 Concentration [*10 8 particles/ml] 8.88 3.44 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. 10-12nm uncoated QDot Biomolecule Polymer Shell Core 15-20nm biomolecule-coated QDot Figure 4. Analysis of non-functionalised QD655 QDot nanocrystals in fluorescence mode. Page 2
Vesicles x 10 8 /ml Particle Concentration 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. 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. 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. Particle Size Figure 5. Analysis of functionalised QDot sample. 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. 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. 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. [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. Page 3
Particles X 10 8 /ml 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. 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. Page 4
NanoSight Example 5 Fluorescently Labelled Cellular Vesicles Using Syringe Pump 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. 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. Scatter mode Fluorescence mode 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. 430nm Longpass Filter- Cut-on: 430+/-3nm 80%min 440-750nm Block: >/=OD4 @ 420nm 500nm Longpass *Contact NanoSight for alternative filter sets. Filter- Cut-on: 500 +/-3nm 80%min 504-750nm Block: >/=OD4 @ 490nm 565nm Longpass Filter- Cut-on: 565 +/-3nm 80%min 570-750nm Block: >/=OD4 @ 550nm 650nm Longpass Filter- Cut-on: 650 +/-3nm 80%min 654-750nm Block: >/=OD4 @ 640nm Contact Details For further information, contact NanoSight or your local distributor, listed at www.nanosight.com Key Features Summary 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 Page 5