To demonstrate SPOT analysis, we synthesized two ~18 nm dual-reporting (magnetic resonance and optical imaging) silica coated iron oxide nanoparticles (SCION) containing Cy5.5 (SCION(Cy5.5)) or Alexa555 (SCION(Alexa555)) fluorophores. Previously, the size, surface charge, structure, and magnetic properties of these nanoparticles were characterized [5
However, quantum yield analysis of the SCION particles was not feasible due to the lack of suitable reference fluorophores and confounding optical effects such as scattering and absorbance of the iron oxide core. With a home-built SPOT instrument the number and properties of fluorophores encapsulated in individual SCIONs were determined and compared to free fluorophore. Fluorescence from particles was well above background and photobleaching was clearly visible as quantized steps (). SCION(Cy5.5) had a mean of 2.07 fluorophores/particle (s.d. = 1.32, n = 1324) whereas SCION(Alexa555) contained 1.31 fluorophores/particle (s.d. = 0.69, n = 2957). Note, these means correspond to the number of fluorophores per labeled particle, not the average number of fluorophores per particle over the entire sample. To understand the distribution of the number of fluorophores per nanoparticle (), we considered a simple model in which incorporation of fluorophores in the nanoparticle during synthesis is a random, non-cooperative process. Under these assumptions, the number of fluorophores per particle will follow a Poisson distribution [17
) is the probability of there being n
fluorophores incorporated in the particle and λ is the average number of incorporated fluorophores. The distributions of incorporated fluorophores were reasonably well fit by Poisson distributions for both particles (). However, control experiments that measured the bleaching of free fluorophores in solution revealed that the free fluorophores did not all bleach in a single step as expected. Rather, a distribution of bleaching steps was observed indicative of aggregation of free fluorophores in solution (). Aggregation of fluorophores is a well-known phenomenon of significant practical importance [18
]. The number distributions of free fluorophore aggregates were similar to those of the labeled particles suggesting that clusters of fluorophores, rather than individual fluorophores, were incorporated into the nanoparticles. In this scenario, the distribution of the number of incorporated fluorophores would be a Poisson distribution for the number of clusters incorporated and the number of fluorophores in each cluster would be given by the measured distribution for the free fluorophore. To account for the incorporation of clusters of fluorophores rather than individuals, we fit the nanoparticle bleaching step distributions with a Poisson distribution for the number of clusters (c) incorporated:
and a second Poisson distribution to describe the number of fluorophores in each incorporated cluster,
Since a closed form of this expression is not readily obtained, the fitting was done via simulations. First, the distribution of free fluorophore cluster sizes (Pcluster
) was fit with a Poisson distribution, which was assumed to be the same for the clusters incorporated into the nanoparticles. The distribution of the number of fluorophores in the nanoparticles was then simulated by assuming a Poisson distribution of incorporated fluorophore clusters, the size of which was drawn from the measured cluster size distribution. This process was repeated over a range of average clusters per particle (λcluster
) and the chi squared deviation between the simulated and experimental distributions was computed. The λcluster
value with minimal chi squared deviation was taken as the best fit for the average number of incorporated clusters. SCION(Cy5.5) and SCION(Alexa555) bleaching step distributions were well fit by this function, resulting in lower chi squared values than the single Poisson fits (Eq. (1)
, ). The average probabilities of fluorophore incorporation were somewhat lower for this fit than for the single Poisson fit.
The labeling efficiency is the probability that a particle will contain one or more clusters:
For SCION(Alexa555) and SCION(Cy5.5), the labeling efficiency obtained from the double Poisson fits were 22 ± 2% and 54 ± 2%, respectively. To experimentally confirm the calculated labeling efficiency, we directly compared the number of particles observed in fluorescence and phase contrast images of SCION(Cy5.5) (
). Both labeled and unlabeled particles were observed by phase contrast imaging, whereas only fluorescently labeled particles were observed by confocal fluorescence imaging. Using this method, 40 ± 5% of SCION(Cy5.5) particles (n = 382) were labeled with at least one fluorophore, which is in reasonable agreement with the labeling efficiency of 54 ± 2% determined from the double Poisson fit, and is far less than the 79 ± 2% labeling efficiency obtained from a single Poisson fit to the data (). Thus, statistical analysis of SPOT data as presented above can be used as a tool to measure the fluorophore labeling efficiency.
Fig. 3 Experimental confirmation of particle labeling efficiency. (a) Spinning disk confocal fluorescence image gives the number of nanoparticles with fluorophores. (b) Phase contrast image of the same field of view gives the total number of nanoparticles with (more ...)
Using SPOT, we observed that encapsulating fluorophores in SCION increased their brightness. The distribution of intensity steps of free fluorophore (
) versus encapsulated fluorophore () reveals that fluorophores in SCION silica shells were brighter than free fluorophores. The weighted mean photo-intensities for encapsulated Alexa555 were 3612, 2698, and 2199 respectively at excitation powers of 12, 6, and 3 mW. This corresponded to a 94.3%, 14.7%, and 6.6% increase over free fluorophore at the respective powers. A similar enhancement of brightness was noted by Burns et al
] who used fluorescence correlation
Fig. 4 Photophysical properties of free and encapsulated Alexa555. (a) Distribution of fluorophore intensity drops for free fluorophore and (b) for SCION particles from individual intensity traces (). (c) Fluorescence decays and fits for free fluorophore (more ...)
spectroscopy to study silica particles containing Cy5. To further understand the origin of this brightness increase we measured the fluorescence lifetimes (). The number of excited fluorophores, F, decay by:
where the fluorescence lifetime, τ, is the inverse of the sum of the rates of individual decay pathways, ki
. In the case of free Alexa555, a single fluorescence lifetime of 228 ps was measured, whereas encapsulated fluorophores decayed with a fluorescence lifetime of 228 ps, as well as through a second pathway with a lifetime of 1.5 ns (). Decrease [21
] and increase [22
] in emission intensity from fluorophores incorporated in silica have been reported, but were associated with a decrease of the fluorescence lifetime. Therefore, the observation of increased brightness of incorporated fluorophores with the simultaneous appearance of an additional 1.5 ns-long decay pathway is interesting. Further studies are required to verify the origin of the second decay pathway.
We further observed that encapsulating fluorophore improved photostability as evidenced by increased photobleaching times. The distributions of photobleaching times for Alexa555 at excitation powers of 3-12 mW were well fit by exponential curves (), as expected for an uncorrelated Poisson process. The average photobleaching time of SCION(Alexa555) encapsulated fluorophores was greater than three-fold longer than the bleaching time of free Alexa fluorophore (). It is possible that photo-oxidation is reduced by encasing fluorophore in silica as there is a decreased amount of free oxygen. When molecular oxygen quenches a fluorophore’s dark triplet excited state, highly reactive singlet oxygen is produced that can react with and bleach the fluorophore. Thus, reducing the oxygen concentration prolongs fluorophore photobleaching time [23
]. Silica is porous; hence, a non-porous coating material may further enhance the photostability of the encapsulated fluorophore.