The mechanism of two photon luminescence is similar to single photon luminescence in that it is due to the electron-hole pair recombination. In the case of gold nanoparticles, the electron-hole pair is formed when the electrons are excited from the d
-level to s-p
level. In general, the two photon luminescence signal is weaker than single photon luminescence but can be enhanced by several orders of magnitude through increased field enhancement effect. Multiphoton absorption cross section is higher when these noble metal structures have a degree of asymmetry associated with them in their structure. It has been reported that luminescence efficiency of these particles is size dependent and is higher from larger particles which have a higher degree of asymmetry associated with them. However, luminescence efficiency can be improved from smaller nanoparticles by exploiting the field enhancement effect. This phenomenon is stronger at the edges and intersection of two or more particles. Therefore, by having a higher concentration of small-sized particles, luminescence intensity or efficiency can be significantly improved. Multiphoton luminescence has also been observed from gold nanoparticles of different shapes such as nanorods and nanostars. Recently, multiphoton luminescence from gold nanosystems has been exploited as a biological imaging modality (Wang et al 2005
In , a 10 μl drop of a 10 nm gold nanoparticles suspension was placed on a coverslip, and drying was facilitated using a “smear” technique. The incident excitation wavelength was 790 nm which gave the best signal to noise ratio. The image is a representative slice (10 fr/ave.) of a 10, 1 μm step z-stack. The bright spots correspond to luminescence from a cluster of gold NPs. The optical resolution of the light microscope limits the detection of luminescence from individual gold NPs and is therefore detected only from clusters of NPs. is a corresponding transmission electron micrograph (TEM) of gold NPs on a copper TEM grid. The individual gold NPs are 10 nm in size and surrounded by stabilizer molecules. However, during the sample preparation step while drying, gold NPs tend to form aggregates.
Photoluminescence and structural characterization of gold nanoparticles. (a) Multi photon absorption induced luminescence from gold nanoparticles dried on a cover slip and (b) Transmission electron microscope (TEM) image of 10 nm gold NPs.
The internalization of nanoparticles, through endocytosis, is an important process that has great potential in several areas including drug and gene delivery, particle tracking, and metabolic characterization. The method allows transport of small particles and other biomolecules across the cell membrane into the cells and can be made cell-specific by exploiting the receptors on the cell surface to induce receptor-mediated endocytosis. A gold nanoparticle-based system using a heterobifunctional PEG spacer was used for intracellular tracking and delivery in model cancer cell lines (Shenoy et al 2006
). Using thiol chemistry, the surface of these gold nanoparticles was readily modified to attach Coumarin and their internalization was monitored through fluorescence microscopy. Recently, gold nanoparticles have been synthesized to achieve organ-specific localization (Kannan et al 2006
). In the work reported here, gold NPs were internalized and subsequent two photon luminescence was observed from two different cell types – Dictyostelium and mES cells.
Dictyostelium was selected since this is a model organism used for the visualization of binding, internalization, and metabolic breakdown of various substrates including tungsten beads, fluorescent latex beads, and externally and internally labeled bacteria (Vogel et al 1980
; Cornillon et al 2000
; Maselli et al 2002
; Montet et al 2006
). The kinetics of these procedures is well characterized. Vegetative Dictyostelium cells in suspension were exposed to Au-NPs for 60 minutes on a rotary shaker to facilitate internalization. Dictyostelium cells rapidly bind and internalize Au-NPs. When excited with 80-fs pulses at a repetition rate of 80 MHz with a center wavelength of 790 nm, Au-NPs provides a bright internal bioluminescent signal that is not degradable in the phagolysosomal pathway, does not bleach, and does not blink.
shows the results of internalization after repeated washings, to visualize internalized particles in Dictyostelium. In the left brightfield image, aggregates of particles are clearly seen within the cell. The right two-photon panel is a maximum intensity z-projection of the acquired two-photon stack (using Image J) (Rasband 1997–2000). Previous studies have determined that the azide method of washing clearly releases bound particles, and allows only internalized particles to be visualized (Maselli 2002). In addition, further analysis revealed almost 80% of the cells internalized some amount of particle (data not shown). It is clear from this image that the presence of gold NPs does not have any adverse reaction to normal function of these Dictyostelium.
Internalization of Au-NPs in Dictyostelium. Brightfield image (left) and corresponding z-projection of the acquired 2-photon stack (right).
The process of phagocytosis is defined as the internalization of particles greater than 0.5 μm in diameter. Receptor-mediated endocytosis has been shown to be an effective method to label several different types of cells, including stem cells, tumor cells, and acinar cells of the pancreas (Gupta et al 2003
; Kim et al 2006
). Fluidic uptake of nutrients from the surrounding medium occurs via the process of pinocytosis. The Au NPs we used in this study are 10 nm in diameter. Therefore, by definition, the process of internalization must be considered either receptor-mediated endocytosis or pinocytosis.
Mouse Embryonic Stem Cells (mESCs) were incubated with gold NPs and were visualized, in vitro, via two-photon luminescence using an excitation wavelength of 790 nm. Luminescence was observed in both the mouse embryonic fibroblasts and undifferentiated mouse embryonic stem cells. To our knowledge, this is the first report of two-photon detection of gold nanoparticle uptake by mammalian stem cells. The cells were passaged every two days and medium replaced along with fresh gold NPs. It is necessary to have a high concentration of gold NPs in the medium to facilitate endocytosis. A similar method was reported in experiments for MRI tracking of stem cells using iron oxide NPs.
The embryonic stem cells grow as a multi-layered colony on top of a layer of nondividing embryonic fibroblast cells (flatter, larger cells) that act as a feeder layer for the stem cells. Since mESCs and fibroblast are both in the same medium gold NPs were internalized in both these cells. The images in were taken as a z-stack on multiple focal planes but only two focal planes from the stack are shown. Top images of focus on the fibroblast feeder layer, while the bottom images of focus on the stem cell colony and therefore, luminescence from the out of focus cells is not visible.
Figure 3 Gold nanoparticles detected via two photon luminescence in mouse embryonic stem cells and mouse embryonic fibroblast cells. In the top panels, the focal plane is on the fibroblast feeder layer, which readily endocytose the particles. A fainter, yet detectable (more ...)
The embryonic stem cells were cultured in the presence of gold nanoparticles for several passages and continued to divide and grow, indicating cell viability. The fibroblasts were labeled to a much greater extent than the mES cells, presumably due to greater endocytosis of the unlabeled gold nanoparticles. The gold nanoparticles are clearly visible within the fibroblast cells and are therefore not merely bound to their surface (). The luminescence signal by the gold nanoparticles within the fibroblast cells was bright in comparison to the signal detected within the embryonic stem cells.
Gold nanoparticles could also be detected via two-photon luminescence throughout the multi-layered embryonic stem cell colony, indicating cellular uptake of the particles by the embryonic stem cells. Within the z-stack images obtained (), the images in a higher focal plane corresponded to the multi-layer mouse embryonic stem cells, which grow as multi-layer colonies on top of the fibroblast feeder layer. Although we have not proven endocytosis as the method of internalization of the gold nanoparticles, it is likely that the gold nanoparticles were endocytosed by the mouse embryonic stem cells.
There are several advantages to using multi photon luminescence from gold NPs as an imaging tool. Embryonic stem cells labeled with gold NPs may allow in vitro tracking of these cells as they differentiate into more specialized cell types. For example, mESCs could be labeled with gold NPs and cultured in the presence of unlabeled cells (either different populations of embryonic stem cells or a different cell type). Using the capabilities of the Keck 3D Fusion microscope, growth rate and morphology of live, dividing cells could be analyzed and different cell types distinguished via two-photon detection of these gold NPs. Recent observations have shown that undifferentiated embryonic stem cells may have phagocytic characteristics and gene expression similar to macrophages (Charriere et al 2006
). Two-photon tracking of gold NPs uptake within embryonic stem cells may be useful in further analysis of this observation.