Metrology of Nanoscale Biophotonic Materials

Optical Metrology of Single Qdots

Since semiconductor nanocrystals (quantum dots or Qdots) were introduced in the early 1990s, researchers have found that Qdots posses a number of unique fluorescent properties that open novel ways of using them in various biological applications. Compelling advantages of Qdots over traditional organic dyes include long photostability, broad spectral coverage, easy excitation, and suitability for multiplexed sensing of nano-environment in systems. In addition, proper surface functionalization allows the production of quantum dots that are water soluble and bioconjugation-ready enabling their employment in the biological sciences.

The fluorescent properties of Qdots have been observed to be strongly dependent upon their immediate nano-environment, which can induce spectral shifts, blinking, and intensity variations. For instance, the fluorescence intermittency phenomenon associated with single quantum dots is thought to occur due to the separation/recombination kinetics of an exciton upon absorption. Upon laser excitation, a charge carrier (an electron or a hole), is ejected from the quantum dot core via pure tunneling or Auger-assisted ejection to a trap state at the quantum dot surface or surrounding environment resulting in an ionized quantum dot. Switching from dark to bright occurs through recombination of excitonic charge carriers. Changes in the length of time the quantum dot fluoresces or is dark, is a direct result of the electron trapping period and the charge carrier separation/recombination kinetics which depends on the immediate nano-environment of the Qdots. To assess precisely on how the functionalization of the Qdot surface affect their optical properties, we developing a combined chemical force microscopy and a single molecule confocal microscopy. Using this technique, the surface functionalities and optical characteristics of single Qdots can be correlated.   (J. Krogmeier, P. Yim, D. Kim, I. Mandelbaum, A.R. Hight Walker, and J. Hwang)

Movie 1 (13 MB AVI movie)

Figure 1. The movie shows the blinking behavior of streptavidin-coated Qdots on a glass substrate (7 µm × 7 µm). The time transient data of fluorescence emissions from a single isolated CdSe/ZnS quantum dot in under continuous laser illumination demonstrate two distinct types of emission signatures indicative of dynamic nano-environment of the Qdot.

Nanocomplex Sensors and Detectors to Target Pathogens

In a time of bio-terrorism threats it is necessary to have new methods available for specifically targeted biological sample such as bacterial pathogens. Different challenges need to be addressed when trying to identify pathogens in the ambient, non-laboratory situation. A detection system needs to be rapid, highly sensitive, and specific. Our previous research involves bioengineering of bacteriophage-Qdot nanocomplexes, demonstrating that bacteriophage can be engineered to express a biotin-binding peptide in the capsid head region so that the biotin linker and streptavidin-functionalized Qdots target the bacteriophage which then binds to a specific strain(s) of E. coli. We are currently extending this approach in an effort to develop a series of phage-Qdot nanocomplexes, not limited to the streptavidin-biotin interaction. We expect that this study will result in a generic method to enable quantitative detection of specific biological pathogens from clinical or environmental isolates such as E. coli O157:H7 which occasionally causes massive meat product recalls, and human illness and sometimes death.   (G. Giulian and J. Hwang)

Movie 2 (50 MB AVI movie)

Figure 2. A schematic of the overall strategy for the detection of pathogens using phage/Qdot nanocomplexes. Genetically engineered phage and bio-functionalized Qdots are self-assembled into nano-manufacture fluorescent nanocomplexes capable of targeting and destroying specific pathogens. The movie displays fluorescence signal from Qdots bound to phage particles infecting E. Coli cells (65 µm × 65 µm).

Nanoscale Molecular Delivery Systems

Novel approaches for the delivery of nanomaterials into complex biological systems are needed for biochemical and biomedical applications including single cell diagnostics, drug targeting and delivery, and tumor imaging and diagnostics. We are developing techniques to manufacture liposomes encapsulated fluorescent nanocrystals for delivery of nanocrystals into cells. In this approach, we encapsulate Qdots with different surface functional coatings within approximately 100 nm unilamellar liposomes prepared by a variety of methods such as injection, extrusion, and electro-formation. Fluorescent lipid analogs were used to label liposomes to verify the encapsulation by assessing fluorescence resonance energy transfer from the Qdots donors to the acceptors (lipid analogs) in total internal reflection fluorescence microscopy (TIRFM). The TIRFM allows us to excite only liposomes near the substrate surface therefore substantially enhances the signal to noise ratio in detection. We also characterize the components of nanodelivery, liposomes and functionalized Qdots, and their complexes from results obtained by a variety of other methods including confocal fluorescence microscopy, real-time polarization modulation optical microscopy, and electron microscopy. This technique will be of immediate use towards targeting, labeling, and analyzing biological cells. Upon being triggered, the liposomes fuse to the plasma membrane of a targeted cell and release their contents into the cytoplasm of the cell.   (I. Mandelbaum, G.G. Giulian, P. Yim, J. Krogmeier, A.R. Hight Walker, and J. Hwang)

Movie 3 (1.3 MB AVI movie)

Figure 3. A schematic of the real-time polarization modulation fluorescence microscopy. The dipole orientations of fluorescence lipid analogs are measured in real-time by direct or FRET excitation using fast-rotating polarization modulating light. The movies of fluorescently labeled liposomes shows that, as the linearly polarized excitation sweeps over 360 degrees, the maximum fluorescence emission is measured from fluorescent lipid analog molecules with the dipole aligned in the direction of the excitation polarization, resulting the rotating fluorescence pattern as the excitation polarization is rotated (15 µm × 15 µm). Current investigations involve a variety of fluorescence measurements in understanding of interactions among a variety of biological and biomimetic molecules and nanoscale molecular delivery systems such as cells, liposomes, nanoparticles and nanoshells, and nanotubes. One of our approaches is using Qdots as FRET donors to encapsulate them in liposomes labeled with fluorescent lipid analogs as FRET acceptors to investigate liposome-mediated nanodelivery mechanisms.