We present real-time, full-field, fluorescence polarization microscopy and its calibration and validation methods to
monitor the absorption dipole orientation of fluorescent molecules. A quarter-wave plate, in combination with a liquid
crystal variable retarder (LCVR), provides a tunable method to rotate a linear polarized light prior to being coupled into
a fluorescence microscope. A series of full-field fluorescence polarization images are obtained of fluorescent molecules
interleaved into the lipid bilyaer of liposomes. With this system, the dynamic dipole orientation of the fluorescent lipid
analog tetramethylindocarbocyanine (DiI)-labeled lipids inserted in liposomes are probed and found to be aligned with
the liposome in a tangential manner. The dipole orientation of 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY)-
labeled lipids are expected to be aligned perpendicularly in the liposome membrane. Spectral separation of fluorescent
lipid analogs into separate images provide an internal control and the ability to quantitatively correlate the membrane
structure and fluctuations, within an optical section, in real-time. Application of this technique to the identification of
characteristic features of cellular processes such as adhesion, endocytosis, and apoptosis are being investigated.
This work describes the development and fabrication of a novel nanofluidic flow-through sensing chip that utilizes a
plasmonic resonator to excite fluorescent tags with sub-wavelength resolution. We cover the design of the microfluidic
chip and simulation of the plasmonic resonator using Finite Difference Time Domain (FDTD) software. The fabrication
methods are presented, with testing procedures and preliminary results.
This research is aimed at improving the resolution limits of the Direct Linear Analysis (DLA) technique developed by
US Genomics . In DLA, intercalating dyes which tag a specific 8 base-pair sequence are inserted in a DNA sample.
This sample is pumped though a nano-fluidic channel, where it is stretched into a linear geometry and interrogated with
light which excites the fluorescent tags. The resulting sequence of optical pulses produces a characteristic "fingerprint"
of the sample which uniquely identifies any sample of DNA. Plasmonic confinement of light to a 100 nm wide metallic
nano-stripe enables resolution of a higher tag density compared to free space optics. Prototype devices have been
fabricated and are being tested with fluorophore solutions and tagged DNA. Preliminary results show evanescent
coupling to the plasmonic resonator is occurring with 0.1 micron resolution, however light scattering limits the S/N of
the detector. Two methods to reduce scattered light are presented: index matching and curved waveguides.
We have fabricated a combined measurement system capable of confocal microscopy and fluorescence spectroscopy to
simultaneously evaluate multiple optical characteristics of single fluorescent nanocrystals. The single particle detection
sensitivity is demonstrated by simultaneously measuring the dynamic excitation-time-dependent fluorescence
intermittency and the emission spectrum of single cadmium selenide/zinc sulfide (CdSe/ZnS) nanocrystals (quantum
dots, QDs). Using this system, we are currently investigating the optical characteristics of single QDs, the surface of
which are conjugated with different ligands, such as trioctylphosphine oxide (TOPO), mercaptoundecanoicacid (MDA),
and amine modified DNA (AMDNA). In this paper, we present the progress of our measurements of the time-dependent
optical characteristics (fluorescence intermittency, photostability, and spectral diffusion) of single MDA-QDs and
AMDNA-MDA-QDs in air in an effort to understand the effects of surface-conjugated biomolecules on the optical
characteristics at single QD sensitivities.
The fluorescence intermittency of single, bare, CdSe/ZnS quantum dots was probed using single molecule confocal microscopy and found to demonstrate power law kinetics. Various threshold values and line fitting parameters are employed in the data analysis and their effects on the extracted power law exponents, moff and mon, are presented. The threshold is found to be critical in determining moff while having no significant effect on mon. The mean plus 2σ threshold, calculated from the background noise in the measurement, results in a more negative moff slope in comparison to the mean plus 3σ or mean plus 4σ thresholds. This is likely due to the mean plus 2σ threshold lying within the background noise outliers which mimic short on events. In contrast, the mean plus 4σ threshold is above 99.99% of the background noise while adequately below the fluorescence signal. Additionally, it is found that fitting only the ten most probable data points rather than all the data points in the log-log probability density graphs results in no significant change in moff and mon.