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.
Photothermal therapy employing nanomaterials is a promising approach to selectively treat targeted tissues with
abnormal characteristics such as tumors. While vital research has focused on the use of these materials in biomedical
applications, net effects of these materials in biological environments are still not well understood. For reliable
biomedical applications, it is crucial to quantitatively evaluate thermal properties of these materials in biological and
physiological environments. To this end, we have developed a highly integrated measurement platform and examined
local thermal properties of single gold shell nanocrystals in biomimetic environments. These nanoshells consist of a
silica core with an outer gold coating. For quantitative measurement of the local thermal profile of gold nanoshells, we
monitor lipid phase transitions triggered by gold nanoshell thermal excitation. Dried lipid layers with adsorbed gold
nanoshells were placed in an aqueous environment. Photothermal excitation of the gold nanoshells induced localized
liposome budding as the lipids were raised above their transition temperature. Single particle tracking of gold
nanoshells in solution and within liposomes revealed larger diffusion rates for the confined nanoparticles, likely due to a
raised local temperature.
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.
Flow cytometry has been instrumental in rapid analysis of single cells since the 1970s. One of the common approaches is
the immunofluorescence study involving labeling of cells with antibodies conjugated to organic fluorophores. More
recently, as the application of flow cytometry extended from simple cell detection to single-cell proteomic analysis, the
need of determining the actual number of antigens in a single cell has driven the flow cytomery technique towards a
quantitative methodology. However, organic fluorophores are challenging to use as probes for quantitative detection
due to the lack of photostability and of quantitative fluorescence standards. National Institute of Standards and
Technologies (NIST) provides a set of fluorescein isothiocyanate (FITC) labeled beads, RM 8640, which is the only
nationally recognized fluorescent particle standard. On the other hand, optical characteristics of semiconductor
nanocrystals or quantum dots or QDs are superior to traditional dye molecules for the use as tags for biological and
chemical fluorescent sensors and detectors. Compelling advantages of QDs include long photostability, broad spectral
coverage, easy excitation, and suitability for multiplexed sensing. Recently, novel surface coatings have been
developed to render QDs water soluble and bio-conjugation ready, leading to their use as fluorescent tags and sensors for
a variety of biological applications including immunolabeling of cells. Here, we describe our approach of using
fluorescent semiconductor QDs as a novel tool for quantitative flow cytometry detection. Our strategy involves the
development of immuno-labeled QD-conjugated silica beads as "biomimetic cells." In addition to flow cytometry, the
QD-conjugated silica beads were characterized by fluorescence microscopy to quantitate the number of QDs attached to
a single silica bead. Our approach enables flow cytometry analysis to be highly sensitive, quantitative, and encompass
a wide dynamic range of fluorescence detection. Quantitative aspects of the proposed flow cytometery-based approach
for measurement of the QD-based biomimetic samples are discussed.
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