Applications of Multiphoton Imaging Techniques To The Study Of Protein Interactions

Several recent improvements in microscopy have been driven by advances in ultrafast laser technology. The goal of the research described in this dissertation was to develop noninvasive, optically based methods to measure the mobility of macromolecules in biologically relevant systems. These methods exploit advances in ultrafast laser science and recent developments in multiphoton spectroscopy techniques. Each of the techniques described in this dissertation is validated and standardized using well characterized systems. We have explored the following techniques: First, 2-photon 2-color Fluorescence Cross Correlation Spectroscopy (FCCS) a powerful technique to measure dilute protein interactions in living cells. We have used FCCS to probe AR-Tif2 (Androgen Receptor - activating cofactor) interactions in the presence of casodex, an antagonist yielding decreased binding affinity. On a much faster timescale, exploring rotational rather than translational diffusion, we used molecular dynamics simulations of the model probe perylene to show that there is `room to wiggle' (sub-ps libration) within pure hexadecane. Third, combining picosecond and microsecond scales, we built a system to measure both rotational and translational motion in one experiment, using advanced Time Correlated Single Photon Counting (TCSPC) techniques. We have tested our ability to measure and link simultaneously the translational rates and decay rates of Alexa488 dye and other biologically relevant fluorophores. Next, exploiting non-linear vibrational spectroscopy, we have imaged the non-fluorescent molecule NAD+ in DPPC vesicles, the C-H stretch of lipids in vesicles and polystyrene beads, and the O-H stretch of water inside living cells (vs. O-D) to demonstrate the chemically selective imaging capabilities of Coherent Anti-Stokes Raman Spectroscopy (CARS) Microscopy. Most recently, we have built a STED (STimulated Emission Depletion) Microscope capable of extracting fluorescent images well below the diffraction limit. The STED microscope was tested using both 170nm fluorescent beads and a novel photochromic dye

FRET microscopy autologous tumor lysate processing in mature dendritic cell vaccine therapy

Abstract Background: Antigen processing by dendritic cells (DC) exposed to specific stimuli has been well characterized in biological studies. Nonetheless, the question of whether autologous whole tumor lysates (as used in clinical trials) are similarly processed by these cells has not yet been resolved. Methods: In this study, we examined the transfer of peptides from whole tumor lysates to major histocompatibility complex class II molecules (MHC II) in mature dendritic cells (mDC) from a patient with advanced melanoma. Tumor antigenic peptides-MHC II proximity was revealed by F\uf6rster Resonance Energy Transfer (FRET) measurements, which effectively extends the application of fluorescence microscopy to the molecular level (<100?). Tumor lysates were labelled with Alexa-488, as the donor, and mDC MHC II HLA-DR molecules were labelled with Alexa-546-conjugated IgG, as the acceptor. Results: We detected significant energy transfer between donor and acceptor-labelled antibodies against HLA-DR at the membrane surface of mDC. FRET data indicated that fluorescent peptide-loaded MHC II molecules start to accumulate on mDC membranes at 16 hr from the maturation stimulus, steeply increasing at 22 hr with sustained higher FRET detected up to 46 hr. Conclusions: The results obtained imply that the patient mDC correctly processed the tumor specific antigens and their display on the mDC surface may be effective for several days. These observations support the rationale for immunogenic efficacy of autologous tumor lysates

Intracellular oxygen mapping using a myoglobin-mCherry probe with fluorescence lifetime imaging.

Oxygen (O2) is one of the most important biometabolites. In abundance, it serves as the limiting terminus of aerobic respiratory chains in the mitochondria of higher organisms; in deficit, it is a potent determinant of development and regulation of other physiological and therapeutic processes. Most knowledge on intracellular and interstitial concentration ([O2]) is derived from mitochondria isolated from cells or tissue biopsies, providing detailed but nonnative insight into respiratory chain function. The possible loss of essential metabolites during isolation and disruption of the normal interactions of the organelle with the cytoskeleton may cause these data to misrepresent intact cells. Several optical methodologies were also developed, but they are often unable to detect heterogeneity of metabolic characteristics among different individual cells in the same culture, and most cannot detect heterogeneous consumption within different areas of a single cell. Here, we propose a noninvasive and highly sensitive fluorescence lifetime microscopy probe, myoglobin-mCherry, appropriate to intracellular targeting. Using our probe, we monitor mitochondrial contributions to O2 consumption in A549 nonsmall cell lung cancer cells and we reveal heterogeneous [O2] within the intracellular environments. The mitochondrial [O2] at a single-cell level is also mapped by adding a peptide to target the probe to the mitochondria

Novel imaging biomarkers for mapping the impact of mild mitochondrial uncoupling in the outer retina in vivo.

PURPOSE: To test the hypothesis that imaging biomarkers are useful for evaluating in vivo rod photoreceptor cell responses to a mitochondrial protonophore. METHODS: Intraperitoneal injections of either the mitochondrial uncoupler 2,4 dinitrophenol (DNP) or saline were given to mice with either higher [129S6/eVTac (S6)] or lower [C57BL/6J (B6)] mitochondrial reserve capacities and were studied in dark or light. We measured: (i) the external limiting membrane-retinal pigment epithelium region thickness (ELM-RPE; OCT), which decreases substantially with upregulation of a pH-sensitive water removal co-transporter on the apical portion of the RPE, and (ii) the outer retina R1 (= 1/(spin lattice relaxation time (T1), an MRI parameter proportional to oxygen / free radical content. RESULTS: In darkness, baseline rod energy production and consumption are relatively high compared to that in light, and additional metabolic stimulation with DNP provoked thinning of the ELM-RPE region compared to saline injection in S6 mice; ELM-RPE thickness was unresponsive to DNP in B6 mice. Also, dark-adapted S6 mice given DNP showed a decrease in outer retina R1 values compared to saline injection in the inferior retina. In dark-adapted B6 mice, transretinal R1 values were unresponsive to DNP in superior and inferior regions. In light, with its relatively lower basal rod energy production and consumption, DNP caused ELM-RPE thinning in both S6 and B6 mice. CONCLUSIONS: The present results raise the possibility of non-invasively evaluating the mouse rod mitochondrial energy ecosystem using new DNP-assisted OCT and MRI in vivo assays