Laura Marcu

In vitro and in vivo evaluation and a case report of intense nanosecond pulsed electric field as a local therapy for human malignancies

When delivered to cells, very short duration, high electric field pulses (nanoelectropulses) induce primarily intracellular events. We present evidence that this emerging modality may have a role as a local cancer therapy. Five hematologic and 16 solid tumor cell lines were pulsed in vitro. Hematologic cells proved particularly sensitive to nanoelectropulses, with more than a 60% decrease in viable cells measured by MTT assay 96 hr after pulsing in 4 of 5 cell lines. In solid tumor cell lines, 10 out of 16 cell lines had more than a 10% decrease in viable cells. AsPC‐1, a pancreatic cancer cell line, demonstrated the greatest in vitro sensitivity among solid tumor cell lines, with a 64% decrease in viable cells. When nanoelectropulse therapy was applied to AsPC‐1 tumors in athymic nude mice, responses were seen in 4 of 6 tumors, including clinical complete responses in 3 of 6 animals. A single human subject applied nanoelectropulse therapy to his own basal cell carcinoma and had a complete pathologic response. In summary, we demonstrate that electric pulses 20 ns or less kill a wide variety of human cancer cells in vitro, induce tumor regression in vivo, and show efficacy in a single human patient. Therefore, nanoelectropulse therapy deserves further study as a potentially effective cancer therapy.

Time-domain laser-induced fluorescence spectroscopy apparatus for clinical diagnostics

We report the design and development of a compact optical fiber-based apparatus for in situ time-resolved laser-induced fluorescence spectroscopy (tr-LIFS) of biological systems. The apparatus is modular, optically robust, and compatible with the clinical environment. It incorporates a dual output imaging spectrograph, a gated multichannel plate photomultiplier (MCP-PMT), an intensified charge-coupled-device (ICCD) camera, and a fast digitizer. It can accommodate various types of light sources and optical fiber probes for selective excitation and remote light delivery/collection as required by different applications. The apparatus allows direct recording of the entire fluorescence decay with high sensitivity (nM range fluorescein dye concentration with signal-to-noise ratio of 46) and with four decades dynamic range. It is capable of resolving a broad range of fluorescence lifetimes from hundreds of picoseconds (as low as 300 ps) using the MCP-PMT coupled to the digitizer to milliseconds using the ICCD. T...

Ultrashort pulsed electric fields induce membrane phospholipid translocation and caspase activation: differential sensitivities of Jurkat T lymphoblasts and rat glioma C6 cells

Megavolt-per-meter electric pulses with durations shorter than charging time constants associated with external cell membrane dielectric properties can generate significant voltages on the membranes of intracellular structures. Nanosecond-duration, high-field (2-4 MV/m) pulses are not immediately lethal to cells and do not produce the conductive openings in the cytoplasmic membrane associated with long-pulse, low-field electroporation, but can induce profound physiological changes, including apoptosis (programmed cell death). We demonstrate rapid, non-destructive, field-dependent translocation of the plasma membrane inner leaflet phospholipid phosphatidylserine in Jurkat T lymphocytes, and we show that cells which exhibit a similar geometry in suspension, rat glioma C6 cells, are highly resistant to these pulses and respond differently even to much higher doses.

Fluorescence Lifetime Techniques in Medical Applications

This article presents an overview of time-resolved (lifetime) fluorescence techniques used in biomedical diagnostics. In particular, we review the development of time-resolved fluorescence spectroscopy (TRFS) and fluorescence lifetime imaging (FLIM) instrumentation and associated methodologies which allow in vivo characterization and diagnosis of biological tissues. Emphasis is placed on the translational research potential of these techniques and on evaluating whether intrinsic fluorescence signals provide useful contrast for the diagnosis of human diseases including cancer (gastrointestinal tract, lung, head and neck, and brain), skin and eye diseases, and atherosclerotic cardiovascular disease.

Fast model-free deconvolution of fluorescence decay for analysis of biological systems

For complex biological systems, conventional analysis of fluorescence intensity decay in terms of discrete exponential components cannot readily provide a true representation of the underlying fluorescence dynamics. We investigate an alternative nonparametric method for the analysis of time-resolved fluorescence data from biochemical and biological systems based on the expansion of fluorescence decay in a discrete Laguerre basis. We report that a unique Laguerre expansion can be found for fluorescence intensity decays of arbitrary form with convergence to a correct solution significantly faster than conventional multiexponential approximation methods. The Laguerre expansion coefficients are shown to be highly correlated with intrinsic fluorescence lifetimes and allow direct characterization of the fluorescence dynamics. A novel method for prediction of concentrations in mixtures of biochemical components using these coefficients is developed and successfully tested (prediction error <2%) using data from different mixtures of fluorescence lifetime standards. These findings suggest that the use of Laguerre expansion coefficients is a fast approach for the characterization and discrimination of complex biological systems such as tissues and cells, and that the method has potential for applications of fluorescence lifetime techniques to tissue diagnostics and imaging microscopy of living cells.

Fluorescence lifetime imaging microscopy for brain tumor image-guided surgery

We demonstrate for the first time the application of an endoscopic fluorescence lifetime imaging microscopy (FLIM) system to the intraoperative diagnosis of glioblastoma multiforme (GBM). The clinically compatible FLIM prototype integrates a gated (down to 0.2 ns) intensifier imaging system with a fiber-bundle (fiber image guide of 0.5 mm diameter, 10,000 fibers with a gradient index lens objective 0.5 NA, and 4 mm field of view) to provide intraoperative access to the surgical field. Experiments conducted in three patients undergoing craniotomy for tumor resection demonstrate that FLIM-derived parameters allow for delineation of tumor from normal cortex. For example, at 460±25-nm wavelength band emission corresponding to NADH/NADPH fluorescence, GBM exhibited a weaker fluorescence intensity (35% less, p-value<0.05) and a longer lifetime τGBM-Amean=1.59±0.24 ns than normal cortex τNC-Amean=1.28±0.04 ns (p-value<0.005). Current results demonstrate the potential use of FLIM as a tool for image-guided surgery of brain tumors.

Fluorescence Lifetime Spectroscopy of Glioblastoma Multiforme

Abstract Fluorescence spectroscopy of the endogenous emission of brain tumors has been researched as a potentially important method for the intraoperative localization of brain tumor margins. We investigated the use of time-resolved, laser-induced fluorescence spectroscopy for demarcation of primary brain tumors by studying the time-resolved spectra of gliomas. The fluorescence of human brain samples (glioblastoma multiforme, cortex and white matter: six patients, 23 sites) was induced ex vivo with a pulsed nitrogen laser (337 nm, 3 ns). The time-resolved spectra were detected in a 360–550 nm wavelength range using a fast digitizer and gated detection. Parameters derived from both the spectral- (intensities from narrow spectral bands) and the time domain (average lifetime) measured at 390 and 460 nm were used for tissue characterization. We determined that high-grade gliomas are characterized by fluorescence lifetimes that varied with the emission wavelength (>3 ns at 390 nm, <1 ns at 460 nm) and their emission is overall longer than that of normal brain tissue. Our study demonstrates that the use of fluorescence lifetime not only improves the specificity of fluorescence measurements but also allows a more robust evaluation of data collected from brain tissue. Combined information from both the spectral- and the time domain can enhance the ability of fluorescence-based techniques to diagnose and detect brain tumor margins intraoperatively.

Nanosecond pulsed electric fields perturb membrane phospholipids in T lymphoblasts.

Nanosecond, megavolt‐per‐meter pulsed electric fields scramble the asymmetric arrangement of phospholipids in cell membranes without the permeabilization associated with longer, lower‐field pulses. A single 30 ns, 2.5 MV/m pulse produces perturbations consistent with phosphatidylserine (PS) externalization in Jurkat T lymphoblasts within milliseconds, polarized in the direction of the applied field, indicating an immediate interaction between membrane components and the electric field. This disturbance occurs only at the anode pole of the cell, supporting the hypothesis that the pulsed field drives the negatively charged PS head group toward the positive electrode, directly providing the energy for crossing the membrane dielectric barrier.

Pulse generators for pulsed electric field exposure of biological cells and tissues

This paper describes three pulse generators: a spark gap switched coaxial cable, a spark gap switched Blumlein, and a solid state modulator, developed for applying ultrashort electrical pulses to biological materials in culture. Research has shown that ultrashort pulsed electric fields can induce apoptosis in biological cells, and that pulses as short as 10 ns with field amplitude greater than 1 W/m cause membrane phospholipid rearrangement and activation of the effector enzymes of apoptosis. Pulses of very short duration use only tens of mJ per mL per pulse to induce apoptosis and other intracellular effects without causing thermal trauma. The pulse generators discussed here, each of a different topology, deliver ns pulsed electric fields (nsPEF) to cells in liquid suspension, and can be modified to drive electrodes for external, surgical, or endoscopic treatment of tissues in situ.

Detection of rupture-prone atherosclerotic plaques by time-resolved laser-induced fluorescence spectroscopy

OBJECTIVE Plaque with dense inflammatory cells, including macrophages, thin fibrous cap and superficial necrotic/lipid core is thought to be prone-to-rupture. We report a time-resolved laser-induced fluorescence spectroscopy (TR-LIFS) technique for detection of such markers of plaque vulnerability in human plaques. METHODS The autofluorescence of carotid plaques (65 endarterectomy patients) induced by a pulsed laser (337 nm, 0.7 ns) was measured from 831 distinct areas. The emission was resolved spectrally (360-550 nm range) and temporally (0.3 ns resolution) using a prototype fiber-optic TR-LIFS apparatus. Lesions were evaluated microscopically and quantified as to the % of different components (fibrous cap, necrotic core, inflammatory cells, foam cells, mature and degraded collagen, elastic fibers, calcification, and smooth muscle cell of the vessel wall). RESULTS We determined that the spectral intensities and time-dependent parameters at discrete emission wavelengths (1) allow for discrimination (sensitivity >81%, specificity >94%) of various compositional and pathological features associated with plaque vulnerability including infiltration of macrophages into intima and necrotic/lipid core under a thin fibrous cap, and (2) show a linear correlation with plaque biochemical content: elastin (P<0.008), collagen (P<0.02), inflammatory cells (P<0.003), necrosis (P<0.004). CONCLUSION Our results demonstrate the feasibility of TR-LIFS as a method for the identification of markers of plaque vulnerability. Current findings enable future development of TR-LIFS-based clinical devices for rapid investigation of atherosclerotic plaques and detection of those at high-risk.