Nicholas I. Smith

Label-free Raman observation of cytochrome c dynamics during apoptosis

We performed label-free observation of molecular dynamics in apoptotic cells by Raman microscopy. Dynamic changes in cytochrome c distribution at the Raman band of 750 cm-1 were observed after adding an apoptosis inducer to the cells. The comparison of mitochondria fluorescence images and Raman images of cytochrome c confirmed that changes in cytochrome c distribution can be distinguished as release of cytochrome c from mitochondria. Our observation also revealed that the redox state of cytochrome c was maintained during the release from the mitochondria. Monitoring mitochondrial membrane potential with JC-1 dye confirmed that the observed cytochrome c release was associated with apoptosis.

Linear phase imaging using differential interference contrast microscopy.

We propose an extension to Nomarski differential interference contrast microscopy that enables isotropic linear phase imaging. The method combines phase shifting, two directions of shear and Fourier‐space integration using a modified spiral phase transform. We simulated the method using a phantom object with spatially varying amplitude and phase. Simulated results show good agreement between the final phase image and the object phase, and demonstrate resistance to imaging noise.

Raman microscopy for dynamic molecular imaging of living cells.

We demonstrate dynamic imaging of molecular distribution in unstained living cells using Raman scattering. By combining slit-scanning detection and optimizing the excitation wavelength, we imaged the dynamic molecular distributions of cytochrome c, protein beta sheets, and lipids in unstained HeLa cells with a temporal resolution of 3 minutes. We found that 532-nm excitation can be used to generate strong Raman scattering signals and to suppress autofluorescence that typically obscures Raman signals. With this technique, we reveal time-resolved distributions of cytochrome c and other biomolecules in living cells in the process of cytokinesis without the need for fluorescent labels or markers.

Dynamic SERS Imaging of Cellular Transport Pathways with Endocytosed Gold Nanoparticles

Dynamic SERS imaging inside a living cell is demonstrated with the use of a gold nanoparticle, which travels through the intracellular space to probe local molecular information over time. Simultaneous tracking of particle motion and SERS spectroscopy allows us to detect intracellular molecules at 65 nm spatial resolution and 50 ms temporal resolution, providing molecular maps of organelle transport and lisosomal accumulation. Multiplex spectral and trajectory imaging will enable imaging of specific dynamic biological functions such as membrane protein diffusion, nuclear entry, and rearrangement of cellular cytoskeleton.

Generation of calcium waves in living cells by pulsed-laser-induced photodisruption

Here, we present an optical technique that can induce waves of calcium ion concentration in live biological cells. Ca2+ waves were induced by femtosecond pulsed-laser illumination. Living HeLa cells were exposed to focused 140 fs pulses of 780 nm wavelength at 30 mW average power. Ca2+ waves were imaged by fluorescence and were observed to propagate from the laser focal point inside the cell. Photoinduced generation of Ca2+ waves can be performed at any point inside the cell, an improvement over previous mechanical or biochemical stimulation techniques.

Time-resolved observation of surface-enhanced Raman scattering from gold nanoparticles during transport through a living cell

We perform time-resolved observation of living cells with gold nanoparticles using surface-enhanced Raman scattering (SERS). The position and SERS spectra of 50-nm gold nanoparticles are simultaneously observed by slit-scanning Raman microscopy with high spatial and temporal resolution. From the SERS observation, we confirm the attachment of the particles on the cell surface and the entry into the cell with the subsequent generation of SERS signals from nearby molecules. We also confirm that the strong dependence of SERS spectra on the position of the particle during the transportation of the particle through the cell. The obtained SERS spectra and its temporal fluctuation indicate that the molecular signals observable by this technique are given only from within a limited volume in close proximity to the nanoparticles. This confirms the high spatial selectivity and resolution of SERS imaging for observation of biomolecules involved in cellular events in situ.

Nanoscale heating of laser irradiated single gold nanoparticles in liquid.

Biological applications where nanoparticles are used in a cell environment with laser irradiation are rapidly emerging. Investigation of the localized heating effect due to the laser irradiation on the particle is required to preclude unintended thermal effects. While bulk temperature rise can be determined using macroscale measurement methods, observation of the actual temperature within the nanoscale domain around the particle is difficult and here we propose a method to measure the local temperature around a single gold nanoparticle in liquid, using white light scattering spectroscopy. Using 40-nm-diameter gold nanoparticles coated with thermo-responsive polymer, we monitored the localized heating effect through the plasmon peak shift. The shift occurs due to the temperature-dependent refractive index change in surrounding polymer medium. The results indicate that the particle experiences a temperature rise of around 10 degrees Celsius when irradiated with tightly focused irradiation of ~1 mW at 532 nm.

Arguments against modulational instabilities of Langmuir waves in Earth's foreshock

The nonlinear dispersion equation for monochromatic pump Langmuir waves is solved analytically and numerically and the results are applied to Langmuir-like waves growing in Earths foreshock. It is shown that modulational instabilities and parametric decay instabilities occur in distinct regions of W - kλ D space, confirming and extending earlier work. Here W is the ratio of electric field to thermal energy density and k is the pump wavenumber. In particular, for W ≤ 10 -3 and T e ≥ 3T i modulational instability occurs only for waves with kλ D ≤ (m e /9 M ) 1/2 , where m e and M i are electron and ion masses; decay is relevant at higher wavenumbers. Beam-driven waves in the foreshock are shown to lie well outside the region of parameter space for which modulational instability can proceed. In contrast, the beam-driven waves have wavenumbers appropriate for the decay. The beam-driven waves are also shown theoretically and observationally to have bandwidths much larger than the nonlinear growth rate for modulational instability and the parametric decay. The absence of a random-phase version of modulational instability and the decrease in growth rate caused by finite bandwidth effects provide two more arguments against modulational instability occurring frequently in the foreshock. Modulational instability may, however, be possible for very rare, intense (W ≥ 10 -2 ) wave packets with small wavenumbers (kλ D ≤ (m e /9M i ) 1/2 ) and small bandwidths (Δ w / wp ≤ 10 -3 ). Furthermore, the large Langmuir bandwidths predicted and observed require that the parametric decay cannot proceed, except perhaps for very intense wave packets with W > 10 -2 . Instead the random-phase, weak turbulence decay is predicted to occur, consistent with some previous observations and theoretical suggestions. Nonlinear plasma theory and our current understanding of foreshock electron beams and waves thus argue strongly against modulational instability and/or parametric decay proceeding or being important for the great majority of beam-driven Langmuir wave packets in Earths foreshock.

A femtosecond laser pacemaker for heart muscle cells

The intracellular effects of focused near-infrared femtosecond laser irradiation are shown to cause contraction in cultured neonatal rat cardiomyocytes. By periodic exposure to femtosecond laser pulse-trains, periodic contraction cycles in cardiomyocytes could be triggered, depleted, and synchronized with the laser periodicity. This was observed in isolated cells, and in small groups of cardiomyocytes with the laser acting as pacemaker for the entire group. A window for this effect was found to occur between 15 and 30 mW average power for an 80 fs, 82 MHz pulse train of 780 nm, using 8 ms exposures applied periodically at 1 to 2 Hz. At power levels below this power window, laser-induced cardiomyocyte contraction was not observed, while above this power window, cells typically responded by a high calcium elevation and contracted without subsequent relaxation. This laser-cell interaction allows the laser irradiation to act as a pacemaker, and can be used to trigger contraction in dormant cells as well as synchronize or destabilize contraction in spontaneously contracting cardiomyocytes. By increasing laser power above the window available for laser-cell synchronization, we also demonstrate the use of cardiomyocytes as optically-triggered actuators. To our knowledge, this is the first demonstration of remote optical control of cardiomyocytes without requiring exogenous photosensitive compounds.

Location-dependent photogeneration of calcium waves in HeLa cells.

The calcium ion (Ca2+) concentrations in a cell are responsible for the control of vital cellular functions and have been widely studied as a means to investigate and control cell activities. Here, we demonstrate Ca2+ wave generation in HeLa cells by femtosecond laser irradiation and show unexpected properties of the Ca2+ release and propagation. When the laser was focused in the cell cytoplasm, Ca2+ release was independent of both external Ca2+ influx and the phosphoinositide-phospholipase C (PLC) signaling pathway. The nucleus was not a susceptible target for laser-induced Ca2+ release, whereas irradiation of the plasma membrane produced evidence of transient poration, through which the extracellular solution could enter the cell. By chelating extracellular Ca2+, we found that laser-induced influx of ethylene glycol tetra-acetic acid (EGTA) can compete with calcium-induced calcium release and significantly delay or suppress the onset of the Ca2+ wave in the target cell. Intercellular Ca2+ propagation was adenosine triphosphate-dependent and could be observed even when the target cell cytosolic Ca2+ rise was suppressed by influx of EGTA. The irradiation effect on overall cell viability was also tested and found to be low (85% at 6h after irradiation by 60 mW average power). Laser-induced Ca2+ waves can be reliably generated by controlling the exposure and focal position and do not require the presence of caged Ca2+. The technique has the potential to replace other methods of Ca2+ stimulation, which either require additional caged molecules in the cell or do not have an interaction that is as well localized.