Maia Brunstein

Thermo-optical dynamics in an optically pumped Photonic Crystal nano-cavity

Linear and non-linear thermo-optical dynamical regimes were investigated in a photonic crystal cavity. First, we have measured the thermal relaxation time in an InP-based nano-cavity with quantum dots in the presence of optical pumping. The experimental method presented here allows one to obtain the dynamics of temperature in a nanocavity based on reflectivity measurements of a cw probe beam coupled through an adiabatically tapered fiber. Characteristic times of 1.0+/-0.2 micros and 0.9+/-0.2 micros for the heating and the cooling processes were obtained. Finally, thermal dynamics were also investigated in a thermo-optical bistable regime. Switch-on/off times of 2 micros and 4 micros respectively were measured, which could be explained in terms of a simple non-linear dynamical representation.

Full-field dual-color 100-nm super-resolution imaging reveals organization and dynamics of mitochondrial and ER networks

Most structured illumination microscopes use a physical or synthetic grating that is projected into the sample plane to generate a periodic illumination pattern. Albeit simple and cost-effective, this arrangement hampers fast or multi-color acquisition, which is a critical requirement for time-lapse imaging of cellular and sub-cellular dynamics. In this study, we designed and implemented an interferometric approach allowing large-field, fast, dual-color imaging at an isotropic 100-nm resolution based on a sub-diffraction fringe pattern generated by the interference of two colliding evanescent waves. Our all-mirror-based system generates illumination pat-terns of arbitrary orientation and period, limited only by the illumination aperture (NA = 1.45), the response time of a fast, piezo-driven tip-tilt mirror (10 ms) and the available fluorescence signal. At low µW laser powers suitable for long-period observation of life cells and with a camera exposure time of 20 ms, our system permits the acquisition of super-resolved 50 µm by 50 µm images at 3.3 Hz. The possibility it offers for rapidly adjusting the pattern between images is particularly advantageous for experiments that require multi-scale and multi-color information. We demonstrate the performance of our instrument by imaging mitochondrial dynamics in cultured cortical astrocytes. As an illustration of dual-color excitation dual-color detection, we also resolve interaction sites between near-membrane mitochondria and the endoplasmic reticulum. Our TIRF-SIM microscope provides a versatile, compact and cost-effective arrangement for super-resolution imaging, allowing the investigation of co-localization and dynamic interactions between organelles--important questions in both cell biology and neurophysiology.

Small volume excitation and enhancement of dye fluorescence on a 2D photonic crystal surface

We demonstrate an easy-to-implement scheme for fluorescence enhancement and observation volume reduction using photonic crystals (PhCs) as substrates for microscopy. By normal incidence coupling to slow 2D-PhC guided modes, a 65 fold enhancement in the excitation is achieved in the near field region (100 nm deep and 1 microm wide) of the resonant mode. Such large enhancement together with the high spatial resolution makes this device an excellent substrate for fluorescence microscopies.

Radiation patterns from coupled photonic crystal nanocavities

We report on far field measurements on two coupled photonic crystal nanocavities. The distinct features of the antisymmetric modes (minima of intensity at zero-emission angles) allow us to demonstrate a π-phase difference between the cavity fields, a clear signature of evanescent coupling. Good agreement between experimental results and simulated radiation patterns has been found.

Eliminating Unwanted Far-Field Excitation in Objective-Type TIRF. Part I. Identifying Sources of Nonevanescent Excitation Light

Total internal reflection fluorescence microscopy (TIRFM) achieves subdiffraction axial sectioning by confining fluorophore excitation to a thin layer close to the cell/substrate boundary. However, it is often unknown how thin this light sheet actually is. Particularly in objective-type TIRFM, large deviations from the exponential intensity decay expected for pure evanescence have been reported. Nonevanescent excitation light diminishes the optical sectioning effect, reduces contrast, and renders TIRFM-image quantification uncertain. To identify the sources of this unwanted fluorescence excitation in deeper sample layers, we here combine azimuthal and polar beam scanning (spinning TIRF), atomic force microscopy, and wavefront analysis of beams passing through the objective periphery. Using a variety of intracellular fluorescent labels as well as negative staining experiments to measure cell-induced scattering, we find that azimuthal beam spinning produces TIRFM images that more accurately portray the real fluorophore distribution, but these images are still hampered by far-field excitation. Furthermore, although clearly measureable, cell-induced scattering is not the dominant source of far-field excitation light in objective-type TIRF, at least for most types of weakly scattering cells. It is the microscope illumination optical path that produces a large cell- and beam-angle invariant stray excitation that is insensitive to beam scanning. This instrument-induced glare is produced far from the sample plane, inside the microscope illumination optical path. We identify stray reflections and high-numerical aperture aberrations of the TIRF objective as one important source. This work is accompanied by a companion paper (Pt.2/2).

Anomalous Dynamics of Melanosomes Driven by Myosin-V in Xenopus laevis Melanophores

The organization of the cytoplasm is regulated by molecular motors, which transport organelles and other cargoes along cytoskeleton tracks. In this work, we use single particle tracking to study the in vivo regulation of the transport driven by myosin-V along actin filaments in Xenopus laevis melanophores. Melanophores have pigment organelles or melanosomes, which, in response to hormones, disperse in the cytoplasm or aggregate in the perinuclear region. We followed the motion of melanosomes in cells treated to depolymerize microtubules during aggregation and dispersion, focusing the analysis on the dynamics of these organelles in a time window not explored before to our knowledge. These data could not be explained by previous models that only consider active transport. We proposed a transport-diffusion model in which melanosomes may detach from actin tracks and reattach to nearby filaments to resume the active motion after a given time of diffusion. This model predicts that organelles spend approximately 70% and 10% of the total time in active transport during dispersion and aggregation, respectively. Our results suggest that the transport along actin filaments and the switching from actin to microtubule networks are regulated by changes in the diffusion time between periods of active motion driven by myosin-V.

Eliminating Unwanted Far-Field Excitation in Objective-Type TIRF. Part II. Combined Evanescent-Wave Excitation and Supercritical-Angle Fluorescence Detection Improves Optical Sectioning

Azimuthal beam scanning makes evanescent-wave (EW) excitation isotropic, thereby producing total internal reflection fluorescence (TIRF) images that are evenly lit. However, beam spinning does not fundamentally address the problem of propagating excitation light that is contaminating objective-type TIRF. Far-field excitation depends more on the specific objective than on cell scattering. As a consequence, the excitation impurities in objective-type TIRF are only weakly affected by changes of azimuthal or polar beam angle. These are the main results of the first part of this study (Eliminating unwanted far-field excitation in objective-type TIRF. Pt.1. Identifying sources of nonevanescent excitation light). This second part focuses on exactly where up beam in the illumination system stray light is generated that gives rise to nonevanescent components in TIRF. Using dark-field imaging of scattered excitation light we pinpoint the objective, intermediate lenses and, particularly, the beam scanner as the major sources of stray excitation. We study how adhesion-molecule coating and astrocytes or BON cells grown on the coverslip surface modify the dark-field signal. On flat and weakly scattering cells, most background comes from stray reflections produced far from the sample plane, in the beam scanner and the objective lens. On thick, optically dense cells roughly half of the scatter is generated by the sample itself. We finally show that combining objective-type EW excitation with supercritical-angle fluorescence (SAF) detection efficiently rejects the fluorescence originating from deeper sample regions. We demonstrate that SAF improves the surface selectivity of TIRF, even at shallow penetration depths. The coplanar microscopy scheme presented here merges the benefits of beam spinning EW excitation and SAF detection and provides the conditions for quantitative wide-field imaging of fluorophore dynamics at or near the plasma membrane.

Transient thermoreflectance imaging of active photonic crystals

Transient thermoreflectance imaging is used to study the dynamics of the temperature inside active two-dimensional photonic crystals (PhCs). We developed a pump-probe setup suited for optically pumped devices that presents submicrosecond time resolution and submicrometer spatial resolution. Characteristic thermal dissipation times of 429 ns in a PhC Bloch mode cavity and of 999 ns in a PhC membrane are measured. This technique gives also access to the diffusivity of the suspended PhC.

All-Optical, All-Fibered Ultrafast Switching in 2-D InP-Based Photonic Crystal Nanocavity

The ultrafast optical switching capabilities of an InP-based photonic crystal cavity with quantum dots (QDs) are studied. The signal is evanescently coupled into the cavity at 1.5 μm through a tapered fiber. Both surface and fiber-coupled pumping schemes are investigated and compared. It is shown that the carrier-induced nonlinear response allows the signal to be switched within time windows ranging from a few picoseconds to 20 ps for the switch ON process and from 30 to 170 ps for the switch OFF process depending on the pumping configuration and power level. The carrier-induced nonlinear effects in the bulk, the wetting layer, and the QDs are discussed and compared. From a fit of the switching dynamics to the solutions of a rate equation for carrier relaxation, we show that the main contribution to carrier dynamics governing the switching processes comes from both nonradiative and radiative carrier recombination within the InAsP wetting layer.

Enhancement of a nano cavity lifetime by induced slow light and nonlinear dispersions

We start from a 2D photonic crystal nanocavity with moderate Q-factor and dynamically increase it by two order of magnitude by the joint action of coherent population oscillations and nonlinear refractive index.