Stanko N. Nikolić

A Single-Photon Avalanche Camera for Fluorescence Lifetime Imaging Microscopy and Correlation Spectroscopy

Confocal laser scanning microscopy (CLSM) is commonly used to observe molecules of biological relevance in their native environment, the live cell, and study their spatial distribution and interactions nondestructively. CLSM can be easily extended to measure the lifetime of the excited state, the concentration and the diffusion properties of fluorescently labeled molecules, using fluorescence lifetime imaging microscopy (FLIM) and fluorescence correlation spectroscopy (FCS), respectively, in order to provide information about the local environment and the kinetics of molecular interaction in live cells. However, these parameters cannot be measured simultaneously using conventional CLSM due to damaging effects that are associated with strong illumination, including phototoxicity, photobleaching, and saturation of the fluorescence signal. To overcome these limitations, we have developed a new camera consisting of 1024 single-photon avalanche diodes that is optimized for multifocal microscopy, FLIM and FCS. We show proof-of-principle measurements of fluorescence intensity distribution and lifetime of the enhanced green fluorescent protein expressed in live cells and measurement of quantum dot diffusion in solution by FCS using the same detector.

Maximal intensity higher-order Akhmediev breathers of the nonlinear Schrödinger equation and their systematic generation

Abstract It is well known that Akhmediev breathers of the nonlinear cubic Schrodinger equation can be superposed nonlinearly via the Darboux transformation to yield breathers of higher order. Surprisingly, we find that the peak height of each Akhmediev breather only adds linearly to form the peak height of the final breather. Using this peak-height formula, we show that at any given periodicity, there exists a unique high-order breather of maximal intensity. Moreover, these high-order breathers form a continuous hierarchy, growing in intensity with increasing periodicity. For any such higher-order breather, a simple initial wave function can be extracted from the Darboux transformation to dynamically generate that breather from the nonlinear Schrodinger equation.

Probing the kinetic landscape of Hox transcription factor-DNA binding in live cells by massively parallel Fluorescence Correlation Spectroscopy.

Hox genes encode transcription factors that control the formation of body structures, segment-specifically along the anterior-posterior axis of metazoans. Hox transcription factors bind nuclear DNA pervasively and regulate a plethora of target genes, deploying various molecular mechanisms that depend on the developmental and cellular context. To analyze quantitatively the dynamics of their DNA-binding behavior we have used confocal laser scanning microscopy (CLSM), single-point fluorescence correlation spectroscopy (FCS), fluorescence cross-correlation spectroscopy (FCCS) and bimolecular fluorescence complementation (BiFC). We show that the Hox transcription factor Sex combs reduced (Scr) forms dimers that strongly associate with its specific fork head binding site (fkh250) in live salivary gland cell nuclei. In contrast, dimers of a constitutively inactive, phospho-mimicking variant of Scr show weak, non-specific DNA-binding. Our studies reveal that nuclear dynamics of Scr is complex, exhibiting a changing landscape of interactions that is difficult to characterize by probing one point at a time. Therefore, we also provide mechanistic evidence using massively parallel FCS (mpFCS). We found that Scr dimers are predominantly formed on the DNA and are equally abundant at the chromosomes and an introduced multimeric fkh250 binding-site, indicating different mobilities, presumably reflecting transient binding with different affinities on the DNA. Our proof-of-principle results emphasize the advantages of mpFCS for quantitative characterization of fast dynamic processes in live cells.

Peak-height formula for higher-order breathers of the nonlinear Schrödinger equation on nonuniform backgrounds

Given any background (or seed) solution of the nonlinear Schrödinger equation, the Darboux transformation can be used to generate higher-order breathers with much greater peak intensities. In this work, we use the Darboux transformation to prove, in a unified manner and without knowing the analytical form of the background solution, that the peak height of a high-order breather is just a sum of peak heights of first-order breathers plus that of the background, irrespective of the specific choice of the background. Detailed results are verified for breathers on a cnoidal background. Generalizations to more extended nonlinear Schrödinger equations, such as the Hirota equation, are indicated.

The connection between electromagnetically induced transparency in the Zeeman configuration and slow light in hot rubidium vapor

We experimentally studied Zeeman electromagnetically induced transparency (EIT) resonances and slow light propagation in hot Rb vapor. Propagation of weak σ− polarized light pulses in the presence of stronger σ+ polarized background through Rb vapor was realized using a single laser beam and the Pockels cell. The dependences of slow light group velocity and fractional pulse delay on the overall laser beam intensity and temporal pulse length showed that lower optical power and longer light pulses lead to improved EIT and slow light features. The connection between EIT and slow light was also investigated showing that narrower and more contrasted EIT resonances are necessary for further decreasing a group velocity.

Quantitative confocal fluorescence microscopy of dynamic processes by multifocal fluorescence correlation spectroscopy

Quantitative confocal fluorescence microscopy imaging without scanning is developed for the study of fast dynamical processes. The method relies on the use of massively parallel Fluorescence Correlation Spectroscopy (mpFCS). Simultaneous excitation of fluorescent molecules across the specimen is achieved by passing a single laser beam through a Diffractive Optical Element (DOE) to generate a quadratic illumination matrix of 32×32 light sources. Fluorescence from 1024 illuminated spots is detected in a confocal arrangement by a matching matrix detector consisting of the same number of single-photon avalanche photodiodes (SPADs). Software was developed for data acquisition and fast autoand cross-correlation analysis by parallel signal processing using a Graphic Processing Unit (GPU). Instrumental performance was assessed using a conventional single-beam FCS instrument as a reference. Versatility of the approach for application in biomedical research was evaluated using ex vivo salivary glands from Drosophila third instar larvae expressing a fluorescently-tagged transcription factor Sex Combs Reduced (Scr) and live PC12 cells stably expressing the fluorescently tagged mu-opioid receptor (MOPeGFP). We show that quantitative mapping of local concentration and mobility of transcription factor molecules across the specimen can be achieved using this approach, which paves the way for future quantitative characterization of dynamical reaction-diffusion landscapes across live cells/tissue with a submillisecond temporal resolution (presently 21 μs/frame) and single-molecule sensitivity.

Optical Ramsey fringes observed during temporal evolution of Zeeman coherences in Rb buffer gas cell

We experimentally studied the temporal evolution of Zeeman electromagnetically induced transparency (EIT) resonances induced by the laser resonant to hyperfine transition of 87Rb in a rubidium buffer gas cell. We simultaneously modulated the laser beam intensity and polarization to achieve the repeated interaction of the laser beam with coherently prepared atoms. Our cell was placed in a homogenous magnetic field to obtain the Larmor precession of the phase of coherences. The weak laser beam was used to probe the atoms at the end of the Ramsey sequence. We measured the transparency of the probe pulse at different magnetic fields for a given excitation pulse and the period of free evolution of Zeeman coherences in the dark. From these data, we reconstructed the temporal evolution of EIT resonances. The Ramsey fringes that appeared on the EIT curves at the beginning of the second probing pulse disappeared at later moments due to various decay processes.

Effects of laser beam diameter on electromagnetically induced transparency due to Zeeman coherences in Rb vapor

We experimentally studied the effects of laser beam diameter on electromagnetically induced transparency (EIT) due to Zeeman coherences induced by a laser resonant with the hyperfine transition Fg = 2 → Fe = 1 of 87Rb in a rubidium buffer gas cell. We use two laser beams of Gaussian intensity radial profile for laser beam diameters of 6.5 and 1.3 mm, laser intensities in the range of 0.1–35 mW cm−2 and cell temperatures between 60 and 82 °C. The results show that the amplitude of the normalized EIT resonance has a maximum at a laser intensity which depends on laser beam diameter and cell temperature. The laser intensity corresponding to the maximum EIT amplitude is higher for a smaller laser beam and higher cell temperature. The linewidth of Zeeman EIT resonance varies nearly linearly with laser intensity, almost independent of cell temperature and laser beam diameter.

Dark Hanle resonances from selected segments of the Gaussian laser beam cross-section.

We present the Hanle EIT resonances obtained from the various segments of the Gaussian laser beam cross-section, selected by moving the small aperture (placed in front of the detector) radially along the laser beam. Significant differences in the Hanle lineshapes are observed depending on whether the central or outer parts of the Gaussian laser beam are detected. The line narrowing and two counter-sign peaks occur at outer, less intense parts of the beam. The theoretical model suggests that the EIT lineshapes in the laser wings are result of the interference of the laser light and coherently prepared atoms coming from the central part of the beam. By blocking the central part of the laser beam in front of the detector, narrower, and for high laser intensities, even more contrasted Hanle resonances are obtained.