Aleksandar J. Krmpot

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.

Protective effect of autophagy in laser-induced glioma cell death in vitro.

Laser phototherapy could be potentially used for cancer treatment, but the mechanisms of laser‐induced cell death are not completely understood. Autophagy is the process in which the damaged cellular proteins and organelles are engulfed by and destroyed in acidified multiple‐membrane vesicles. The aim of the present study was to investigate the role of autophagy in laser‐induced tumor cell death in vitro.

Imaging Caenorhabditis elegans embryogenesis by third-harmonic generation microscopy.

In this study, third-harmonic generation (THG) imaging measurements were performed to characterize different developmental stages of the nematode Caenorhabditis elegans (C. elegans) embryos. Femtosecond laser pulses (1028 nm) were utilized for excitation. THG image contrast modality proved as a powerful diagnostic tool, providing valuable information and offering new insights into the complex developmental process of C. elegans embryogenesis.

Efficient parametric non-degenerate four-wave mixing in hot potassium vapor

We have observed high gains of the probe and the conjugate beams in non-degenerate four-wave mixing in hot potassium vapor, using a double-Λ configuration at the D1 line of the 39 K isotope. Gains of up to 82 for the conjugate beam and 63 for the probe beam have been achieved. Higher gains were obtained than with other alkali atoms under comparable experimental conditions due to lower ground state hyperfine splitting in the potassium atom. Experimental parameters for maximal gain have been determined. Notable gains are achieved at low pump intensities (~10 W cm−2) that are attainable even by conventional laser diodes. Due to their high gains, the probe and the conjugate beams may be suitable for utilization in quantum correlation and relative intensity squeezing experiments.

Nonlinear microscopy of chitin and chitinous structures: a case study of two cave-dwelling insects

Abstract. We performed a study of the nonlinear optical properties of chemically purified chitin and insect cuticle using two-photon excited autofluorescence (TPEF) and second-harmonic generation (SHG) microscopy. Excitation spectrum, fluorescence time, polarization sensitivity, and bleaching speed were measured. We have found that the maximum autofluorescence signal requires an excitation wavelength below 850 nm. At longer wavelengths, we were able to penetrate more than 150-μm deep into the sample through the chitinous structures. The excitation power was kept below 10 mW (at the sample) in order to diminish bleaching. The SHG from the purified chitin was confirmed by spectral- and time-resolved measurements. Two cave-dwelling, depigmented, insect species were analyzed and three-dimensional images of the cuticular structures were obtained.

Mapping of hemoglobin in erythrocytes and erythrocyte ghosts using two photon excitation fluorescence microscopy

Abstract. The present study describes utilization of two photon excitation fluorescence (2PE) microscopy for visualization of the hemoglobin in human and porcine erythrocytes and their empty membranes (i.e., ghosts). High-quality, label- and fixation-free visualization of hemoglobin was achieved at excitation wavelength 730 nm by detecting visible autofluorescence. Localization in the suspension and spatial distribution (i.e., mapping) of residual hemoglobin in erythrocyte ghosts has been resolved by 2PE. Prior to the 2PE mapping, the presence of residual hemoglobin in the bulk suspension of erythrocyte ghosts was confirmed by cyanmethemoglobin assay. 2PE analysis revealed that the distribution of hemoglobin in intact erythrocytes follows the cells’ shape. Two types of erythrocytes, human and porcine, characterized with discocyte and echinocyte morphology, respectively, showed significant differences in hemoglobin distribution. The 2PE images have revealed that despite an extensive washing out procedure after gradual hypotonic hemolysis, a certain amount of hemoglobin localized on the intracellular side always remains bound to the membrane and cannot be eliminated. The obtained results open the possibility to use 2PE microscopy to examine hemoglobin distribution in erythrocytes and estimate the purity level of erythrocyte ghosts in biotechnological processes.

On non-vanishing amplitude of Hanle electromagnetically induced absorption in Rb.

Amplitude and linewidts of the Hanle EIA, obtained from transmission of the laser locked to closed F(g) ? F(e) = F(g) +1 transitions in (85)Rb and(87)Rb, have maximum values at few mW/cm2. Amplitude of the EIA reaches steady value different from zero for higher laser intensities, even for laser intensities of 40 mW/cm(2). Theoretical model of EIA, for the same atomic system as in the experiment, show that the laser intensity, at which maximum of amplitudes and widths occur, depends on the laser detuning. For smaller laser detuning of a few tens of MHz, EIA has a maximum and then vanishes at higher laser intensities. For larger laser detuning of the order of hundreds MHz (but still in the range of Doppler broadening) amplitude of the EIA has very broad maximum and remains above zero for intensities above 40 mW/cm(2). Such theoretical results indicate that Hanle absorption peak remains in the experimental results, regardless of the laser intensities, due to Doppler effect.

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.

Scattering-enhanced absorption and interference produce a golden wing color of the burnished brass moth, Diachrysia chrysitis

Here we report how interference and scattering-enhanced absorption act together to produce the golden wing patches of the burnished brass moth. The key mechanism is scattering on rough internal surfaces of the wing scales, accompanied by a large increase of absorption in the UV-blue spectral range. Unscattered light interferes and efficiently reflects from the multilayer composed of the scales and the wing membranes. The resulting spectrum is remarkably similar to the spectrum of metallic gold. Subwavelength morphology and spectral and absorptive properties of the wings are described. Theories of subwavelength surface scattering and local intensity enhancement are used to quantitatively explain the observed reflectance spectrum.

Transient development of Zeeman electromagnetically induced transparency during propagation of Raman–Ramsey pulses through Rb buffer gas cell

We investigate, experimentally and theoretically, time development of Zeeman electromagnetically induced transparency (EIT) during propagation of two time separated polarization laser pulses, preparatory and probe, through Rb vapour. The pulses were produced by modifying laser intensity and degree of elliptical polarization. The frequency of the single laser beam is locked to the hyperfine transition of the D1 line in 87Rb. Transients in the intensity of component of the transmitted light are measured or calculated at different values of the external magnetic field, during both preparatory and probe pulse. Zeeman EIT resonances at particular time instants of the pulse propagation are reconstructed by appropriate sampling of the transients. We observe how laser intensity, Ramsey sequence and the Rb cell temperature affect the time dependence of EIT line shapes, amplitudes and linewidths. We show that at early times of the probe pulse propagation, several Ramsey fringes are present in EIT resonances, while at later moments a single narrow peak prevails. Time development of EIT amplitudes are determined by the transmitted intensity of the component during the pulse propagation.