Bioimaging最新文献

The requirement to functionally probe biological structures, with ever increasing selectivity and three-dimensional resolution is a frontier area in microscopy. Non-linear optics has a unique potential in this regard with numerous studies focused on the potential of three-dimensional imaging with super-resolution. In this paper we demonstrate that non-linear optical phenomena, such as second harmonic (SH) generation, which is very sensitive to the membrane potential, can be locally enhanced by complexing or approaching a SH generating molecular probe to a nanoantenna of a silver or gold nanoparticle. This gives complexes with gigantic optical non-linearities. These contrast enhancing non-linear optical complexes have the potential to be directed selectively to specific nanometric regions in cells in order to report on alterations on the structure and the function in such regions while overcoming the inherent inefficiency of non-linear optical interactions.

High-quantum-efficiency photodetection, millisecond pixel dwell time stage scanning and image restoration by maximum-likelihood estimation are synergetically combined and shown to improve the resolution of two-photon excitation microscopy 2–4 fold in all directions. Measurements of the two-photon excitation point-spread function (PSF) of a 1.4 aperture oil immersion lens are carried out by imaging fluorescence beads with a diameter of one seventh of the excitation wavelength (830 nm) and subsequent deconvolution with the bead object function. The proposed method of resolution increase is applied to beads as well as to rhodamine labelled actin fibres in mouse fibroblast cells. As the resolution improvement is not based on the non-linear effect of two-photon excitation, the results imply a comparable resolution increase in single-photon excitation confocal microscopy. In the fibroblasts, we established a three-fold improvement in axial resolution, namely from 840 nm before, to 280 nm after restoration (full-width at half-maximum). Actin fibres with axial distances of 850 nm, otherwise difficult to discern, are fully separated. In the lateral direction, images of fluorescence beads of about 110 nm diameter are restored to the real dimensions of the beads with an accuracy of better than one pixel (41 nm).

The interferometric spatial overlap of two laterally offset focal field distributions of a high numerical aperture lens in combination with confocal detection lead to improved resolution in confocal imaging. Experimental data of the achieved signal, the point spread autocorrelation function (PSAF), are presented for both the lateral and axial directions. Numerical simulations of lateral PSAF imaging of selected fluorescent objects show an increased resolution of up to 30% with moderate ringing, obtainable also in the presence of moderate spherical aberrations.

We demonstrate that three-photon excitation images of both fixed and living biological specimens can be readily obtained using an all-solid-state Nd:YLF laser excitation source. Optically sectioned images of fixed Caenorhabditis elegans embryos stained with DAPI and embryos triple-labeled with DAPI, fluorescein and Texas Red are presented. Time series images of a living LLC-PK cell stained with Hoechst 33342 during the progression from metaphase to telophase are also presented. The mode of excitation was inferred from the power-law of anthracene and Hoechst 33342 fluorescence versus incident laser power and an axial resolution comparison of anthracene fluorescence with two-photon excited Calcium Crimson fluorescence. Multiphoton excitation imaging is an attractive method for optically sectioning live specimens because of the lower levels of phototoxicity produced compared to other optical sectioning techniques. The combination of two- and three-photon excitation extends the capabilities of a multiple- photon imaging system since a single wavelength can provide localized excitation of a wide variety of fluorophores whose collective emission spectra can span the entire visible spectrum.

A comparatively simple laser scanning microscopic method for the determination of lateral diffusion coefficients at high temporal and spatial resolution is described. Combining two previously developed methods, continuous fluorescence microphotolysis and scanning microphotolysis, the new method is referred to as continuous scanning microphotolysis (continuous SCAMP). The principle of the method is simply to operate a commercial laser scanning microscope in the line scanning mode while monitoring the fluorescence emitted from the continuously scanned line as an x–t ‘image’. Fluorescence excitation can be effected by either single- or two-photon absorption. In the former case a standard, low power ion laser is sufficient while for two-photon excitation a femtosecond-pulsed titan sapphire laser can be employed. In both cases the laser beam power is adjusted such that a substantial but not excessive degree of photobleaching is induced. x–t images are evaluated so as to determine the dependence of the scanned line intensities on the scanning time. The fluorescence decay curves obtained in this manner are evaluated in terms of diffusion coefficients and photobleaching rate constants by numerical simulation of appropriate diffusion-reaction systems. The validity of the experimental and theoretical procedures was tested by measurements on a simple well-defined model system. The results suggested that the continuous SCAMP, when using single-photon excitation, is a particularly simple and sensitive method for determining lateral diffusion in two-dimensional systems such as cell membranes. Employing two-photon excitation, on the other hand, provides the continuous SCAMP with the capability for studying three-dimensional diffusion within cells, cell organelles and similar systems by still comparatively simple means. Keywords: confocal microscopy, continuous fluorescence microphotolysis, fluorescence photobleaching, lateral diffusion, two-photon excitation