17,521 research outputs found

    Confocal and multiphoton imaging of intracellular Ca<sup>2+</sup>

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    This chapter compares the imaging capabilities of a range of systems including multiphoton microscopy in regard to measurements of intracellular Ca&lt;sup&gt;2+&lt;/sup&gt; within living cells. In particular, the excitation spectra of popular fluorescent Ca&lt;sup&gt;2+&lt;/sup&gt; indicators are shown during 1P and 2P excitation. The strengths and limitations of the current indicators are discussed along with error analysis which highlights the value of matching the Ca&lt;sup&gt;2+&lt;/sup&gt; affinity of the dye to a particular aspect of Ca&lt;sup&gt;2+&lt;/sup&gt; signaling. Finally, the combined emission spectra of Ca&lt;sup&gt;2+&lt;/sup&gt; and voltage sensitive dyes are compared to allow the choice of the optimum combination to allow simultaneous intracellular Ca&lt;sup&gt;2+&lt;/sup&gt; and membrane voltage measurement

    Wide-Field Multi-Parameter FLIM: Long-Term Minimal Invasive Observation of Proteins in Living Cells.

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    Time-domain Fluorescence Lifetime Imaging Microscopy (FLIM) is a remarkable tool to monitor the dynamics of fluorophore-tagged protein domains inside living cells. We propose a Wide-Field Multi-Parameter FLIM method (WFMP-FLIM) aimed to monitor continuously living cells under minimum light intensity at a given illumination energy dose. A powerful data analysis technique applied to the WFMP-FLIM data sets allows to optimize the estimation accuracy of physical parameters at very low fluorescence signal levels approaching the lower bound theoretical limit. We demonstrate the efficiency of WFMP-FLIM by presenting two independent and relevant long-term experiments in cell biology: 1) FRET analysis of simultaneously recorded donor and acceptor fluorescence in living HeLa cells and 2) tracking of mitochondrial transport combined with fluorescence lifetime analysis in neuronal processes

    Detection of Single Molecules Illuminated by a Light-Emitting Diode

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    Optical detection and spectroscopy of single molecules has become an indispensable tool in biological imaging and sensing. Its success is based on fluorescence of organic dye molecules under carefully engineered laser illumination. In this paper we demonstrate optical detection of single molecules on a wide-field microscope with an illumination based on a commercially available, green light-emitting diode. The results are directly compared with laser illumination in the same experimental configuration. The setup and the limiting factors, such as light transfer to the sample, spectral filtering and the resulting signal-to-noise ratio are discussed. A theoretical and an experimental approach to estimate these parameters are presented. The results can be adapted to other single emitter and illumination schemes.Comment: 7 pages, 5 figure

    Visible spectrum extended-focus optical coherence microscopy for label-free sub-cellular tomography

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    We present a novel extended-focus optical coherence microscope (OCM) attaining 0.7 {\mu}m axial and 0.4 {\mu}m lateral resolution maintained over a depth of 40 {\mu}m, while preserving the advantages of Fourier domain OCM. Our method uses an ultra-broad spectrum from a super- continuum laser source. As the spectrum spans from near-infrared to visible wavelengths (240 nm in bandwidth), we call the method visOCM. The combination of such a broad spectrum with a high-NA objective creates an almost isotropic 3D submicron resolution. We analyze the imaging performance of visOCM on microbead samples and demonstrate its image quality on cell cultures and ex-vivo mouse brain tissue.Comment: 15 pages, 7 figure
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