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      Label-free imaging of neurotransmitters in live brain tissue by multi-photon ultraviolet microscopy

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          Visualizing small biomolecules in living cells remains a difficult challenge. Neurotransmitters provide one of the most frustrating examples of this difficulty, as our understanding of signaling in the brain critically depends on our ability to follow the neurotransmitter traffic. Last two decades have seen considerable progress in probing some of the neurotransmitters, e.g. by using false neurotransmitter mimics, chemical labeling techniques, or direct fluorescence imaging. Direct imaging harnesses the weak UV fluorescence of monoamines, which are some of the most important neurotransmitters controlling mood, memory, appetite, and learning. Here we describe the progress in imaging of these molecules using the least toxic direct excitation route found so far, namely multi-photon (MP) imaging. MP imaging of serotonin, and more recently that of dopamine, has allowed researchers to determine the location of the vesicles, follow their intracellular dynamics, probe their content, and monitor their release. Recent developments have even allowed ratiometric quantitation of the vesicular content. This review shows that MP ultraviolet (MP-UV) microscopy is an effective but underutilized method for imaging monoamine neurotransmitters in neurones and brain tissue.

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          We introduce a method for optically imaging intracellular proteins at nanometer spatial resolution. Numerous sparse subsets of photoactivatable fluorescent protein molecules were activated, localized (to approximately 2 to 25 nanometers), and then bleached. The aggregate position information from all subsets was then assembled into a superresolution image. We used this method--termed photoactivated localization microscopy--to image specific target proteins in thin sections of lysosomes and mitochondria; in fixed whole cells, we imaged vinculin at focal adhesions, actin within a lamellipodium, and the distribution of the retroviral protein Gag at the plasma membrane.
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            A new generation of Ca2+ indicators with greatly improved fluorescence properties.

            A new family of highly fluorescent indicators has been synthesized for biochemical studies of the physiological role of cytosolic free Ca2+. The compounds combine an 8-coordinate tetracarboxylate chelating site with stilbene chromophores. Incorporation of the ethylenic linkage of the stilbene into a heterocyclic ring enhances the quantum efficiency and photochemical stability of the fluorophore. Compared to their widely used predecessor, "quin2", the new dyes offer up to 30-fold brighter fluorescence, major changes in wavelength not just intensity upon Ca2+ binding, slightly lower affinities for Ca2+, slightly longer wavelengths of excitation, and considerably improved selectivity for Ca2+ over other divalent cations. These properties, particularly the wavelength sensitivity to Ca2+, should make these dyes the preferred fluorescent indicators for many intracellular applications, especially in single cells, adherent cell layers, or bulk tissues.
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              Breaking the diffraction resolution limit by stimulated emission: stimulated-emission-depletion fluorescence microscopy.

              We propose a new type of scanning fluorescence microscope capable of resolving 35 nm in the far field. We overcome the diffraction resolution limit by employing stimulated emission to inhibit the fluorescence process in the outer regions of the excitation point-spread function. In contrast to near-field scanning optical microscopy, this method can produce three-dimensional images of translucent specimens.

                Author and article information

                Neuronal Signal.
                Neuronal Signaling
                Neuronal Signal.
                Portland Press Ltd.
                12 November 2018
                03 December 2018
                21 December 2018
                : 2
                : 4
                Department of Chemical Sciences, Tata Institute of Fundamental Research, Homi Bhabha Road, Colaba, Mumbai 400005, India
                Author notes
                Correspondence: Sudipta Maiti ( maiti@ )
                © 2018 The Author(s).

                This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution License 4.0 (CC BY).

                Pages: 14
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