Effective synthesis, development and application of a highly fluorescent cyanine dye for antibody conjugation and microscopy imaging.

An asymmetric cyanine-type fluorescent dye was designed and synthesized via a versatile, multi-step process, aiming to conjugate with an Her2+ receptor specific antibody by an azide-alkyne click reaction. The aromaticity and the excitation and relaxation energetics of the fluorophore were characterized by computational methods. The synthesized dye exhibited excellent fluorescence properties for confocal microscopy, offering efficient applicability in in vitro imaging due to its merits such as a high molar absorption coefficient (36 816 M-1 cm-1), excellent brightness, optimal wavelength (627 nm), larger Stokes shift (26 nm) and appropriate photostability compared to cyanines. The conjugated cyanine-trastuzumab was constructed via an effective, metal-free, strain-promoted azide-alkyne click reaction leading to a regulated number of dyes being conjugated. This novel cyanine-labelled antibody was successfully applied for in vitro confocal imaging and flow cytometry of Her2+ tumor cells.

Synthesis and Application of Two-Photon Active Fluorescent Rhodol Dyes for Antibody Conjugation and In Vitro Cell Imaging.

A novel family of julolidine-containing fluorescent rhodols equipped with a wide variety of substituents was synthesized in a versatile two-step process. The prepared compounds were fully characterized and exhibited excellent fluorescence properties for microscopy imaging. The best candidate was conjugated to the therapeutic antibody trastuzumab through a copper-free strain-promoted azide-alkyne click reaction. The rhodol-labeled antibody was successfully applied for in vitro confocal and two-photon microscopy imaging of Her2+ cells.

Large-Scale 3D Two-Photon Imaging of Molecularly Identified CA1 Interneuron Dynamics in Behaving Mice.

Cortical computations are critically reliant on their local circuit, GABAergic cells. In the hippocampus, a large body of work has identified an unprecedented diversity of GABAergic interneurons with pronounced anatomical, molecular, and physiological differences. Yet little is known about the functional properties and activity dynamics of the major hippocampal interneuron classes in behaving animals. Here we use fast, targeted, three-dimensional (3D) two-photon calcium imaging coupled with immunohistochemistry-based molecular identification to retrospectively map in vivo activity onto multiple classes of interneurons in the mouse hippocampal area CA1 during head-fixed exploration and goal-directed learning. We find examples of preferential subtype recruitment with quantitative differences in response properties and feature selectivity during key behavioral tasks and states. These results provide new insights into the collective organization of local inhibitory circuits supporting navigational and mnemonic functions of the hippocampus.

Synthesis and spectroscopic characterization of novel GFP chromophore analogues based on aminoimidazolone derivatives.

In order to improve the fluorescence properties of the green fluorescent protein chromophore, p­HOBDI ((5­(4­hydroxybenzylidene)­2,3­dimethyl­3,5­dihydro­4H­imidazol­4­one), sixteen dihydroimidazolone derivates were synthesized from thiohydantoin and arylaldehydes. The synthesis developed is an efficient, novel, one-pot procedure. The study provides a detailed description of the spectroscopic characteristics of the newly synthesized compounds, using p­HOBDI as a reference. The new compounds all exhibited significantly stronger fluorescence than p­HOBDI, up to 28 times higher quantum yields. An experimental and theoretical investigation of the relationship of the fluorescence properties with the molecular structure was also carried out. A good correlation was found between the emission wavenumber and the Hammett constant of the functional group, which suggests the intermolecular charge transfer (ICT) mechanism between the aromatic groups.