Multimodal nonlinear-optical imaging of nucleoli.

Multimodal nonlinear microscopy combining third-harmonic generation (THG) with two- and three-photon-excited fluorescence (2PEF and 3PEF) is shown to provide a powerful resource for high-fidelity imaging of nucleoli and nucleolar proteins. We demonstrate that, with a suitably tailored genetically encoded fluorescent stain, the 2PEF/3PEF readout from specific nucleolar proteins can be reliably detected against the extranucleolar 2PEF/3PEF signal, enabling high-contrast imaging of the key nucleolar ribosome biogenesis components, such as fibrillarin. THG is shown to provide a versatile readout for unstained nucleolus imaging in a vast class of biological systems as different as neurons in brain slices and cultured HeLa cells.

Cell-specific three-photon-fluorescence brain imaging: neurons, astrocytes, and gliovascular interfaces.

We present brain imaging experiments on rat cortical areas, demonstrating that, when combined with a suitable high-brightness, cell-specific genetically encoded fluorescent marker, three-photon-excited fluorescence (3PEF), enables subcellular-resolution, cell-specific 3D brain imaging that is fully compatible and readily integrable with other nonlinear-optical imaging modalities, including two-photon-fluorescence and harmonic-generation microscopy. With laser excitation provided by sub-100-fs, 1.25-µm laser pulses, cell-specific 3PEF from astrocytes and their processes detected in parallel with a three-photon-resonance-enhanced third harmonic from blood vessels is shown to enable a high-contrast 3D imaging of gliovascular interfaces.

Stain-free subcellular-resolution astrocyte imaging using third-harmonic generation.

We demonstrate stain-free, high-contrast, subcellular-resolution imaging of astroglial cells using epi-detected third-harmonic generation (THG). The astrocyte-imaging capability of THG is verified by colocalizing THG images with fluorescence images of astrocytes expressing a genetically encodable fluorescent reporter. We show that THG imaging with an optimized point-spread function can reliably detect significant subcellular features of astrocytes, including cell nuclei, as well as the soma shape and boundaries.

[Genetically Encoded Fluorescent Redox Sensors].

Redox processes play a key role in cells of all.organisms. These processes imply directed flows of electrons via so-called redox pairs: substances that exist in both reduced and oxidized states simultaneously within the cell. Examples of redox pairs are NAD+/NADH, NADP+/NADPH, GSSG/2GSH. Until recently, studies of redox processes in the living cells were challenged by the lack of suitable methods. Genetically encoded fluorescent biosensors provide a new way to study biological processes including redox ones. Biosensors allow real-time detection of messengers, metabolites and enzymatic activities in living systems of different complexity from cultured cells to transgenic animals. In this review, we describe the main types of known redox biosensors with examples of their use.

[Main Cellular Redox Couples].

Most of the living cells maintain the continuous flow of electrons, which provides them by energy. Many of the compounds are presented in a cell at the same time in the oxidized and reduced states, forming the active redox couples. Some of the redox couples, such as NAD+/NADH, NADP+/NADPH, oxidized/reduced glutathione (GSSG/GSH), are universal, as they participate in adjusting of many cellular reactions. Ratios of the oxidized and reduced forms of these compounds are important cellular redox parameters. Modern research approaches allow setting the new functions of the main redox couples in the complex organization of cellular processes. The following information is about the main cellular redox couples and their participation in various biological processes.

SypHer3s: a genetically encoded fluorescent ratiometric probe with enhanced brightness and an improved dynamic range.

We designed a genetically encoded ratiometric fluorescent probe, SypHer3s, with enhanced brightness and optimized pKa, which responds to pH changes in different cellular compartments. SypHer3s was successfully utilized for imaging the pH dynamics in mitochondria of living neurons and in quantitative pH measurement in zebrafish embryos.