The combination of multiphoton microscopy under two- and three-photon excitation of specially selected fluorescent protein sensors as well as second and third harmonic generation microscopy opened up the possibility to study the dynamics of oxidative processes with subcellular resolution in a mouse brain in vivo.
At present the technology of identifying the objects by their radiation in radio band is developing rapidly. The use of submillimeter band allowed approaching the radio bright image to the photographic one. The use of decimeter and centimeter bands allowed identifying the objects under ground. In the article the theoretical foundation of identifying and recognizing objects on the ground, on water and under ground is presented. Estimated values of radio bright contrasts of various objects on the surface and under surface of the ground are presented. In the article the results of flight (aircraft and space) experiments of measuring radio bright image over ground, above water and under ground are given.
We demonstrate label-free imaging of genetically induced hepatocellular carcinoma (HCC) in a murine model provided by two- and three-photon fluorescence microscopy of endogenous fluorophores excited at the central wavelengths of 790, 980 and 1250 nm and reinforced by second and third harmonic generation microscopy. We show, that autofluorescence imaging presents abundant information about cell arrangement and lipid accumulation in hepatocytes and hepatic stellate cells (HSCs), harmonics generation microscopy provides a versatile tool for fibrogenesis and steatosis study. Multimodal images may be performed by a single ultrafast laser source at 1250 nm falling in tissue transparency window. Various grades of HCC are examined revealing fibrosis, steatosis, liver cell dysplasia, activation of HSCs and hepatocyte necrosis, that shows a great ability of multimodal label-free microscopy to intravital visualization of liver pathology development.
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.
Abstract We demonstrate a versatile single‐laser platform for single‐beam dual‐color two‐photon spectroscopy that combines a short‐pulse laser source with a tunable broadband wavelength converter based on a highly nonlinear photonic‐crystal fiber (PCF). We show that the short‐pulse PCF output can be tailored, via dispersion and nonlinearity management, to deliver a broadband optical driver whose spectral structure is ideally suited for a single‐beam two‐photon absorption (2PA) spectroscopy of the next‐generation genetically encodable fluorescent‐protein (FP) sensors of pH and redox‐responsive contrast agents. As a promising spectroscopic resource for redox biology, the short‐pulse PCF output can be finely sculpted to alternately drive an FP system via a one of its two 2PA‐allowed quantum pathways, yielding a high‐contrast fluorescence readout for a highly sensitive detection of redox reactions and signaling, pH sensing, and oxidative‐stress diagnosis.
Materials about possibilities of object detection, correlated with its radiobrightness contrast to surroundings, are presented. The results of investigation of possibilities of biological object detection for objects, shaded by vegetation, are presented. The conclusion was made, that biological object (a man), shaded by vegetation, can be detected from a distance up to 15 km, if attenuation of electromagnetic waves, correlated with vegetation, is not more -25 dB. The experimental results of object detection are presented.
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.
Abstract Methods of nonlinear optics provide a vast arsenal of tools for label‐free brain imaging, offering a unique combination of chemical specificity, the ability to detect fine morphological features, and an unprecedentedly high, subdiffraction spatial resolution. While these techniques provide a rapidly growing platform for the microscopy of neurons and fine intraneural structures, optical imaging of astroglia still largely relies on filament‐protein‐antibody staining, subject to limitations and difficulties especially severe in live‐brain studies. Once viewed as an ancillary, inert brain scaffold, astroglia are being promoted, as a part of an ongoing paradigm shift in neurosciences, into the role of a key active agent of intercellular communication and information processing, playing a significant role in brain functioning under normal and pathological conditions. Here, we show that methods of nonlinear optics provide a unique resource to address long‐standing challenges in label‐free astroglia imaging. We demonstrate that, with a suitable beam‐focusing geometry and careful driver‐pulse compression, microscopy of second‐harmonic generation (SHG) can enable a high‐resolution label‐free imaging of fibrillar structures of astrocytes, most notably astrocyte processes and their endfeet. SHG microscopy of astrocytes is integrated in our approach with nonlinear‐optical imaging of red blood cells based on third‐harmonic generation (THG) enhanced by a three‐photon resonance with the Soret band of hemoglobin. With astroglia and red blood cells providing two physically distinct imaging contrasts in SHG and THG channels, a parallel detection of the second and third harmonics enables a high‐contrast, high‐resolution, stain‐free stereoimaging of gliovascular interfaces in the central nervous system. Transverse scans of the second and third harmonics are shown to resolve an ultrafine texture of blood‐vessel walls and astrocyte‐process endfeet on gliovascular interfaces with a spatial resolution within 1 μm at focusing depths up to 20 μm inside a brain .
We demonstrate an accurate quantitative characterization of absolute two- and three-photon absorption (2PA and 3PA) action cross sections of a genetically encodable fluorescent marker Sypher3s. Both 2PA and 3PA action cross sections of this marker are found to be remarkably high, enabling high-brightness, cell-specific two- and three-photon fluorescence brain imaging. Brain imaging experiments on sliced samples of rat's cortical areas are presented to demonstrate these imaging modalities. The 2PA action cross section of Sypher3s is shown to be highly sensitive to the level of pH, enabling pH measurements via a ratiometric readout of the two-photon fluorescence with two laser excitation wavelengths, thus paving the way toward fast optical pH sensing in deep-tissue experiments.
'/%3e%3cpath id='b' d='M658.84 441.31c-7.618 16.446-22.845 19.271-36.164 5.995 0 0 5.354-3.783 11.086-4.826-1.075-4.574 1.13-10.902 4.235-14.324 3.258 2.227 7.804 6.689 8.96 11.874 8.03-1.04 11.883 1.282 11.883 1.282z'/%3e%3cuse xlink:href='%23b' transform='rotate(9.37 700 804)'/%3e%3cuse xlink:href='%23b' transform='rotate(18.74 700 804)'/%3e%3cpath fill='none' stroke='%23f8c300' stroke-width='16' d='M603 478a340 340 0 0 1 194 0'/%3e%3cg transform='translate(700 380)'%3e%3cg transform='translate(0 -140)'%3e%3cpath id='c' d='m0-513674 301930 929245-790463-574305h977066l-790463 574305z' transform='scale(.00005)'/%3e%3c/g%3e%3cg id='d'%3e%3cuse xlink:href='%23c' transform='translate(-70 -121.244)'/%3e%3cuse xlink:href='%23c' transform='translate(-121.244 -70)'/%3e%3cuse xlink:href='%23c' transform='translate(-140)'/%3e%3c/g%3e%3cuse xlink:href='%23d' transform='scale(-1 1)'/%3e%3c/g%3e%3c/g%3e%3c/svg%3e)