In this study, second harmonic generation (SHG) and third harmonic generation (THG) spectroscopic imaging were performed on biological samples using a femtosecond laser source in the third near-infrared (NIR) optical window (NIR-III). Using a visible-NIR spectrometer, the SHG and THG signals were simultaneously detected and were extracted using spectral analysis. Visualization of biological samples such as cultured cells (HEK293 T), mouse brain slices, and the nematode Caenorhabditis elegans was performed in a label-free manner. In particular, in an SHG image of an entire coronal brain section (8 × 6 mm2), we observed mesh-like and filamentous structures in the arachnoid mater and wall of the cerebral ventricle, probably corresponding to the collagen fibers, cilia, and rootlet. Moreover, the THG images clearly depicted the densely packed axons in the white matter and cell nuclei at the cortex of the mouse brain slice sample and lipid-rich granules such as lipid droplets inside the nematode. The observations and conclusions drawn from this technique confirm that it can be utilized for various biological applications, including in vivo label-free imaging of living animals.
A multiplex CARS imaging system, equipped with an EM-CCD camera, was developed to improve the sensitivity of backward CARS imaging in biological analysis using an inverted microscope. The signal-to-noise ratio was improved by a factor of ca. 3 compared to a conventional CCD mode through the use of EM gain. When imaging cultured epithelial cells in the backward CARS configuration, intracellular organelles such as lipid droplets and nuclei were spectroscopically identified with an exposure time of only 100 ms/pixel.
Opalescence
of therapeutic antibody solutions is one of the concerns
in drug formulation. However, the mechanistic insights into the opalescence
of antibody solutions remain unclear. Here, we investigated the assembly
states of antibody molecules as a function of antibody concentration.
The solutions of bovine gamma globulin and human immunoglobulin G
at around 100 mg/mL showed the formation of submicron-scale network
assemblies. The network assembly resulted in the appearance of opalescence
with a transparent blue color without the precipitates of antibodies.
Furthermore, the addition of trehalose and arginine, previously known
to act as protein stabilizers and protein aggregation suppressors,
was able to suppress the opalescence arising from the network assembly.
These results will provide an important information for evaluating
and improving protein formulations.
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