Hydrodynamic focusing capable of readily producing and controlling laminar flow facilitates drug treatment of cells in existing microfluidic culture devices. However, to expand applications of such devices to multiparameter drug testing, critical limitations in current hydrodynamic focusing microfluidics must be addressed. Here we describe hydrodynamic focusing and shifting as an advanced microfluidics tool for spatially selective drug delivery and integrative cell-based drug testing. We designed and fabricated a co-flow focusing, three-channel microfluidic device with a wide cell culture chamber. By controlling inlet flow rates of sample and two side solutions, we could generate hydrodynamic focusing and shifting that mediated precise regulation of the path and width of reagent and drug stream in the microfluidic device. We successfully validated a hydrodynamic focusing and shifting approach for spatially selective delivery of DiI, a lipophilic fluorophore, and doxorubicin, a chemotherapeutic agent, to tumor cells in our device. Moreover, subsequent flowing of a trypsin EDTA solution over the cells that were exposed to doxorubicin flow allowed us to selectively collect the treated cells. Our approach enabled downstream high-resolution microscopy of the cell suspension to confirm the nuclear delivery of doxorubicin into the tumor cells. In the device, we could also evaluate in situ the cytotoxic effect of doxorubicin to the tumor cells that were selectively treated by hydrodynamic flow focusing and shifting. These results show that hydrodynamic focusing and shifting enable a fast and robust approach to spatially treat and then collect cells in an optimized microfluidic device, offering an integrative assay tool for efficient drug screening and discovery.
Nowadays, as a such big number of countries pay more attention to the scientific research strength, The importance of high and new technology is more and more recognized, the proportion in the securities market is increasing, technology stocks in the eyes of investors. This paper selects 5 Chinese technology growth stocks in Hang Seng Index as the research object and establish the dataset which contains the monthly time series of price in Hang Seng index. Fama experiment is used to well diversify the idiosyncratic risk and make sure that the portfolio is affected by the system risk. Through the mean-standard deviation plot, it can check the independence of the data we collect and check the normality. The portfolio can be divided into two situations: one includes risk-free assets and non risk-free assets. Finally, the performances of the portfolios are accessed including return volatility and weight of assets. The results show that, first, based on the Fama experiment, at least 5 stocks must be chosen to deal with idiosyncratic risk. Second, according to the mean-standard deviation plot,all the assets data are i.i.d. Third, the result show that both maximum return portfolios have the high return, and the portfolio contains risk-free assets is higher than portfolios without risk-free assets in the maximum return portfolio, but lower in the maximum Sharpe ratio portfolio. The findings may be useful to stimulate investors pay attention to invest in the technology stocks and create efficient portfolio. Meanwhile, this paper can suggest people how to rationally allocate stocks from multiple dimensions, build an effective combination, and try to obtain higher returns as far as possible in the condition of low risk.
Multiplexing in immunofluorescence imaging is important for the spatial profiling of cells and molecules in tumor tissue samples. Cyclic immunofluorescence (IF) methods using oxidants (e.g. hydrogen peroxide) and enzymes (e.g. DNase) localize a great number of cellular makers and proteins in a tissue section while repeating a process of IF staining, imaging, and fluorescence deactivation. However, the repeated use of chemicals and enzymes might cause artifacts in tissue and cell morphologies. Furthermore, these methods are restricted to thin tissue sections (~5 μm thick) which are inappropriate to provide comprehensive structural information on tissue samples. Although reconstruction of two-dimensional (2D) images from serial tissue sections can provide a certain volumetric tissue image, it takes a huge amount of time and effort. Here we introduce a three-dimensional (3D) multiplex IF imaging method using LED photobleaching. We built high-power LED illuminators with 100W warm (emission wavelength: 480-700 nm), green (430-520 nm), and red (600- 680 nm) LED chips, which can efficiently bleach a broad or selected wavelength of fluorescence signals in tissue samples. We integrated this LED photobleaching with the Transparent Tissue Tomography (T3) protocol and created a 3D cyclic IF method involving tissue macrosectioning (400 μm), three-color IF staining, D-fructose-based tissue clearing, 3D confocal fluorescence microscopy, LED photobleaching, tissue washing, and three-color IF staining for other biomarkers, and repeating the process. By applying this method to mouse mammary tumor tissues, we could perform 8-plex fluorescence microscopy for visualizing cell nuclei (DAPI), vascular (CD31, SMA) and structural (ER-TR7) cells, immune cells (CD3, CD8, CD45), and cancer cells (CK8) in the tumor macrosections in 3D at tissue and cellular resolution. To validate the method as an evaluation tool for immunotherapy, we treated the mouse mammary tumor with a STING agonist (DMAXX) intratumorally and collected the tumor tissue 1 day after the treatment, and processed it for the 3D cyclic IF protocol. The quantitative multiplex image data showed immune-driven-cancer eradication and high tumor infiltration of a large number of CD3+CD8+CD45+ cytotoxic T cells. We also examined that Red and Green LED illumination can selectively bleach fluorophores in tissues, which would be useful for patterning fluorescence in tissue as well as studying fluorescent drug-cell interaction in a tissue. In summary, this chemical and enzyme-free 3D cyclic IF imaging method will be a powerful tissue assay tool to provide comprehensive spatial information of tissue (tumor) samples including cell types, cellular and molecular location, and their 3D organization in a tissue sample. Citation Format: Jingtian Zheng, Evan Phillips, Yi-Chien Wu, Steve Seung-Young Lee, Vytautas Bindokas. LED photobleaching-based multiplex 3D microscopy of the tumor microenvironment. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4707.
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