| Optically transparent technology mitigates light scattering in complex biological tissue samples,enabling optical imaging of large/high-thickness tissue samples without physical slicing for high-resolution,high-depth visualization of 3D detail structures.The optically transparent method is classified into a water-based transparent reagent and an organic transparent reagent depending on the components of the transparent reagent.According to the mesoscopic hierarchical tissue sample(mm-cm level),combined with the target molecular technology in the fluorescent labeled tissue sample,the imaging target contains the fluorescent protein in the tissue sample,considering the practical application of safety,simplicity,cheapness,easy operation and other factors.The water-based optically transparent method was selected for the study of mesoscopic biological samples and further development.The high-refractive index,high-permeability water-based optically transparent reagent can make biological tissue samples more transparent and transparent,and the transparency effect is better.Based on the basic principle of optical transparency,based on the physicochemical properties of chemical reagents,polyhydroxy sugars are selected.An optically transparent reagent library is established by a sugar alcohol reagent,a small molecule reagent,a surface detergent,etc.,and an optically transparent method with a fast and transparent effect on the target sample is established.(1)To compare the transparency effects of different polyhydroxy sugars and sugar alcohol reagents on isolated rat ears,and to study the optical transparency of the coordination of different sugars with small molecules and detergents.Developed a water-based optically transparent reagent that is highly transparent and safe for the skin of living mouse ears.It has been developed to achieve rapid and transparent micro-vessels and skin in a short time(10 minutes);blood circulation in the main blood vessels of the rat ear is smooth,and the research on the structure of the mouse ear,the structure of microvessels,and the blood oxygen concentration in blood vessels can be achieved.Application prospects.(2)The urea-based optically transparent reagent will cause a large expansion of the sample due to the hydration of urea during the optical transparency of the tissue sample.Due to the structural complexity of the biological tissue sample,the uneven deformation during the expansion and contraction stages increases the difficulty of registration between the measurement data before and after the sample is transparent,and is also proven to affect the detailed structure and organization of the sample.By adding the selected polyhydroxy sugars,we developed the DASMU reagent to achieve the shrinkage phase equilibrium caused by the expansion and concentration differences caused by hydration during the optical transparency of tissue samples,and to achieve a sample expansion throughout the optically transparent process.100%.Our research and development can quickly make the tissue sample transparent,the tissue expansion ratio is nearly 100%,the tissue detail structure is well preserved,and the optically transparent treatment of 1mm sample for 12 h,the fluorescent protein preservation degree is nearly 75%,and the 1mm DiI labeled brain slice is transparently treated for 2 hours.DiI-labeled neurons are observed as water-based optically transparent methods that are relatively compatible with DiI lipophilic dye s.(3)In the field of neuroscience,fluorescent proteins(FPs)are often used together with transgenic labeling methods to express targeted neurons,so FP preservation is very important.From a practical point of view,most experimental systems are non-transgenic,with lipophilic fluorescent dyes.Therefore,there is a need to achieve compatibility of optically clear methods with lipophilic fluorescent dyes.For DASMU optically clear reagents that do not preserve fluorescent proteins for long periods of time,we have studied water-based optically transparent methods that preserve fluorescent proteins for up to several days.The DASMU reagent for detergent addition does have a certain cleaning ability for lipophilic dyes.The compatibility of optically clear reagents with different detergent types and concentrations with different lipophilic fluorescent dyes DiI/CM-DiI labeled neurons was studied.A water-based optically transparent method that completely preserves the neuronal structure of the DiI/CM-DiI label.Our new optical transparent reagent DASMT can achieve transparent treatment of 1d tissue samples with fluorescence preservation of up to 95%,transparent treatment of 7d tissue samples with fluorescence preservation of 85%;and compatibilit y with DiI/CM-DiI labeled neurons is very good,basically not Will damage the membrane structure.In summary,this paper studies water-based optical transparency methods for mesoscopic tissue samples.It can realize the transparent treatment of living mouse ears,realize the speckle optical imaging of rat ear blood vessels,and conduct related research.Studying urea-based optical transparency method to solve the tissue expansion caused by urea,achieving the expansion ratio of tissue samples in the optical transparent process is nearly 100%,and it is better to preserve the fluorescent protein within 12 h.This transparent technology is suitable for Alzheimer's disease-related medicine.R&D and smooth muscle cell staining have been used to study the structural information of biological tissue samples.In the practical research related to biomedicine,good experimental results have been obtained.To study the compatibility of new detergents with different detergent types and concentrations with different lipophilic fluore scent dyes DiI/CM-DiI labeled neurons,and to develop a protein growth ratio of nearly 85% in 7d.Maintaining nearly 100% of new optically transparent reagents,this transparent technology has achieved good experimental results in biomedical research and has broad application prospects in biomedical research. |
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