Imaging The Enzymatic Activity At Tissues With Single-cell Resolution

Considering the individual heterogeneity of cells,it is impossible to provide information about the enzymatic activity at individual cells from the analysis of tissues or cell populations.Therefore,enzymatic activity at single cell level is helpful to reveal the role of enzymes in cell heterogeneity,and is of great significance for early prevention and diagnosis of diseases.Although a lot of progress has been made in detecting the enzymatic activity of single cells,the information of single cultured cells cannot explain the cell behavior in real organs or tissues where cells are interconnected.In view of the fact that tissue sections can retain all kinds of biological information in vivo,single-cell resolution imaging of enzyme activity at tissue sections is conducive to clinical disease diagnosis.Currently,a lot of progress has been made in tissue imaging;however,it is still meaningful to develop relatively easy and high-resolution methods for tissue imaging.In the first work,a method was designed to measure the enzyme activity in tissue sections with single cell resolution,which provided a new way to explain single cell behavior in organs or tissues.Based on the previous research,a microkit with a diameter of about 20 ?m was designed and developed to measure ACh E activity in mouse brain slices with single cell resolution.The corresponding kit components were poured into the capillary with 1 micron tip opening,and dropped on the designated positions of tissue slices by an electrochemical pump.The kit components contained in the droplet reacted with the target enzyme in the cell to produce hydrogen peroxide.At the same time,the enzymatic activity of multiple cells in a slice can be measured in parallel.Using fluorescence imaging technology to measure the content of hydrogen peroxide in the series of microdroplets,the distribution of the enzyme activity in tissue sections with single cell resolution is achieved.The results showed that the distribution of ACh E activity in tissues was uneven.Compared with the existing single-cell analysis,the single cell resolution analysis in tissue can provide the information closer to the real physiological state,and generate scientific data to clarify the heterogeneity of enzymes at the tissue level.In the second work,in order to further obtain the quantitative information about the distribution of enzyme activity on tissue sections,the ACh E activity in mouse brain sections was mapped by electrochemiluminescence(ECL)imaging.The high vacuum coating apparatus was used to spray gold on tissue slices to ensure the conductivity.Under the condition of an applied voltage,the ECL produced by the reaction of L012 and hydrogen peroxide on the ITO electrode was recorded in an image,so that the content of hydrogen peroxide on the tissue surface can be analyzed.Acetylcholine reacted with ACh E on the membrane to form choline,which reacted with choline oxidase to form hydrogen peroxide.In view of the positive correlation between the intensity ratio of luminescence difference and the concentration of hydrogen peroxide,the enzyme activity in tissue sections can be obtained based on the ECL intensity in the image.The successful completion of this work indicates that ECL can offer molecular distribution imaging with biological information on tissue sections,which opens up a new application prospect for ECL imaging.