Methodology of using raman imaging microscopy for evaluating drug action within living cells

Abstract

A method of using Raman imaging microscopy to evaluate drug actions in living cells is disclosed. Specifically the invention describes the methods of using Raman imaging microscopy to detect drug uptake, distribution, binding, and metabolism in a single cell, and to study drug pharmacokinetics at the cellular level. The method involves measuring the Raman image of both the drug and the cell. Control images and post-treatment images of the cell were studied. Ratio images were calculated and the requisite information was obtained from a study of the intensity of the bright areas in the ratio images.

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         15 . A method of using Raman imaging microscopy for evaluating drug action within living cells comprising the steps of: 
 first measuring a Raman spectrum of said drug to determine a fingerprint of said drug;    second measuring a first direct Raman image of said living cells at a preselected Raman peak of said drug to determine a pretreatment background of said living cells and recording a corresponding visible image of said living cells;    treating a culture of said living cells with said drug to obtain treated living cells;    next measuring a second direct Raman image of said treated living cells at said preselected Raman peak of said drug to obtain post-treatment images of said living cells and recording a corresponding visible image of said living cells;    processing said post-treatment images and said pretreatment background to obtain processed post-treatment images and processed pretreatment backgrounds, respectively, to reduce imaging artifacts;    comparing said processed post-treatment images with said processed pretreatment backgrounds to resolve the changes due to drug distribution within said living cells after said treatment and    determining said drug distribution relative to cellular organelles of said living cells by comparing said Raman images with said corresponding visible images, whereby said drug distribution is used to evaluate said drug actions within said living cells.    
     
     
         16 . The method of using Raman imaging microscopy for evaluating drug action within living cells of  claim 15  wherein said processing step further comprises one or more of the following: 
 a) smoothing noise;    b) reducing blurring by the optical system;    c) correcting non-uniform illumination effects; and/or    d) eliminating fluorescence background from said post-treatment images.    
     
     
         17 . The method of using Raman imaging microscopy for evaluating drug action within living cells of  claim 15  wherein using said drug distribution to evaluate said drug action within said living cells comprises one or more of the following process steps: 
 a) estimating the cellular uptake of said drug;    b) detecting said drug distribution within said living cell;    c) determining the local binding and biochemical pathway of said drug;    d) determining the cellular resistance of said living cells to said drug;    e) analyzing the pharmacokinetics of said drug; and    f) determining the metabolism of said drug.    
     
     
         18 . The method of using Raman imaging microscopy for evaluating drug action within living cells of  claim 15  further comprising the step of plating said living cells on a dish coated with Raman inactive material to prevent Raman signals coming from said dish during said measuring steps.  
     
     
         19 . The method of using Raman imaging microscopy for evaluating drug action within living cells of  claim 15  wherein said treating step incorporates the use of a drug delivery system.