Method for detecting the impacts of interfering effects on experimental data

Abstract

The invention provides a method for identifying the impacts of interfering effects on experimental data. In particular, a method is described for identifying the impacts of unwanted auto-fluorescence, fluorescence quenching, and deterioration of a fluorescent sample under study on the collected experimental data. The data are analyzed whether or not said data fulfill certain criteria with respect to a threshold which is indicative for said interfering effect.

1 . A method for detecting the impacts of interfering effects on data, such as experimental data, comprising the steps of: 
 (i) providing the data,    (ii) determining values of one or a plurality of identification parameters from said data,    (iii) creating a histogram or distribution of the values of the identification parameters,    (iv) determining one or a plurality of thresholds for the values of identification parameters from said histogram or distribution, which thresholds are indicative for the interfering effects,    (v) analyzing the values of one or a plurality of identification parameters whether or not these values fulfill one or a plurality of criteria with respect to the thresholds, and    (vi) determining those data which are influenced and/or those data which are not affected by the interfering effects.    
     
     
         2 . The method of  claim 1  wherein the data provided comprise a plurality of data sets, wherein values of one or a plurality of identification parameters from each of the data sets are determined, and wherein those data sets which are influenced and/or those data sets which are not affected by the interfering effects are determined.  
     
     
         3 . The method according to  claim 2  wherein the histogram or distribution of the values of the identification parameters is created from all of the data sets.  
     
     
         4 . The method according to  claim 2  wherein the histogram or distribution of the values of the identification parameters is created from a subgroup of the data sets.  
     
     
         5 . The method according to any one of  claims 1  to  4  wherein values of a single identification parameter are determined and a one-dimensional histogram or distribution of these values of the identification parameter is created.  
     
     
         6 . The method according to any one of  claims 1  to  4  wherein values of a plurality of identification parameters are determined and a multi-dimensional histogram or distribution of these values of the identification parameters is created.  
     
     
         7 . The method according to any one of  claims 1  to  6  wherein the experimental data result from fluorescence measurements on fluorescent samples.  
     
     
         8 . The method according to  claim 7  wherein the interfering effects result from unwanted auto-fluorescence of material present in the samples and/or from unwanted fluorescence quenching.  
     
     
         9 . The method according to any one of  claims 1  to  8  wherein the interfering effects result from measuring errors or errors in the preparation of the samples used to gather the experimental data, in particular from dispensing or pipetting errors.  
     
     
         10 . The method according to any one of  claims 1  to  9  wherein the identification parameter is selected from the group consisting of a fluorescence intensity, a ratio of fluorescence intensities at selected wavelengths, a ratio of fluorescence intensities at different polarization directions, a fluorescence anisotropy, a fluorescence polarization, a fluorescence lifetime, a rotational correlation time, a diffusion constant, a concentration of fluorophores, and a specific fluorescence brightness.  
     
     
         11 . The method according to any one of  claims 1  to  9  wherein the identification parameter is a function of a fluorescence intensity, a ratio of fluorescence intensities at selected wavelengths, a ratio of fluorescence intensities at different polarization directions, a fluorescence anisotropy, a fluorescence polarization, a fluorescence lifetime, a rotational correlation time, a diffusion constant, a concentration of fluorophores, or a specific fluorescence brightness.  
     
     
         12 . The method according to any one of  claims 1  to  9  wherein the identification parameter is a parameter resulting from a fit to a lifetime histogram, a fluorescence correlation function, a FIDA histogram, a 2D-FIDA histogram, a FIMDA histogram or a FILDA histogram.  
     
     
         13 . The method according to any one of  claims 1  to  9  wherein the identification parameters result from a moment-analysis to a lifetime histogram, a fluorescence correlation function, a FIDA-, 2D-FIDA-, FIMDA-, or FILDA-histogram by calculating moments, correlations, cumulants, or functions of these.  
     
     
         14 . The method according to any one of  claims 1  to  13  further comprising the performance of a correction step on those data which have been identified as being influenced by the interfering effects.  
     
     
         15 . The method according to  claim 14  further comprising the performance of a control procedure on the corrected data to check whether or not the correction procedure has succeeded.  
     
     
         16 . The method according to  claim 14  wherein the control procedure comprises the steps of: 
 (i) providing data including, but not limited to, the corrected data,  
 (ii) determining values of one or a plurality of identification parameters from said data,  
 (iii) creating a histogram or distribution of the values of the identification parameters,  
 (iv) determining one or a plurality of thresholds for the values of identification parameters from said histogram or distribution, which thresholds are indicative for the success of the correction,  
 (v) analyzing the values of one or a plurality of identification parameters whether or not these fulfill one or a plurality of criteria with respect to the thresholds, and  
 (vi) determining the data on which the correction procedure has succeeded and/or the data on which the correction procedure has failed.  
 
     
     
         17 . The method according to any one of  claims 1  to  16  wherein the experimental data result from screening of potentially pharmaceutical active compounds and the data which are influenced by the interfering effects represent false-positive or false-negative results.  
     
     
         18 . A method for detecting the impacts of auto-fluorescence and/or fluorescence quenching on experimental data resulting from fluorescence experiments comprising the steps of: 
 (i) providing the experimental data comprising a plurality of data sets,    (ii) determining values of one or a plurality of identification parameters from said data sets,    (iii) creating a histogram or distribution of the values of the identification parameters,    (iv) determining one or a plurality of first thresholds for the values of identification parameters from said histogram or distribution, which first thresholds are indicative for auto-fluorescence, and/or determining one or a plurality of second thresholds for the values of identification parameters from said histogram or distribution, which second thresholds are indicative for fluorescence quenching,    (v) analyzing the values of one or a plurality of identification parameters whether or not these fulfill one or a plurality of criteria with respect to the thresholds, and    (vii) determining those data sets which are influenced and/or those data sets which are not affected by auto-fluorescence and/or fluorescence quenching.    
     
     
         19 . The method according to  claim 18  wherein the histogram or distribution of the values of the identification parameters is created from all of the data sets.  
     
     
         20 . The method according to  claim 18  wherein the histogram or distribution of the values of the identification parameters is created from a subgroup of the data sets.  
     
     
         21 . The method according to any one of  claims 18  to  20  wherein values of a single identification parameter are determined and a one-dimensional histogram or distribution of these values of the identification parameter is created.  
     
     
         22 . The method according to any one of  claims 18  to  20  wherein values of a plurality of identification parameters are determined and a multi-dimensional histogram or distribution of these values of the identification parameters is created.  
     
     
         23 . The method according to any one of  claims 18  to  22  wherein the identification parameter is selected from the group consisting of a fluorescence intensity, a ratio of fluorescence intensities at selected wavelengths, a ratio of fluorescence intensities at different polarization directions, a fluorescence anisotropy, a fluorescence polarization, a fluorescence lifetime, a rotational correlation time, a diffusion constant, a concentration of fluorophores, and a specific fluorescence brightness.  
     
     
         24 . The method according to any one of  claims 18  to  22  wherein the identification parameter is a function of a fluorescence intensity, a ratio of fluorescence intensities at selected wavelengths, a ratio of fluorescence intensities at different polarization directions, a fluorescence anisotropy, a fluorescence polarization, a fluorescence lifetime, a rotational correlation time, a diffusion constant, a concentration of fluorophores, or a specific fluorescence brightness.  
     
     
         25 . The method according to any one of  claims 18  to  22  wherein the identification parameter is a parameter resulting from a fit to a lifetime histogram, a fluorescence correlation function, a FIDA histogram, a 2D-FIDA histogram, a FIMDA histogram or a FILDA histogram.  
     
     
         26 . The method according to any one of  claims 18  to  22  wherein the identification parameters result from a moment-analysis to a lifetime histogram, a fluorescence correlation function, a FIDA-, 2D-FIDA-, FIMDA-, or FILDA-histogram by calculating moments, correlations, cumulants, or functions of these.  
     
     
         27 . A method for detecting false positive and/or false negative results in data, in particular experimental data resulting from screening of potentially pharmaceutical active compounds, comprising the steps of: 
 (i) providing the data,    (ii) determining values of one or a plurality of identification parameters from said data,    (iii) creating a histogram or distribution of the values of the identification parameters,    (iv) determining one or a plurality of first thresholds for the values of identification parameters from said histogram or distribution, which first thresholds are indicative for a false-positive result, and/or determining one or a plurality of second thresholds for the values of identification parameters from said histogram or distribution, which second thresholds are indicative for a false-negative result,    (v) analyzing the values of one or a plurality of identification parameters whether or not these fulfill one or a plurality of criteria with respect to the thresholds, and    (vi) determining those data which represent a false-positive result and/or those data which represent a false-negative result.    
     
     
         28 . The method according to  claim 27  wherein the data provided comprise a plurality of data sets, wherein values of one or a plurality of identification parameters from each of the data sets are determined, and wherein those data sets which represent false-positive results and/or those data sets which represent false-negative results are determined.  
     
     
         29 . The method according to  claim 28  wherein the histogram or distribution of the values of the identification parameters is created from all of the data sets.  
     
     
         30 . The method according to  claim 28  wherein the histogram or distribution of the values of the identification parameters is created from a subgroup of the data sets.  
     
     
         31 . The method according to any one of  claims 27  to  30  wherein values of a single identification parameter are determined and a one-dimensional histogram or distribution of these values of the identification parameter is created.  
     
     
         32 . The method according to any one of  claims 27  to  30  wherein values of a plurality of identification parameters are determined and a multi-dimensional histogram or distribution of these values of the identification parameters is created.  
     
     
         33 . The method according to any one of  claims 27  to  32  wherein the identification parameter is selected from the group consisting of a fluorescence intensity, a ratio of fluorescence intensities at selected wavelengths, a ratio of fluorescence intensities at different polarization directions, a fluorescence anisotropy, a fluorescence polarization, a fluorescence lifetime, a rotational correlation time, a diffusion constant, a concentration of fluorophores, and a specific fluorescence brightness.  
     
     
         34 . The method according to any one of  claims 27  to  32  wherein the identification parameter is a function of a fluorescence intensity, a ratio of fluorescence intensities at selected wavelengths, a ratio of fluorescence intensities at different polarization directions, a fluorescence anisotropy, a fluorescence polarization, a fluorescence lifetime, a rotational correlation time, a diffusion constant, a concentration of fluorophores, or a specific fluorescence brightness.  
     
     
         35 . The method according to any one of  claims 27  to  32  wherein the identification parameter is a parameter resulting from a fit to a lifetime histogram, a fluorescence correlation function, a FIDA histogram, a 2D-FIDA histogram, a FIMDA histogram or a FILDA histogram.  
     
     
         36 . The method according to any one of  claims 27  to  32  wherein the identification parameters result from a moment-analysis to a lifetime histogram, a fluorescence correlation function, a FIDA-, 2D-FIDA-, FIMDA-, or FILDA-histogram by calculating moments, correlations, cumulants, or functions of these.  
     
     
         37 . A system for detecting the impacts of interfering effects on experimental data resulting from optical experiments, said system comprising: 
 (i) means for supporting one or a plurality of samples in an inspection station ( 2 ),    (ii) one or a plurality of photosensitive detectors ( 9 ,  10 ) which are positioned relative to the inspection station ( 2 ) so that electromagnetic radiation emitted from the samples impinges on the detectors ( 9 ,  10 ), and a    (iii) signal processing unit ( 11 ) comprising 
 means for addressing the photosensitive detectors ( 9 ,  10 ) to generate experimental data,  
 means for determining values of one or a plurality of identification parameters from said data,  
 means for storing the values in such a manner that preferably all the values which relate to any one of the samples are linked,  
 means for creating a histogram or distribution of the values of the identification parameters,  
 means for determining one or a plurality of thresholds for the values of identification parameters from said histogram or distribution, which thresholds are indicative for the interfering effects,  
 means for analyzing the values of one or a plurality of identification parameters whether or not these fulfill one or a plurality of criteria with respect to the thresholds, and  
 means for supplying as output information those data which are influenced and/or those data which are not affected by the interfering effects.  
   
     
     
         38 . The system of  claim 37  comprising a fluorescence reader.  
     
     
         39 . The system of  claim 38  wherein the fluorescence reader comprises a confocal optical set-up.  
     
     
         40 . The system according to any one of  claims 37  to  39  wherein the photosensitive detector ( 9 ,  10 ) comprises an avalanche photodiode or a charged coupled device (CCD) camera.  
     
     
         41 . A signal processing unit ( 11 ) for processing experimental data which may be deteriorated by interfering effects comprising 
 means for storing experimental data,    means for determining values of one or a plurality of identification parameters from said data,    means for storing the values in such a manner that preferably all the values which relate to any one of the samples are linked,    means for creating a histogram or distribution of the values of the identification parameters,    means for determining one or a plurality of thresholds for the values of identification parameters from said histogram or distribution, which thresholds are indicative for the interfering effects,    means for analyzing the values of one or a plurality of identification parameters whether or not these fulfill one or a plurality of criteria with respect to the thresholds, and    means for supplying as output information those data which are influenced and/or those data which are not affected by the interfering effects.    
     
     
         42 . Use of the system according to  claim 37  for the conductance of the method according to any one of  claims 1  to  36 .  
     
     
         43 . Use of the signal processing unit according to  claim 41  for the conductance of the method according to any one of  claims 1  to  36 .  
     
     
         44 . The method according to any one of  claims 1  to  36  wherein the data are provided by measuring samples in a measuring device, such as a fluorescence reader.