Spectroscopic methods to detect and characterize microorganisms

Abstract

Methods and systems for Resonant Raman spectroscopy are provided. Methods according to certain embodiments include irradiating a sample with a monochromatic light source at a first irradiation intensity and a second irradiation intensity, determining the intensity of one or more of the Resonant Raman scattering and fluorescence scattering at the first irradiation intensity and second irradiation intensity, calculating a rate of change of one or more of the intensity of Resonant Raman scattering and fluorescence in response to the change in irradiation intensity from the first irradiation intensity to the second irradiation intensity and comparing one or more of the rate of change in the intensity of Resonant Raman scattering and the rate of change in the intensity of fluorescence scattering with the rate of change in the irradiation intensity by the monochromatic light source to determine the Resonant Raman response of the sample. Methods also include determining the presence or absence of a microorganism in a sample and correcting for variations associated with measurement instrumentation (e.g., monochromatic light source) and variations associated with the sample (e.g., fluorescence from non-target compounds). Also provided are methods for determining the antimicrobial susceptibility of a microorganism to an antimicrobial agent as well as methods for characterizing a phenotype of an unknown microorganism in a sample. Systems for practicing the subject methods are also provided.

1 - 22 . (canceled) 
     
     
         23 . A system comprising:
 (a) a monochromatic light source;   (b) an optical adjustment component;   (c) a photodetector; and   (d) a processor comprising memory operably coupled to the processor wherein the memory comprises instructions stored thereon, which when executed by the processor, cause the processor to:
 irradiate a sample in a sample holder with the monochromatic light source with either an invariant light intensity or one that varies over time at an interface between the sample and a surface of the sample holder; 
 measure scattered light from the sample with the photodetector at a plurality of different times; 
 determine the intensity of Resonant Raman scattering and fluorescence scattering; 
 calculate a rate of change in the intensity of Resonant Raman scattering and the intensity of fluorescence scattering; and 
 correct the calculated rate of change of the intensity of Resonant Raman scattering and the intensity of fluorescence scattering to obtain a net signal. 
   
     
     
         24 . The system according to  claim 23 , wherein the optical adjustment component comprises collimating optics mounted on a linear table that is configured to produce an adjustable focal spot. 
     
     
         25 . The system according to  claim 23 , wherein the optical adjustment component further comprises a mechanical shutter. 
     
     
         26 . The system according to  claim 23 , further comprising a rotary table with a plurality of sample chambers. 
     
     
         27 . The system according to  claim 26 , wherein the rotary table comprises 8 sample chambers. 
     
     
         28 . The system according to  claim 27 , further comprising a subsystem for characterizing incoherently scattered light, wherein the subsystem comprises:
 collimating optics; and   optical adjustment components configured to spread the incoherently scattered light onto a linear array of photodetectors.   
     
     
         29 . The system according to  claim 28 , wherein the processor further comprises memory with instructions stored thereon, which when executed by the processor, cause the processor to move the collimating optics to focus light on each of the sample chambers. 
     
     
         30 . The system according to  claim 23 , wherein the sample further comprises an albumin with an incorporated ligand. 
     
     
         31 . The system according to  claim 23 , wherein the memory further comprises instructions which when executed by the processor, cause the processor to:
 determine a rate of change in the intensity of fluorescent scattering (or any other optical output) from the reagent vial;   calculate a net signal as the rate of change in the intensity of the Resonant Raman scattering minus the rate of change in the intensity of fluorescent scattering from the standard sample.   
     
     
         32 . The system according to  claim 23 , wherein the memory further comprises instructions which when executed by the processor, cause the processor to:
 determine a rate of change in total output from a standard reference sample;   calculate a net signal as the rate of change in the intensity of the Resonant Raman scattering minus the rate of change in the intensity of Resonant Raman scattering from the standard sample.   
     
     
         33 . The system according to  claim 31 , wherein the memory further comprises instructions which when executed by the processor, cause the processor to compare the net signal with preset thresholds, and to determine that the sample includes actively metabolizing microorganisms based on the comparison. 
     
     
         34 . The system according to  claim 23 , wherein the sample further comprises a reducing agent. 
     
     
         35 . The system according to  claim 34 , wherein the sample further comprises a disulfide crosslinking agent. 
     
     
         36 . The system according to  claim 35 , where the disulfide crosslinking agent further comprises a core that is cleaved by enzymes or a metabolite produced by the microorganism in the sample. 
     
     
         37 . The system according to  claim 35 , wherein the crosslinking agent comprises a compound of Formula (I): 
       
         
           
           
               
               
           
         
       
     
     
         38 . The system according to  claim 23 , wherein the sample holder is a glass vial. 
     
     
         39 . The system according to  claim 23 , wherein the sample holder further comprises a coating that modifies albumin absorption. 
     
     
         40 . The system according to  claim 39 , wherein the coating comprises a Zwitterionic coating. 
     
     
         41 . The system according to  claim 23 , wherein the processor is configured to expose the sample to the monochromatic light source to minimize the net signal. 
     
     
         42 . (canceled) 
     
     
         43 . The system according to  claim 42 , wherein the system is configured to maximize the net signal.