Method and apparatus for characterizing biological objects

Abstract

In order to quantitatively characterize biological objects, for example individual cells, a stimulus is applied to a biological object in a contactless fashion. A measurement and a further measurement are performed on the biological object in order to ascertain a response of the biological object to the stimulus, wherein the measurement and the further measurement comprise detecting Raman scattering on and/or in the biological object and/or capturing data using digital holographic microinterferometry (DHMI). The biological object is characterized according to a result of the measurement and is sorted if needed. The stimulus can be applied by means of a laser beam that creates optical tweezers or an optical trap, by means of ultrasonic waves or an electric or magnetic radio frequency field.

1 . A method for characterizing a biological object, the method comprising:
 applying a stimulus to the biological object in a contactless fashion;   carrying out plural measurements on the biological object to ascertain a response of the biological object to the stimulus, wherein, to ascertain the response,
 a measurement which comprises a detection of Raman scattering is performed on the biological object before application of the stimulus, and 
 a further measurement which comprises a further detection of Raman scattering is performed on the biological object after or during application of the stimulus; 
   performing a comparison of results of the measurement and the further measurement; and   characterizing the biological object as a function of the comparison of the results of the measurements.   
     
     
         2 . The method according to  claim 1 , wherein the measurement and the further measurement comprise both the detection of a Raman scattering and data acquisition by means of DHMI. 
     
     
         3 . The method according to  claim 1 , wherein a Raman spectrum is captured in the measurement and a further Raman spectrum is captured in the further measurement, wherein a difference spectrum between the Raman spectrum and the further Raman spectrum is determined, and wherein the biological object is characterized based on the difference spectrum. 
     
     
         4 . The method according to  claim 3 , wherein a first excitation beam which is irradiated onto the biological object in the measurement for detecting the Raman spectrum and a further excitation beam which is irradiated onto the biological object for detecting the further Raman spectrum in the further measurement have different polarizations. 
     
     
         5 . The method according to  claim 1 , wherein a background Raman spectrum of a substrate on which the biological object is arranged is detected, and wherein the background Raman spectrum is subtracted from a Raman spectrum detected in the measurement and from a further Raman spectrum detected in the further measurement. 
     
     
         6 . The method according to  claim 1 , wherein the stimulus is applied without use of a marker. 
     
     
         7 . The method according to  claim 1 , wherein electromagnetic radiation is irradiated onto the biological object to apply the stimulus. 
     
     
         8 . The method according to  claim 1 , wherein a laser beam or plural laser beams are irradiated onto the biological object to apply the stimulus. 
     
     
         9 . The method according to  claim 8 , wherein the laser beam or the plural laser beams are irradiated onto the biological object in a pulsed fashion, wherein a repetition rate of the laser beam or of the plural laser beams is varied to excite a resonance vibration of the biological object. 
     
     
         10 . The method according to  claim 8 , wherein the laser beam is scanned over plural different channels of a microfluidic system to apply the stimulus to plural biological objects. 
     
     
         11 . The method according to  claim 8 , wherein a deformation of the biological object is induced using the laser beam, wherein a power density of the laser beam at the biological object is selected such that the biological object is not destroyed. 
     
     
         12 . The method according to  claim 8 , wherein the laser beam holds the biological object at a position in a fluid flow, and wherein the laser beam and a flow speed of the fluid flow are set such that the fluid flow induces a deformation of the biological object. 
     
     
         13 . The method according to  claim 1 , wherein the stimulus is applied using ultrasonic waves or using an electric or magnetic high frequency field. 
     
     
         14 . The method according to  claim 1 , wherein a volume and/or a shape of the biological object in response to the stimulus is determined by means of DHMI. 
     
     
         15 . The method according to  claim 1 , wherein, by detecting the Raman scattering on the biological object an agglomeration of molecules and/or a molecular composition in a nucleus and/or a cytoplasm of a cell is respectively determined. 
     
     
         16 . The method according to  claim 1 , wherein the measurement and the further measurement are performed using a measurement laser beam which is scanned over different positions; and wherein the measurement laser beam is scanned such that the measurement and the further measurement are performed on plural portions of the biological object. 
     
     
         17 . (canceled) 
     
     
         18 . The method according to  claim 1 , wherein the measurement and the further measurement are performed on the biological object while the biological object is positioned in a fluid channel of a microfluidic system. 
     
     
         19 . The method according to  claim 18 , wherein the microfluidic system comprises a closed fluid loop in which biological objects are transported before the measurement is performed. 
     
     
         20 . The method according to  claim 1 , wherein a result of the comparison of results of the measurement and the further measurement is automatically matched with a data base which has entries for different types of biological objects. 
     
     
         21 . The method according to  claim 1 , wherein the biological object is automatically sorted as a function of the result of the comparison. 
     
     
         22 . The method according to  claim 1 , wherein the method is performed automatically with aid of a computer. 
     
     
         23 . An apparatus for characterizing a biological object, comprising:
 a device for generating a stimulus on the biological object; and   a measurement device for performing a measurement on the biological object to ascertain a response of the biological object to the stimulus, the measurement device being configured for data acquisition by means of a detection of Raman scattering on the biological object, the measurement device being configured, for ascertaining the response of the biological object to the stimulus,
 to perform a measurement comprising a detection of the Raman scattering before application of the stimulus, and 
 to perform a further measurement comprising a further detection of the Raman scattering after or during application of the stimulus; and 
   an evaluation logic which is configured to compare results of the measurement performed before application of the stimulus and results of the further measurement performed after or during application of the stimulus, and to characterize the biological object as a function of a comparison of the results of the measurement and of the further measurement.   
     
     
         24 . The apparatus according to  claim 23 , wherein the measurement device comprises a Raman device for detecting a Raman scattering on the biological object and a DHMI device for capturing a DHMI image of the biological object. 
     
     
         25 . The apparatus according to  claim 24 , wherein the apparatus comprises an optical microscope, and wherein the Raman device is configured to controllably move a focus of an excitation beam of the Raman device in three orthogonal spatial directions independently of a focus of a beam path of the optical microscope. 
     
     
         26 . The apparatus according to  claim 25 , wherein the Raman device comprises a lens and a positioning device coupled to the lens and configured to controllably move the lens in three orthogonal spatial directions in a controllable manner, to controllably move the focus of the excitation beam of the Raman device in three orthogonal spatial directions independently of a focus of the optical microscope. 
     
     
         27 . The apparatus according to  claim 23 , comprising an electronically controllable beam deflection device for scanning a laser beam of the device for generating the stimulus and/or a measurement beam of the measurement device over a plurality of positions. 
     
     
         28 . The apparatus according to  claim 27 , wherein the microfluidic system comprises a closed fluid loop for transporting biological objects before the measurement is performed. 
     
     
         29 . (canceled) 
     
     
         30 . The method according to  claim 1 , wherein applying the stimulus comprises exposing the biological object to chemical agents, active substances or drugs. 
     
     
         31 . The method according to  claim 15 , wherein, by detecting the Raman scattering on the biological object an agglomeration of a protein composition in a nucleus and/or a cytoplasm of a cell is respectively determined. 
     
     
         32 . The method according to  claim 19 , wherein the closed loop of the microfluidic system transports the biological objects continuously before the measurement is performed. 
     
     
         33 . The apparatus according to  claim 28 , wherein the closed fluid loop of the microfluidic system transports biological objects continuously before the measurement is performed.