Abstract:
A Raman microscopy imaging device ( 100 ) is described, having: a first laser light source ( 12 ) for emitting a first laser beam ( 16 ) having a first wavelength along a first light path ( 20 ); a second laser light source ( 44 ) for emitting a second laser beam ( 18 ) having a second wavelength, different from the first wavelength, along a second light path ( 22 ) physically separated from the first light path ( 20 ); a beam combining element ( 32 ) for collinearly combining the two laser beams ( 16, 18 ) in one shared light path ( 34 ) directed onto a sample; a detector ( 38 ) for sensing a measured signal on the basis of the two laser beams ( 16, 18 ) interacting with the sample; and an evaluation unit ( 40 ) for evaluating the measured signal sensed by the detector ( 38 ). According to the present invention the first laser light source ( 12 ) is embodied as a pulsed source, and the second laser light source ( 44 ) as a continuous source.

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
       [0001]    The present application is the U.S. national phase of International Application No. PCT/EP2014/075049 filed Nov. 19, 2014, which claims priority of German Application No. 10 2013 112 759.2 filed Nov. 19, 2013, the entirety of which is incorporated herein by reference. 
     
    
     FIELD OF THE INVENTION 
       [0002]    The invention relates to a Raman microscopy imaging device, having a first laser light source for emitting a first laser beam having a first wavelength along a first light path; a second laser light source for emitting a second laser beam having a second wavelength, different from the first wavelength, along a second light path physically separated from the first light path; a beam combining element for collinearly combining the two laser beams in one shared light path directed onto a sample; a detector for sensing a measured signal on the basis of the two laser beams interacting with the sample; and an evaluation unit for evaluating the measured signal sensed by the detector. The invention further relates to a Raman microscopy imaging method. 
       BACKGROUND OF THE INVENTION 
       [0003]    Coherent Raman scattering microscopy (abbreviated “CRSM”) has recently acquired considerable importance in image-producing chemical sample analysis, for example in biology, pharmacy, or food science. A variety of CRSM methods are utilized, for example stimulated Raman scattering (SRS), coherent anti-Stokes Raman scattering (CARS), and Raman-induced Kerr effect scattering (RIKES). The list of documents below will be referred to hereinafter regarding the existing art:
   [1] Nandakumar, P., Kovalev, A., Volkmer, A.: “Vibrational imaging based on stimulated Raman scattering microscopy,” New Journal of Physics, 2009, 11, 033026.   [2] Freudiger, C. W., Roeffaers, M. B. J., Zhang, X., Saar, B. G., Min, W., Xie,   
 
         [0006]    X. S.: “Optical heterodyne-detected Raman-induced Kerr effect (OHD-RIKE) microscopy,” Journal of Physical Chemistry B, 2011, 115, 5574-5581.
   [3] Saar, B. G., Freudiger, C. W., Reichman, J., Stanley, C. M., Holtom, G. R., Vie, X. S.: “Video-rate molecular imaging in vivo with stimulated Raman scattering,” Science, 2010, 330, 1368-1370.   [4] Mikhail N. Slipchenko, Robert A. Oglesbee, Delong Zhang, Wei Wu, Ji-Xin Cheng: “Heterodyne detected nonlinear optical imaging in a lock-in free manner,” J. Biophotonics, 2012, 5, 1-7.   [5] Zumbusch, A., Holtom, G. R., Xie, X. S.: “Three-dimensional vibrational imaging by coherent anti-Stokes Raman scattering,” Phys. Rev. Lett., 1999, 82, 4142-4145.   [6] Cheng, J. X. and Xie, X. S.: “Coherent anti-Stokes Raman scattering microscopy: Instrumentation, theory, and applications,” J. Phys. Chem. B, 2004, 108, 827-840.   [7] Evans, C. L. and Xie, X. S.: “Coherent anti-Stokes Raman scattering microscopy: chemical imaging for biology and medicine,” Annu. Rev. Anal. Chem., 2008, 1, 883-909.   
 
         [0012]    In the CRSM technique, two pulsed light fields having pulse widths in a range from 100 fs to 20 ps, of different wavelengths, are directed through a confocal microscope optical system and focused onto the sample. The pulsed light fields are emitted onto the sample at frequencies that are typically in a range from 1 to 100 MHz. The light fields are spatially and temporally superimposed on one another on the sample via corresponding beam guidance and suitable focusing optics. In the SRS method or the image-producing superimposed RIKES method, for example, one of the two light fields is modulated in terms of intensity, frequency, or polarization at a specific frequency that is typically in the kHz to MHz range, before interacting in the sample with the other light field. For SRS and RIKES image production, the initially unmodulated light field is then sensed and, using a lock-in technique or envelope curve demodulation technique, the intensity modulation is extracted and presented in the form of an image. Reference is made to documents [1], [2], and [3] regarding implementation of the lock-in technique. The envelope curve demodulation technique is described in document [4]. In the case of CARS and CSRS a third light field is sensed as a consequence of interaction with the sample, and displayed as an image. This is described in documents [5], [6], and [7]. 
         [0013]    In all the CRSM techniques recited above, the measured signal is strong only if the difference between the frequencies of the incident light fields coincides with a vibrational resonance frequency in the sample. At present the best images in terms of spectral selectivity, signal intensity, and signal to noise ratio are obtained using picosecond laser light sources and optical parametric oscillators (OPOs) having pulse widths from 5 to 7 ps. 
         [0014]    Among the various image-producing techniques based on coherent Raman scattering, the SRS image-producing method has attracted particular attention in the recent past because of the resonance-free background that is not present in the images. Because of the modulation and demodulation techniques explained above that are utilized in the SRS method, however, this method is also very sensitive in terms of the delay that must be established between the laser beams emitted from the two pulsed laser light source, in terms of the pulse synchronization that must exist during operation of the two pulsed laser light sources, and in terms of the time-related cyclical “jitter” that often occurs when the pulsed laser light sources are operated. A typical SRS image-producing device is moreover comparatively costly, and is limited with regard to its application capabilities. 
         [0015]    It has therefore been proposed that in SRS imaging, the two pulsed laser light sources be replaced with continuous sources. In this case as well, one of the two laser beams is amplitude-modulated by means of a modulator. Although a solution of this kind is inexpensive, the resulting SRS measured signal is at least 10 6  times weaker than the measured signal obtained with the aid of pulsed laser light sources. It is evident from this that a device operating according to the SRS method with two continuous laser light sources is unsuitable for real-time image generation, in which approximately 25 individual images must be generated every second. In addition, continuous laser light sources adjustable over a broad wavelength band are in any case not yet readily available at this time. 
       SUMMARY OF THE INVENTION 
       [0016]    An object of the invention is to describe a Raman microscopy imaging device that can be implemented inexpensively and nevertheless in a reliably operating fashion. A further object of the invention is to describe a corresponding Raman microscopy imaging method. 
         [0017]    The invention achieves this object by way of the subject matter of the independent claims. Advantageous refinements are described in the dependent claims. 
         [0018]    The invention makes provision, in the context of a device of the kind described previously, to embody the first laser light source as a pulsed source that outputs the first laser beam in the form of a pulsed laser beam, while the second laser light source is embodied as a continuous source that emits the second laser beam in the form of a continuous laser beam. 
         [0019]    The first laser beam emitted from the pulsed source according to the present invention is intrinsically amplitude-modulated as a pulsed beam. With the device according to the present invention it is therefore no longer necessary to provide a modulator intended specifically for amplitude modulation. A high-frequency driver required in the existing art for application of control to the modulator can correspondingly also be omitted. 
         [0020]    Embodying the second laser light source as a continuous source, also referred to as a “CW” source, in turn makes it possible to omit the delay stage that is usual in the existing art. The device according to the present invention thus makes do with a smaller number of optical elements, e.g. mirrors, so that light losses are also avoided. 
         [0021]    In the context of application of the approach according to the present invention to the SRS method, the sensed measured signal is based on the originally continuous laser beam. Undesired intensity peaks in the measured signal, which can occur when two pulsed laser light sources are used, are thus avoided. It is therefore also not necessary to provide in the evaluation unit a precise low-pass filtering function with which the intensity peaks in the measured signal can be filtered out. 
         [0022]    Continuously operating laser light sources that exhibit outstanding beam quality as well as high stability over time are obtainable at comparatively low cost. The embodiment according to the present invention of one of the two laser light sources in the form of a continuously operating source is consequently advantageous not only from the standpoint of cost but also in terms of beam quality and stability over time. It can be expected that noise in the sensed measured signal will be low. 
         [0023]    Pulsed laser light sources having an adjustable wavelength are often furnished together with commercially usual optical systems, for example confocal laser scanning microscopes, in order to enable a variety of image-producing applications. Imaging methods based on the principle of frequency doubling (SHG) or frequency tripling (THG) may be mentioned here purely by way of example. Two-photon microscopy (TPEF) also represents a possible application. A system designed for such an application can thus be expanded in order to implement the approach according to the present invention with no need to provide additional laser light sources. 
         [0024]    An optical filter that blocks the first laser beam and directs the second laser beam onto the detector is preferably arranged in front of the detector. The device according to the present invention is thereby configured, for example, to carry out the SRS method, in which an amplitude modulation of the second laser beam, which modulation acts upon the second laser beam as a consequence of the interaction of the two laser beams with the sample, is sensed as a measured signal. 
         [0025]    The evaluation unit is embodied, for example, in such a way that it evaluates the measured signal as a function of a reference signal that corresponds to a pulse repetition rate at which the first laser light source emits the pulsed first laser beam. 
         [0026]    The device according to the present invention preferably contains a reference signal generator that generates the reference signal, corresponding to the pulse repetition rate, on the basis of the first laser beam emitted from the first laser source and delivers it to the evaluation unit. The pulse generator can be contained in the pulsed source itself. Alternatively, it is embodied as a separate subassembly. 
         [0027]    If the reference signal generator constitutes a separate unit it encompasses, for example, a beam splitter arranged in the first light path for splitting off a portion of the first laser beam emitted from the pulsed source; a rate detector for sensing the pulse repetition rate of the pulsed source on the basis of the first laser beam split off by the beam splitter; and a signal shaper for generating the reference signal as a function of the sensed pulse repetition rate. 
         [0028]    A femtosecond laser or a picosecond laser is preferably used as a pulsed source. The device according to the present invention can thus be used profitably in a CRSM method in which, as explained previously, pulse widths in a range from 100 fs to 20 ps are utilized. 
         [0029]    The approach according to the present invention is particularly advantageously suitable for carrying out the SRS method. It is not limited thereto, however, and can likewise be applied to other methods such as CARS, CSRS, or RIKES. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWING VIEWS 
         [0030]    The invention will be explained below on the basis of an exemplifying embodiment with reference to the Figures, in which: 
           [0031]      FIG. 1  shows a device not in accordance with the present invention for carrying out the SRS method, as a comparative example; 
           [0032]      FIG. 2  shows schematic pulse diagrams in order to illustrate how an SRS measured signal is obtained with the comparative device of  FIG. 1 ; 
           [0033]      FIG. 3  shows a device according to the present invention for carrying out the SRS method; and 
           [0034]      FIG. 4  shows schematic pulse diagrams in order to illustrate how an SRS measured signal is obtained with the device of  FIG. 3  according to the present invention. 
       
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
       [0035]    Firstly an SRS imaging device not covered by the invention will be described as a comparative example with reference to  FIGS. 1 and 2 , and then the approach according to the present invention will be explained on the basis of that comparative example. 
         [0036]    The SRS imaging device designated in general as  10  in  FIG. 1  contains a first pulsed laser light source  12  and a second pulsed laser light source  14 . The two pulsed laser light sources  12  and  14  emit pulsed laser beams  16  and  18  respectively along a first light path  20  and a second light path  22 . One of these laser beams  16 ,  18  functions in SRS imaging as a pump beam, and the other laser beam as a Stokes beam. The laser beam that has the lesser wavelength is typically referred to as a “pump beam,” while the laser beam having the greater wavelength is referred to as a “Stokes beam.” 
         [0037]    In the arrangement according to  FIG. 1 , first laser beam  16  emitted along first light path  20  from pulsed laser light source  12  is amplitude-modulated with the aid of a modulator  24  activated via a high-frequency driver  23 , for example an acousto-optic modulator (AOM) or an electro-optic modulator (EOM). The amplitude-modulated laser beam  16  then passes through a delay stage  30  constituted by two mirrors  26 ,  28  and is then incident onto a mirror  31  that reflects first laser beam  16  toward a dichroic beam splitter  32 . Dichroic beam splitter  32  combines first laser beam  16 , emitted along first light path  20 , with second laser beam  18 , emitted along second light path  22 , in a shared light path  34 . The two laser beams  16  and  18  thus become temporally and spatially superimposed onto one another as a result of delay stage  30  and dichroic mirror  32 . 
         [0038]    Arranged in shared light path  34  is an optical system  36  that encompasses a confocal microscope optical system having a laser scanning unit. Optical system  36  directs the two mutually superimposed laser beams  16  and  18  onto a sample (not explicitly depicted in  FIG. 1 ). 
         [0039]    Device  10  according to  FIG. 1  further contains a detector  38  operating in transmission and/or reflection. Arranged in front of detector  38  is an optical filter  39  that blocks first laser beam  16  and directs second laser beam  18  onto detector  38 . Detector  38  is electrically connected to an evaluation unit  40  that is in turn coupled to an image generation unit  42 . 
         [0040]    Laser beams  16  and  18  that are spatially and temporally superimposed on one another interact with the sample in such a way that second laser beam  18  experiences a slight amplitude modulation whose frequency corresponds to the modulation frequency at which modulator  24  amplitude-modulates first laser beam  16 . This situation is depicted in  FIG. 2 . 
         [0041]      FIG. 2  shows in the left part of the image, in the upper region thereof the pulsed first laser beam emitted by pulsed laser light source  12 , and in the lower region thereof the pulsed second laser beam  18  emitted by pulsed laser light source  14 . The two laser beams constitute the input signal. The right part of  FIG. 2 , conversely, shows an SRS measured signal that detector  38  senses as a result of the interaction of the two laser beams  16 ,  18  with the sample. According to this, second laser beam  18  experiences an intensity gain or an intensity loss as a result of this interaction, depending on which of the two laser beams has the greater wavelength. If the wavelength of second laser beam  18  (λ Laser2  in  FIG. 2 ) is less than the wavelength of first laser beam  16  (λ Laser1  in  FIG. 2 ), the result is then an intensity loss that is labeled I SRL  in  FIG. 2 . On the other hand, if the wavelength of second laser beam  18  is greater than that of first laser beam  16 , an intensity gain (indicated as I SRG  in  FIG. 2 ) then occurs. As is evident from  FIG. 2 , the intensity loss I SRL  or intensity gain I SRG  has a modulation frequency that is equal to the frequency of the high-frequency driver signal with which high-frequency driver  23  activates modulator  22 . The measured signal according to  FIG. 2  which is sensed by detector  38  is demodulated by evaluation unit  40 . For this, the high-frequency driver signal with which high-frequency driver  23  activates modulator  22  for the purpose of amplitude modulation of first laser beam  16  is delivered to evaluation unit  40 . A demodulation signal generated in this manner is sent by evaluation unit  40  to image generation unit  42  which generates an image signal therefrom, and/or to additional processing units that further process the demodulation signal, for example for purposes of storage or signal analysis. 
         [0042]    The following problems occur in the context of comparative device  10  according to  FIG. 1 : Because of the two pulsed laser light sources  12  and  14 , comparative device  10  is extremely sensitive in terms of the delay that must be established between the two laser beams  16  and  18 . Stringent requirements also exist in terms of the pulse synchronization that must exist in the context of operation of the two pulsed laser light sources  12 ,  14 . Lastly, arrangements must be made to reliably avoid a time-related cyclical “jitter” that can occur during the operation of pulsed laser light sources  12 ,  14 . 
         [0043]      FIG. 3  shows a device  100  for carrying out the SRS method, which device is modified according to the present invention with respect to the comparative example according to  FIGS. 1 and 2  in order to eliminate the problems recited above. Those aspects in which device  100  according to the present invention shown in  FIG. 3  corresponds to the comparative example shown in  FIGS. 1 and 2  will not be explained again below. Components that correspond to one another in terms of function and/or configuration are accordingly labeled with the same reference characters. 
         [0044]    Device  100  according to the present invention as shown in  FIG. 3  differs from comparative device  10  as shown in  FIG. 1  substantially in that a continuous laser light source  44 , which emits second laser beam  18  in the form of a continuous laser beam, is provided instead of pulsed laser source  14 . Device  100  according to the present invention further differs from comparative device  10  in that the components required therein in order to amplitude-modulate and delay first laser beam  16 , namely respectively modulator  24  and high-frequency driver  23 , and delay stage  30 , are omitted. 
         [0045]    Device  100  according to the present invention furthermore contains a reference signal generator, labeled overall  46  in  FIG. 3 , that is constituted by: a beam splitter  48  arranged in first light path  20 , a rate detector  50 , and a signal shaper  52  that is coupled to evaluation unit  40 . Beam splitter  48  is embodied in such a way that it diverts a portion of the pulsed first laser beam  16  out of first light path  20  and delivers it to rate detector  50 . The latter senses, from the light signal delivered to it, the pulse repetition rate at which pulsed laser light source  12  is emitting first laser beam  16 . Rate detector  50  then outputs an electrical signal, corresponding to the sensed pulse repetition rate, to signal shaper  52 , which generates therefrom a reference signal that evaluation unit  40  requires in order to evaluate the SRS measured signal sensed by detector  38 . 
         [0046]    First laser beam  16  emitted from pulsed laser light source  12  is reflected in first light path  20 , at a mirror  49 , toward dichroic mirror  32 . The latter combines first laser beam  16  and second laser beam  18 , emitted from continuous laser light source  44 , in shared light path  34 . Laser beams  16 ,  18 , spatially superimposed on one another in this manner, are then directed through optical system  36  onto the sample (not explicitly shown in  FIG. 3 ). 
         [0047]    As a consequence of the interaction of the two laser beams  16 ,  18  with the sample, the continuous second laser beam  18  experiences an intensity modulation in the form of an intensity loss or an intensity gain, depending on whether its wavelength is less than or greater than the wavelength of first laser beam  16 , the frequency of this intensity modulation being equal to the pulse modulation rate at which pulsed laser light source  12  emits first laser beam  16 . This situation is illustrated by the pulse diagrams of  FIG. 4 . 
         [0048]      FIG. 4  shows in the left part of the image, in the upper region thereof the pulsed first laser beam  16  that pulsed laser light source  12  emits, and in the lower region thereof the continuous second laser beam  18  that continuous laser light source  44  emits. The right part of  FIG. 2  shows the SRS measured signal sensed by detector  38  as a function of the wavelengths of the two laser beams  16  and  18 . If the wavelength of the continuous second laser beam  18  (λ Laser2  in  FIG. 4 ) is less than the wavelength of the pulsed first laser beam  16  (λ Laser1  in  FIG. 4 ), second laser beam  18  then experiences an intensity loss that is labeled I SRL  in  FIG. 4 . On the other hand, if the wavelength of the continuous second laser beam  18  is greater than the wavelength of the pulsed first laser beam  16 , second laser beam  18  then experiences an intensity gain that is labeled I SRG  in  FIG. 4 . 
         [0049]    Detector  38  outputs the sensed SRS measured signal to evaluation unit  40 . The latter demodulates the SRS measured signal with the aid of the reference signal that is furnished to it from reference signal generator  46 . This demodulation can be accomplished, for example, by way of a lock-in amplification operation. It is self-evident that the embodiment explained with reference to  FIGS. 3 and 4  is to be understood merely as an example. This embodiment refers, for example, to utilization of the SRS method. The concept according to the present invention of Raman microscopy imaging with the aid of both a pulsed laser beam and a continuous laser beam is likewise applicable to other methods, for example CARS, CSRS, or RIKES. Upon application of these methods, suitable filters and polarizing elements in the light paths depicted are to be supplemented as applicable in accordance with the specifically selected experimental configuration. 
       PARTS LIST 
       [0050]      10  Comparative device 
         [0051]      12  Pulsed source 
         [0052]      14  Pulsed source 
         [0053]      16  First laser beam 
         [0054]      18  Second laser beam 
         [0055]      20  First light path 
         [0056]      22  Second light path 
         [0057]      23  High-frequency driver 
         [0058]      24  Modulator 
         [0059]      26 ,  28  Mirror 
         [0060]      30  Delay stage 
         [0061]      32  Dichroic mirror 
         [0062]      34  Shared light path 
         [0063]      36  Optical system 
         [0064]      38  Detector 
         [0065]      39  Optical filter 
         [0066]      40  Evaluation unit 
         [0067]      42  Image generation unit 
         [0068]      44  Continuous source 
         [0069]      46  Reference signal generator 
         [0070]      48  Beam splitter 
         [0071]      50  Rate detector 
         [0072]      52  Signal shaper 
         [0073]      100  Device