Abstract:
The present invention provides a plural third harmonic generation (THG) microscopic system and method. The system includes a laser device, a microscopic device, a beam splitter device and a photodetective device. By utilizing lasers with different central wavelengths or a broad band light source to simultaneously analyze THG response with respect to different wavelengths, a plurality of THG images and THG spectrum of the material or bio-tissue under stimulation of different wavelengths are obtained, thereby retrieving distributed microscopic images and resonant characteristics of the observational material or bio-molecules.

Description:
BACKGROUND OF THE INVENTION 
       [0001]    1. Field of the Invention 
         [0002]    The invention relates to a plural third harmonic generation (THG) microscopic system and method, in particular to a plural third harmonic generation (THG) microscopic system and method applied to bio-molecules in vivo and medical engineering. 
         [0003]    2. Description of Related Art 
         [0004]    The technique of third harmonic generation (THG), with the advantages of having no light harm, no light bleaching, high penetrating depth and having resolution of sub-micron, has been widely applied in research of bio-images. The harmonic generation microscopy disclosed in U.S. Pat. No. 6,922,279 is directed to a microscopic system making use of a third harmonic generation incorporated with a second harmonic generation and having no fluorescent light, and is focused on the application in bio-medicine. As revealed in an essay published in “Resonant Frequency-domain Interferometry via Third Harmonic Generation,” Optics Letter 32, 1265-1267 (2007) by D. S. Stoker, J. W. Keto, J. Baek and J. Ma, the THG is also applied in the spectrum science to understand the structure and characteristics of the material or bio-molecules. However, such research is more or less insufficient with respect to imaging of bio-molecules, due to that the existing THG microscopic system merely provides a harmonic generation image with a single wavelength. That is, measuring the strength of the THG and then mapping the same to form an intensity image. As a result, only the morphalogy of the bio-cells and bio-tissues can be known. Further, the existing research of applying the THG in the spectrum science makes use of directly modulating the wavelength of a laser beam to obtain a spectrum of the THG, while unable to provide spatially distributed related information and unable to provide the function of simultaneously presenting a plurality of THG images. 
       SUMMARY OF THE INVENTION 
       [0005]    The present invention is mainly purposed to provide a plural third harmonic generation (THG) microscopic system, utilizing lasers with different central wavelengths or a broad band light source to simultaneously analyze THG response with respect to different wavelengths, while to obtain a plurality of THG images and THG spectrum of the material or bio-tissues, and then to retrieve microscopic images of the bio-molecules, such that it is adapted to provide molecule imaging ability in THG microscopy of clinical medicine. 
         [0006]    A further object of the invention is to provide a plural third harmonic generation (THG) microscopic system, comprising: 
         [0007]    a laser device for irradiating a laser beam with a plurality of different wavelengths; 
         [0008]    a microscopic device for receiving the laser beam with a plurality of different wavelengths and projecting the same onto an observational sample, thereby producing a third harmonic generation observational beam with a plurality of different wavelengths; 
         [0009]    a beam splitter device for splitting the third harmonic generation observational beam into a first third harmonic generation portion and a second third harmonic generation portion; and 
         [0010]    a photodetective device for respectively detecting the first third harmonic generation portion and the second third harmonic generation portion and converting into a first third harmonic generation image signal or a first third harmonic generation spectrum signal and a second third harmonic generation image signal or a second third harmonic generation spectrum signal. 
         [0011]    According to the plural third harmonic generation (THG) microscopic system of the invention, the photodetective device is used for respectively detecting the first third harmonic generation portion and the second third harmonic generation portion and simultaneously converting the same into a first third harmonic generation image signal or a first third harmonic generation spectrum signal and a second third harmonic generation image signal or a second third harmonic generation spectrum signal. 
         [0012]    According to the plural third harmonic generation (THG) microscopic system of the invention, the photodetective device is used for further detecting a third THG portion or more THG portions, while simultaneously converting into a third THG image signal or a third THG spectrum signal and more THG image signals or more THG spectrum signals. 
         [0013]    The plural third harmonic generation (THG) microscopic system of the invention further comprises a computer system and an image processing software, for receiving the first THG image signal or the first THG spectrum signal and the second THG image signal or the second THG spectrum signal, while simultaneously processing a first THG image or a first THG spectrum and a second THG image or a second THG spectrum of the observational sample. 
         [0014]    According to the plural third harmonic generation (THG) microscopic system of the invention, the laser device is preferably a single laser for producing a broad band short pulse laser. 
         [0015]    According to the plural third harmonic generation (THG) microscopic system of the invention, the laser device is preferably a short pulse laser to irradiate onto an optical fiber or non-linear material for producing a laser beam with broad band or having a plurality of various wavelengths. 
         [0016]    According to the plural third harmonic generation (THG) microscopic system of the invention, preferably, the laser device includes a plurality of short pulse lasers having different central wavelengths. 
         [0017]    According to the plural third harmonic generation (THG) microscopic system of the invention, preferably, the photodetective device includes a plurality of photomultiplier tube photodetectors. 
         [0018]    According to the plural third harmonic generation (THG) microscopic system of the invention, preferably, the photodetective device includes a spectrometer. 
         [0019]    According to the plural third harmonic generation (THG) microscopic system of the invention, the microscopic device includes: 
         [0020]    a scanning system for scanning the laser beam; 
         [0021]    a mirror for receiving the scanned laser beam and reflecting the same; and 
         [0022]    an object lens for focusing the reflected laser beam and irradiating the laser beam onto the sample. 
         [0023]    According to the plural third harmonic generation (THG) microscopic system of the invention, the beam splitter device is preferably a dichroic beam splitter. 
         [0024]    A still further object of the invention is to provide a plural third harmonic generation (THG) microscopic method, comprising the following steps: 
         [0025]    providing a laser device for irradiating a laser beam with a plurality of different wavelengths; 
         [0026]    using a microscopic device for projecting the laser beam onto an observational sample to produce a THG observing beam having a plurality of various wavelengths, in which the THG observing beam includes a THG light having a first wavelength and a THG light having a second wavelength; 
         [0027]    separating the THG light having a first wavelength with the THG light having a second wavelength; 
         [0028]    respectively converting the THG light having a first wavelength and the THG light having a second wavelength into a corresponding first electrical signal and a second electrical signal; and 
         [0029]    processing the first electrical signal and the second electrical signal to simultaneously forming a plural THG image or a plural THG spectrum of the observational sample. 
         [0030]    According to the plural third harmonic generation microscopic method of the invention, the THG beam further includes a THG light with a third wavelength or more wavelengths to be respectively converted into a corresponding third electrical signal or more electrical signals. 
         [0031]    According to the plural third harmonic generation microscopic method of the invention, preferably, the laser device is a single laser for producing a broad band short pulse laser. 
         [0032]    According to the plural third harmonic generation microscopic method of the invention, preferably, the laser device is a short pulse laser to irradiate onto an optical fiber or non-linear material for producing a laser beam with broad band or having a plurality of various wavelengths. 
         [0033]    According to the plural third harmonic generation microscopic method of the invention, preferably, the laser device includes a plurality of short pulse lasers having different central wavelengths. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0034]      FIG. 1  shows a plural third harmonic generation (THG) microscopic system according to a first preferred embodiment of the invention; 
           [0035]      FIG. 2  shows a plural third harmonic generation (THG) microscopic system according to a second preferred embodiment of the invention; 
           [0036]      FIG. 3  shows a plural third harmonic generation (THG) microscopic system according to a third preferred embodiment of the invention; 
           [0037]      FIG. 4  shows a plural third harmonic generation (THG) microscopic system according to a fourth preferred embodiment of the invention; 
           [0038]      FIG. 5  shows a function diagram of strength versus wavelength of a laser source. 
       
    
    
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT 
       [0039]    Accompanied by the drawings, it will be explained below the preferred embodiments of the plural third harmonic generation (THG) microscopic system of the invention. 
         [0040]    Please refer to  FIG. 1 . It shows a plural third harmonic generation (THG) microscopic system  10  according to a first preferred embodiment of the invention. The system  10  comprises a scanning device  112 , capable of receiving a laser beam  116  from a laser device or laser source  114 . The scanning device  112  provides the function of producing a two-dimensional scanning laser beam, and is one, such as being installed with a rotating two-dimensional lens set (not shown), for directing the laser beam onto to a reflective mirror  113 , in which the laser beam is reflected and directed to a dichroic beam splitter  126  and then incident downward to a microscopic device (not shown). The microscopic device includes an object lens  122 . After that, the laser beam is focused by the object lens  122  and irradiated onto an observational sample  120  to produce a third harmonic generation (THG) signal. A third harmonic generation beam (THG beam)  116 ′, being reversely incident on the object lens  122  from the observational sample and irradiated to the dichroic beam splitter  126 , is split and irradiated onto a lens  128 . The third harmonic generation beam  116 ′ is processed by a spectrometer  132 , a signal of the third harmonic generation beam  116 ′ is converted into an analog signal by such as a photodetector, and the analog signal is amplified by an electrical amplifier and converted into a digital image signal or spectrum signal by an Analog/Digital converter, which is then transferred to a computer system  140  to produce a plurality of THG image signals with different wavelengths or a THG spectrum, and then to show a plurality of THG images with different wavelengths or a THG spectrum.  FIG. 1  is directed to a collection approach of backward collection. 
         [0041]    Please refer to  FIG. 2 .  FIG. 2  shows a plural third harmonic generation (THG) microscopic system  20  according to a second preferred embodiment of the invention. The system  20  comprises a scanning device  212 , capable of receiving a laser beam  216  from a laser device or laser source  214 . The scanning device  212  provides the function of producing a two-dimensional scanning laser beam, and is one, such as being installed with a rotating two-dimensional lens set (not shown), for directing the laser beam onto to a reflective mirror  2131 . The laser beam  216  is then directed to a microscopic device (not shown), being provided with an object lens  222 . After that, the laser beam is focused by the object lens  222  and irradiated onto an observational sample  220  to produce a third harmonic generation (THG) signal. A third harmonic generation beam (THG beam)  216 ′ after collection is reflected by a reflective mirror  2132 , passes a light filter  229  and a lens  228 , and is directed to a spectrometer  232 . A signal of the third harmonic generation beam is converted into an analog signal by such as a photodetector, and the analog signal is amplified by an electrical amplifier and converted into a digital image signal or spectrum signal by an Analog/Digital converter, which is then transferred to a computer system  240  to produce a plurality of THG image signals with different wavelengths or a THG spectrum, and then to show a plurality of THG images with different wavelengths or a THG spectrum.  FIG. 2  is directed to a collection approach of forward collection. 
         [0042]    Please refer to  FIG. 3 .  FIG. 3  shows a plural third harmonic generation (THG) microscopic system  30  according to a third preferred embodiment of the invention. The system  30  comprises a scanning device  312 , capable of receiving a laser beam  316  from a THG laser device or THG laser source  314 . The scanning device  312  provides the function of producing a two-dimensional scanning laser beam, and is one, such as being installed with a rotating two-dimensional lens set (not shown), for directing the laser beam onto to a reflective mirror  3131 . The laser beam reflected therefrom is directed to a dichroic beam splitter  3261  and then incident downward to a microscopic device (not shown), being provided with an object lens  322 . After that, the laser beam is focused by the object lens  322  and irradiated onto an observational sample  320  to produce a third harmonic generation (THG) signal. A third harmonic generation beam (THG beam)  316 ′ is reversely incident onto the object lens  322 , and incident on a lens  328 . Then, after irradiating on a dichroic beam splitter  3262  and splitting, the THG beam downward passes an interferometric light filter  3311  and irradiates on a photodetector  3301 . A signal of the third harmonic generation beam is converted into an analog electrical signal thereby, and the analog electrical signal is amplified by an amplifier and converted into a digital image signal or spectrum signal by an Analog/Digital converter, which is then transferred to a computer system  340  to show a THG image signals with a first wavelength or a THG spectrum. The THG beam leftward passes an interferometric light filter  3312  and irradiates on a photodetector  3302 . A signal of the third harmonic generation beam is converted into an analog electrical signal thereby, and the analog electrical signal is amplified by an amplifier and converted into a digital image signal or a THG spectrum signal by an Analog/Digital converter, which is then transferred to the computer system  340  to show a THG image signal with a second wavelength or a THG spectrum.  FIG. 3  is directed to a collection approach of backward collection. 
         [0043]    Please refer to  FIG. 4 .  FIG. 4  shows a plural third harmonic generation (THG) microscopic system  40  according to a fourth preferred embodiment of the invention. The system  40  comprises a scanning device  412 , capable of receiving a laser beam  416  from a THG laser device or THG laser source  414 . The scanning device  412  provides the function of producing a two-dimensional scanning laser beam, and is one, such as being installed with a rotating two-dimensional lens set (not shown), for directing the laser beam onto to a reflective mirror  4131  and then incident downward to a microscopic device (not shown), being provided with an object lens  422 . After that, the laser beam is focused by the object lens  422  and irradiated onto an observational sample  420  to produce a third harmonic generation (THG) signal. A third harmonic generation beam (THG beam) downward therefrom passes a beam collection device  424  for collection and then reflected by a reflective mirror  4132 . after passing a light filter  429 , the beam irradiates and passes a lens  428  and is incident to a dichroic beam splitter  4261 . After splitting, the beam is incident upward to an interferometric light filter  4311  and irradiated on a photodetector  4301 . A signal of the third harmonic generation beam is converted into an analog electrical signal thereby, and the analog electrical signal is amplified by an amplifier and converted into a digital image signal or spectrum signal by an Analog/Digital converter, which is then transferred to a computer system  440  to show a THG image signal with a first wavelength or a THG spectrum. The THG beam leftward passes an interferometric light filter  4312  and irradiates on a photodetector  4302 . A signal of the third harmonic generation beam is converted into an analog electrical signal thereby, and the analog electrical signal is amplified by an amplifier and converted into a digital image signal or a THG spectrum signal by an Analog/Digital converter, which is then transferred to the computer system  440  to show a THG image signals with a second wavelength or a THG spectrum.  FIG. 4  is directed to a collection approach of forward collection. 
         [0044]    According to the invention, the laser device or laser source  114 ,  214 ,  314 ,  414  may be a single laser output device. For example, a broad band short pulse laser may be produced by a single laser to thereby obtain a beam with a plurality of wavelengths. As shown in  FIG. 5 , such a beam has a broader band. Or, the laser device is a short pulse laser to irradiate onto an optical fiber or other non-linear material for producing two or more than two short pulse lasers having two different central wavelengths as shown in  FIG. 5 . The laser source  114 ,  214 ,  314 ,  414  may be a plurality of laser output devices, utilizing a plurality of short pulse lasers having different central wavelengths as a multi-wavelengthed laser source. The laser source may be one revealed in 1.2- to 2.2-μm Tunable Raman Soliton Source Based on a Cr: Forsterite Laser and a Photonic-Crystal Fiber, IEEE PHOTONICS TECHNOLOGY LETTERS, VOL. 20, No. 11, Jun. 1, 2008. 
         [0045]    The photodetector of the invention may be any kind of photodetector for sensing a laser beam, such as a photomultiplier tube photodetector. 
         [0046]    The number of the photodetector in the invention is not limited to two. Three or more than three photodetectors may be allocated to detect three or more than three THG light having different wavelengths. 
         [0047]    In the microscopic device, the molecular images may be inspected. Such an advantage further facilitates an application of noninvasive clinical molecular imaging in vivo. For example, from the flowing situation of the red blood cell in capillaries of human tissue, the distributed microscopic images of the red oxyhemoglobin and dark deoxyhemoglobin may be observed. 
         [0048]    According to the invention, the computer system  140 ,  240 ,  340 ,  440  may be any kind of PC, notebook computer, and small notebook computer. The image processing software may be any kind of THG image processing software, such as Fluoview 300 of Olympus. 
         [0049]    The plural THG microscopic method of the invention may further comprise a scanning step, which may use a THG beam to proceed with a two-dimensional planar scanning and imaging at a certain depth of the sample, so as to retrieve plural THG images and THG spectrum with different wavelengths in a noninvasive manner. 
         [0050]    A further advantage of the invention lies in providing a noninvasive, multi-wavelengthed THG microscopic system, when incorporating with a THG spectrum analysis of different wavelengths produced simultaneously, it is capable of obtaining distributed microscopic images and characteristics of bio-molecules. 
         [0051]    Although the present invention has been explained in relation to its preferred embodiments, it is to be understood that other possible modifications and variations can be made without departing from the scope of the invention as claimed below.