Abstract:
The present invention discloses a method for non-fluorescence higher harmonic generation ground state depletion super-resolution microscopy, it includes the following steps: providing an organic material unit, focusing excitation light and ground state depletion light, generating a higher harmonic signal, performing ground state depletion and performing microscopic imaging. With the implementation of the present invention, the stimulated electrons of the organic material remains majorly on the singlet (S 1 ) state or the triplet (T 1 ) state, instead of the ground (S 0 ) state, to provide modulation of the spatial distribution of the non-fluorescence signal, and make STED microscopy applicable to non-fluorescence signals to promote the resolution of the images.

Description:
BACKGROUND OF THE INVENTION 
       [0001]    1. Technical Field 
         [0002]    The present invention relates to a method for super-resolution microscopy and, more particularly, to a method for non-fluorescence higher harmonic generation ground state depletion super-resolution microscopy. 
         [0003]    2. Description of Related Art 
         [0004]    The recently developed high-resolution STED (stimulated emission depletion) microscopes and STED microscopy have overcome the 200-nm upper limit of resolution imposed by diffraction on the conventional fluorescence microscopes. Using the innovative point-spread function technique, STED microscopes have a resolution more than ten times as high as that of their traditional counterparts and can therefore provide much finer microscopic images. 
         [0005]    One major limitation on the application of STED microscopy, however, is that the STED technique can only be used to modulate, and form microscopic images with, fluorescence signals. The conventional STED ultra-resolution microscopy is used mainly in fluorescence-related applications and achieves ultra-high resolution by modulating fluorescence intensity with STED; it does not work or cannot offer any help when it is desired to modulate, or form microscopic images with, non-fluorescence signals. 
         [0006]    In view of this, it has been a common goal of development and innovation in the fields of cell analysis, spatial domain analysis, and microscopy to extend the currently limited use of STED ultra-resolution microscopy in modulating fluorescence signals alone, and to create a useful and easy-to-implement method for non-fluorescence STED microscopy that features fast and accurate detection, stable imaging, and high spatial domain resolution, thereby expanding the application of STED to the modulation and detection of non-fluorescence signals. 
       BRIEF SUMMARY OF THE INVENTION 
       [0007]    The present invention provides a method for non-fluorescence higher harmonic generation ground state depletion ultra-resolution microscopy, and the method includes the steps of: providing an organic material unit, focusing excitation light and ground state depletion light, generating a higher harmonic signal, performing ground state depletion, and performing microscopic imaging. The present invention expands the application of STED ultra-resolution microscopy to modulating, and forming microscopic images with, non-fluorescence signals and increases the image resolution of microscopic imaging. 
         [0008]    More particularly, the present invention provides a method for non-fluorescence higher harmonic generation ground state depletion ultra-resolution microscopy, and the method includes the steps of: providing an organic material unit, wherein the organic material unit includes a plurality of molecules, each of the molecules has a plurality of electrons, and each of the electrons has an energy band with energy of hv such that, when excited by hv, the electrons jump from the ground state to the singlet state and undergo inter-system crossing from the singlet state to the triplet state, with h being the Planck constant (6.626×10̂−34) and v being a frequency expressed in the unit of hertz (Hz); focusing excitation light and ground state depletion light by collimating excitation light projected by a long-wavelength ultrafast pulse laser and ground state depletion light projected by a short-wavelength continuous-wave laser, combining the collimated excitation light with the collimated ground state depletion light, and focusing the combined light onto a plurality of test positions of the organic material unit sequentially; generating a higher harmonic signal by irradiating and exciting the test positions of the organic material unit with the focused excitation light such that the electrons of the molecules at the test positions are excited and jump to the singlet state and the molecules induce the higher harmonic generation signal; performing ground state depletion by irradiating with the focused ground state depletion light, and thus depleting, the electrons at the test positions that are in the ground state such that the electrons are excited and jump to the singlet state and undergo inter-system crossing to the triplet state; and performing microscopic imaging by receiving the higher harmonic generation signal of the test positions of the organic material unit and generating an ultra-resolution microscopic image corresponding to the organic material unit, with a STED system. 
         [0009]    Implementation of the present invention at least provides the following advantageous effects: 
         [0010]    1. The application of STED microscopy is expanded to modulating, and forming microscopic images with, non-fluorescence signals. 
         [0011]    2. With ground state depletion, non-linear absorption is reduced, and higher harmonic generation signals are suppressed in strength to facilitate the modulation of spatial distribution of such signals. 
         [0012]    3. The method of the present invention can be directly applied to cell analysis and microscopic imaging and is useful, easy to implement, fast and accurate in detection, and stable in terms of imaging. 
         [0013]    The features and advantages of the present invention are detailed hereinafter with reference to the preferred embodiments. The detailed description is intended to enable a person skilled in the art to gain insight into the technical contents disclosed herein and implement the present invention accordingly. In particular, a person skilled in the art can easily understand the objects and advantages of the present invention by referring to the disclosure of the specification, the claims, and the accompanying drawings. 
     
    
     
       BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS 
         [0014]    The invention as well as a preferred mode of use, further objectives and advantages thereof will be best understood by reference to the following detailed description of illustrative embodiments when read in conjunction with the accompanying drawings, wherein: 
           [0015]      FIG. 1  is the flowchart of the method in an embodiment of the present invention for non-fluorescence higher harmonic generation ground state depletion super-resolution microscopy; 
           [0016]      FIG. 2  schematically shows how an electron changes through the ground state, the singlet state, and the triplet state; 
           [0017]      FIG. 3  is a schematic diagram of the optical system in an embodiment of the present invention; 
           [0018]      FIG. 4A  schematically shows how a second harmonic generation signal is generated in an embodiment of the present invention; 
           [0019]      FIG. 4B  schematically shows how a third harmonic generation signal is generated in an embodiment of the present invention; and 
           [0020]      FIG. 5  is a schematic diagram of the optical system in another embodiment of the present invention. 
       
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
       [0021]    Referring to  FIG. 1 , the method S 100  in an embodiment of the present invention for non-fluorescence higher harmonic generation ground state depletion super-resolution microscopy includes the steps of: providing an organic material unit (step S 10 ), focusing excitation light and ground state depletion light (step S 20 ), generating a higher harmonic signal (step S 30 ), performing ground state depletion (step S 40 ), and performing microscopic imaging (step S 50 ). 
         [0022]    Referring to  FIG. 1  and  FIG. 2 , the step S 10  of providing an organic material unit uses an organic material unit  10  which includes a plurality of molecules each having a plurality of electrons e. Each electron e has an energy band with energy of hv, wherein h is the Planck constant (6.626×10̂−34) and v is a frequency expressed in the unit of hertz (Hz). When excited by energy of hv, the electrons e jump from the ground state S 0  to the singlet state S 1 . 
         [0023]    As shown in  FIG. 2 , once the electrons e in the ground state S 0  are excited and jump to the singlet state S 1 , some of the electrons e in the singlet state S 1  are transferred from the singlet state S 1  to the triplet state T 1  through inter-system crossing (ISC). 
         [0024]    More specifically, some of the electrons e in the singlet state S 1  undergo inter-system crossing from the singlet state S 1  to the triplet state T 1  due to the fact that the orbitals in which the electrons e revolve about the atom&#39;s nucleus overlap, or that the spin directions of the electrons e are non-conservative or unstable. There is significant inter-system crossing in the organic material unit  10 . 
         [0025]    Referring to  FIG. 1  and  FIG. 3 , the method S 100  for non-fluorescence higher harmonic generation ground state depletion ultra-resolution microscopy can be carried out via an optical system  100 . The optical system  100  may include a long-wavelength ultrafast pulse laser  20 , a short-wavelength continuous-wave laser  30 , two laser collimation units A 1 , a light combining unit A 2 , an objective lens A 3 , and a photodetector DR. 
         [0026]    The step S 20  of focusing excitation light and ground state depletion light is now described with reference to  FIG. 1  and  FIG. 3 . To begin with, the laser collimation units A 1  respectively collimate the excitation light  21  projected by the long-wavelength ultrafast pulse laser  20  and the ground state depletion light  31  projected by the short-wavelength continuous-wave laser  30 . Then, the collimated excitation light  21  and the collimated ground state depletion light  31  are combined by the light combining unit A 2 , in order for the objective lens A 3  to focus the combined excitation light  21  and ground state depletion light  31  onto a plurality of test positions  11  of the organic material unit  10  sequentially. The test positions  11  are located on the surface of the organic material unit  10  that is irradiated by the focused excitation light  21  and the focused ground state depletion light  31 . 
         [0027]    The term “ultrafast pulse” means that the pulse width of the long-wavelength ultrafast pulse laser  20  is selected to be less than 1 picosecond. 
         [0028]    Referring to  FIG. 1 ,  FIG. 3 ,  FIG. 4A , and  FIG. 4B , the step S 30  of generating a higher harmonic signal involves irradiating the test positions  11  of the organic material unit  10  with the focused excitation light  21 . As a result, the electrons e of the molecules at the test positions  11  are excited and jump from the ground state S 0  to the singlet state S 1 , and the molecules induce a higher harmonic generation signal whose frequency is a multiple of v. 
         [0029]    Referring to  FIG. 1 ,  FIG. 3 , and  FIG. 4A , if the wavelength of the excitation light  21  emitted by the long-wavelength ultrafast pulse laser  20  is selected to be twice as long as the wavelength corresponding to the frequency v, the sum of the energy of a biphoton (two photons) of the excitation light  21  will be hv, which is sufficient to excite the electrons e at the test positions  11  from the ground state S 0  to the singlet state S 1  and cause the molecules of the organic material unit  10  to induce a second harmonic generation signal F 2  whose frequency is twice that of the excitation light  21 . 
         [0030]    Referring to  FIG. 1 ,  FIG. 3 , and  FIG. 4B , if the wavelength of the excitation light  21  emitted by the long-wavelength ultrafast pulse laser  20  is selected to be three times as long as the wavelength corresponding to the frequency v, the sum of the energy of a triphoton (three photons) of the excitation light  21  will be hv, which is sufficient to excite the electrons e at the test positions  11  from the ground state S 0  to the singlet state S 1  and cause the molecules of the organic material unit  10  to induce a third harmonic generation signal F 3  whose frequency is three times that of the excitation light  21 . 
         [0031]    In the following step S 40  of performing ground state depletion, referring to  FIG. 1  to  FIG. 3 , the focused ground state depletion light  31  of the short-wavelength continuous-wave laser  30  is projected to the electrons e at the test positions  11  that are in the ground state S 0 , with a view to depleting the electrons e. The electrons e will be excited and jump to the singlet state S 1 , and some of the electrons e will undergo inter-system crossing to the triplet state T 1 . 
         [0032]    According to physics, the time it takes for an electron e in the triplet state T 1  to return to the ground state S 0  (i.e., the lifetime of the electron) is much longer than the time it takes for an electron e in the singlet state S 1  to return to the ground state S 0  (i.e., the lifetime of the electron). Moreover, as previously mentioned, there is significant inter-system crossing in the organic material unit  10 . 
         [0033]    Therefore, irradiating the organic material unit  10  with the focused ground state depleting light  31  will cause the irradiated electrons e to stay in the triplet state T 1  most of the time such that ground state depletion (GSD) is achieved. 
         [0034]    When the organic material unit  10  undergoes ground state depletion, the depletion of ground-state electrons e reduces non-linear absorption of the organic material unit  10 , thus allowing modulation of strength of the higher harmonic generation signal induced by the organic material unit  10 . 
         [0035]    Herein, the term “short wavelength” means that the wavelength of the short-wavelength continuous-wave laser  30  is selected to be a wavelength corresponding to the frequency v. 
         [0036]    To carry out the step S 50  of performing microscopic imaging, referring back to  FIG. 1  to  FIG. 3 , the higher harmonic generation signal induced by the molecules at the test positions  11  of the organic material unit  10  is received by the photodetector DR in order for a microscopic imaging device of the STED system to generate an ultra-resolution microscopic image corresponding to the organic material unit  10 . 
         [0037]    Modulation of the higher harmonic generation signal helps enhance the resolution of the image of the organic material unit  10  obtained from the step S 50  of performing microscopic imaging, and this contributes to expanding the applicability of STED microscopy substantially. 
         [0038]    Referring to  FIG. 5 , the optical system  100  implementing the method S 100  for non-fluorescence higher harmonic generation ground state depletion ultra-resolution microscopy may further include a spiral phase plate  50  provided between the short-wavelength continuous-wave laser  30  and the light combining unit A 2 . 
         [0039]    Once the collimated ground state depletion light  31  passes through the spiral phase plate  50 , the center of the light is twisted like eccentric spirals that meet in opposite directions every 180 degrees. When subsequently focused by the objective lens A 3 , the spirals at the center of the ground state depletion light  31  cancel each other due to their difference in phase, forming an annular distribution of light. 
         [0040]    On the other hand, referring to  FIG. 3  or  FIG. 5 , the light combining unit A 2  can be a dichroic mirror for combining the collimated excitation light and the collimated ground state depletion light. The selection of the dichroic mirror is based mainly on the mirror&#39;s permeability to the excitation light. The higher the permeability to the excitation light is, the better the microscopic imaging result will be. 
         [0041]    As shown in  FIG. 5 , the excitation light  21 , the ground state depletion light  31 , and the signal light  80  in the optical system  100  lie on the same optical axis (optical path). Hence, a band pass filter  60  can be provided upstream of the photodetector DR, which serves to receive the signal light  80 . The band pass filter  60  will filter out the excitation light  21  and the ground state depletion light  31  so that the photodetector DR receives only the higher harmonic generation signal. 
         [0042]    Since higher harmonic generation signals are difficult to obtain, the photodetector DR in the optical system  100  can be a photomultiplier tube (PMT) for receiving the signal light  80 , converting the received signal light  80  into an electrical signal, and then increasing the strength of the electrical signal with an amplifier to facilitate subsequent imaging. 
         [0043]    The embodiments described above are intended only to demonstrate the technical concept and features of the present invention so as to enable a person skilled in the art to understand and implement the contents disclosed herein. It is understood that the disclosed embodiments are not to limit the scope of the present invention. Therefore, all equivalent changes or modifications based on the concept of the present invention should be encompassed by the appended claims.