Chapter 1Introduction


Optical microscopy is unique in current imaging modalities. It can detect living tissue at subcellular resolution, visualize morphological details in tissue, and cannot be resolved by ultrasound or magnetic resonance imaging£¨MRI£©. However, to date, optical microscopy has not been fully successful in providing high resolution morphological information with chemical specificity. For example, the contrast of the confocal reflectance microscope£Û1£Ý and the optical coherence spectrum£Û2£Ý is based on the refractive index difference and cannot directly detect the chemical composition of the tissue structure. Fluorescence microscopy, although extremely sensitive and widely used, is limited in chemoselectivity due to the small number of intrinsic fluorophores such as Nicotinamide adenine dinucleotide phosphate(NAD£¨P)H£©, riboflavin and elastin£Û3£Ý. The introduction of an exogenous fluorophore provides a specific probe, but often causes undesirable perturbations. Second harmonic generation (SHG) microscopy can be used to visualize wellª²ordered protein components, such as collagen fibers, but with insufficient sensitivity and specificity for other tissue components£Û4£Ý. The vibrational spectrum of a biological sample contains multiple molecular features that can be used to identify biochemical components in the tissue. However, conventional vibration microscopy methods lack the sensitivity required for rapid tissue inspection. Infrared microscopy is limited by the low spatial resolution caused by longª²wavelength infrared light£Û5£Ý and the strong water absorption in biological samples. Although Raman microspectroscopy can distinguish between healthy and diseased tissues in vivo£Û5£Ý, it is hindered by undesired long integration times and/or high laser power in biomedical applications. A stronger vibration signal£Û6£Ý can be obtained by coherent antiª²Stokes Raman scattering (CARS), a nonlinear Raman technique. Typical CARS signals from submicron objects are orders of magnitude stronger than the corresponding spontaneous Raman responses. Since CARS is a nonlinear effect, the signal is only generated at the laser focus, which allows pointª²byª²point threeª²dimensional(3D) imaging of thick samples, similar to a twoª²photon fluorescence microscope£Û7£Ý. Recent developments in laser sources and detection protocols have significantly improved the ability of CARS as a bioimaging model£Û8£Ý. CARS microscopy has been shown to be useful for mapping lipid compartments£Û9£Ý, protein clusters£Û10£Ý and water distribution£Û11£Ý in cell tissue cultures.






Precise imaging and treatment of biological tissues on a microscopic scale is a major requirement of modern biomedicine and clinical medicine. In recent years, optical imaging and diagnosis technology has achieved rapid development and considerable progress. For example, the confocal laser scanning microscope excites the fluorescent label on the twoª²dimensional(2D) plane in the tissue by means of point irradiation, which has become a widely used tool in biomedical imaging£Û12£Ý; the superresolution optical imaging method has broken through the optical diffraction limit and can obtain nanoscale spatial resolution, can observe the dynamic behavior of subcellular structures and biomolecules in living cells, which greatly promotes the development of cell biology£Û13ª²14£Ý. However, these technologies all use fluorescent dyes, fluorescent nanocrystals or genetically encoded fluorescent proteins. The properties of these fluorescent substances have inherent limitations on optical imaging: 

(1) Dye saturation. The maximum number of photons that a fluorescent dye can emit in a given time is limited. Only when an excited electron occupies the excited state for about 5 ns and then returns to the ground state, the fluorescence reª²emission of the dye molecule will occur. 

(2) Dye bleaching. The total number of photons that fluorescent dyes can emit is limited. When the excited dye molecule changes from a 
singlet state to a triplet state, the chemical damage process of the dye will occur. The dye molecules in this state are highly reactive with oxygen molecules, which produces singlet oxygen (i.e. free radicals), which permanently destroy the dye and become the main source of phototoxicity affecting cells and tissues. 

(3) Fluorescence flicker. Most fluorescent dye molecules will ¡°turn on¡± and ¡°turn off¡± intermittently even under continuous light. The existence of the ¡°off¡± period limits the longª²term fluorescence imaging tracing process, causing the labeled molecules unable to be tracked continuously. Flicker and dye saturation greatly limit the number of photons that can be detected in a given period of time, resulting in a decrease in image signalª²toª²noise ratio. In addition, the phototoxicity and photobleaching of fluorescent materials also limit the length of time that biological targets can be observed£Û15£Ý.

With the increasing application of femtosecond laser technology, nonlinear optical imaging technology with fsª²pulsed laser as the light source has aroused great interest of researchers.  Nonlinear  imaging  methods  that have received widespread attention include: twoª²photon excited fluorescence (TPEF) imaging, CARS imaging, and SHG imaging£Û7,16ª²18£Ý. Twoª²photon and multiphoton fluorescence imaging requires the use of fluorescent probes, but the emission wavelength of highª²quantumª²efficiency fluorescent probes is mainly in the visible light region, which is still subject to strong tissue scattering and absorption£Û19£Ý. In CARS imaging, the Raman signal of the molecular vibration spectrum is usually much lower than the fluorescence signal£Û20ª²21£Ý£¬ which makes it more limited in detection sensitivity, acquisition time, laser power, etc., which greatly affects CARS application in living tissues. The second harmonic uses excitation light in the nearª²infrared region (700~1300 nm), with a detection depth of up to 1000 ¦Ìm, low photoª²
toxicity and point excitation, so it is more suitable for living tissue and animal imaging. Secondª²harmonic imaging is a unique nonlinear optical imaging technology. It is fundamentally different from twoª²photon fluorescence and coherent antiª²Stokes Raman in terms of imaging principles. The main features are as follows:

(1) SHG is a secondª²order nonlinear effect. TPEF and CARS are thirdª²order nonlinear effects. In nonª²centrosymmetric materials, the secondª²order nonlinear effects are much larger than the thirdª²order one. 

(2) SHG is a nonlinear scattering process, the sample does not absorb energy, which fundamentally overcomes phototoxicity and photodamage; while in twoª²photon imaging, fluorescent molecules suffer from severe photobleaching, and energy is lost due to processes such as vibration relaxation. 

(3) SHG signal is strictly frequencyª²doubled. Changing the pump wavelength can obtain frequencyª²doubled signals of different wavelengths. TPEF has a red shift relative to the double frequency, and the emission spectrum remains unchanged. Although the imaging principle is different, the second harmonic is fully compatible with the twoª²photon imaging system. Therefore, the twoª²photon confocal system can be easily transformed into a second harmonic imaging system by replacing the filter.

Hematoxylin and eosin (H & E) staining is a standard histopathological method used as a clinical diagnosis£Û22£Ý. Eosin stains proteins and cytoplasm in bright pink, while hematoxylin stains basic structures (such as DNA) in blueª²violet. However, H & E staining is a very slow process that requires biopsy, fixation, sectioning and staining. It usually takes several days, so it cannot be used for intraoperative diagnosis. Intraoperative freezing technology still takes a long time, at least 30 minutes. Therefore, fast and accurate imaging methods are very necessary for intraoperative diagnosis, and it is also very important for the judgment of the resection margin and the making of surgical decisions.

Research on detecting disease states through a series of technologies has been rapidly developed. Nonª²invasive imaging methods include computed tomography (CT), MRI, and positron emission tomography (PET)£Û23£Ý, but they are largely affected by low spatial resolution and intraoperative compatibility limitations. Intraoperative MRI can provide updated images during the operation, indicating that this technique has great potential, but it is limited due to the high cost and extended operation time£Û24£Ý. Ultrasound and optical coherence tomography (OCT) have been proven to provide structural information in real time, but they can only be used on a large scale, and it is difficult to obtain highª²definition tissue structure information with subcellular resolution£Û25£Ý, and lack of molecular specificity£Û26£Ý.

In recent years, fluorescent imaging using molecular markers has made important breakthroughs and improved the sensitivity of intraoperative detection. For example, brain tumor imaging is expected to reveal the edge of brain tumors, but it is still subject to certain restrictions£Û27ª²29£Ý: firstly, only some cancer cells absorb fluorescent molecules£Û29£Ý; secondly, the dye has the disadvantage of nonª²specific labeling; finally, fluorescein often undergoes fluorescent bleaching under laser irradiation. Confocal microscopy has been used for intraoperative imaging of fluorescently labeled tissues, but it also has limitations similar to fluorescent imaging£Û27£Ý. Various nonlinear optical imaging techniques are also used for tissue imaging, such as SHG and third harmonic generation (THG) microscopy. Among them, secondª²harmonic generation microscopy can selectively image structures with nonª²central inversion symmetry (such as collagen fibers and microtubules)£Û30£Ý; THG microscopy is sensitive to the nonuniformity of refractive index, But it cannot provide enough molecular information£Û31£Ý.

Vibration spectrum imaging provides a new method for specific imaging of pathological tissues. The fingerprint vibration spectra of molecules can be recorded by infrared spectroscopy or Raman spectroscopy£Û32ª²34£Ý. However, spontaneous Raman imaging of biological tissues is limited by weak signal strength and slow imaging speed, and it is difficult to directly use in biomedical imaging. Although the surfaceª²enhanced Raman scattering (SERS) method has the advantage of significantly enhancing the Raman signal, it requires the use of nanoparticles for exogenous labeling£Û35£Ý. Stimulated Raman scattering (SRS) microscopic imaging technology has been widely used in the field of biomedicine due to its unique chemical bond specific imaging function, including labelª²free DNA imaging£Û36£Ý, drug molecule tracking£Û37ª²38£Ý, tumor detection£Û39ª²40£Ý, lipid quantitative analysis£Û41ª²42£Ý, molecular metabolism and the mechanism of action of biological enzymes, etc.£Û43ª²44£Ý. Compared with fluorescence imaging, a major advantage of SRS imaging technology is that it 
does not require any markers to help it complete the imaging, and it has the advantages of high sensitivity, molecular selectivity and high resolution. SRS microscopy imaging technology overcomes the potential problems caused by label imaging technology with its characteristics of labelª²free imaging, such as nonª²specific labeling, toxicity, and influence on the biological process of the label£Û45£Ý.

Optical microscope has become an important tool for biomedical research by virtue of its high resolution, nonª²contact, nonª²invasive, and fast imaging advantages. Every advancement in optical microscopy imaging technology has greatly promoted the development of life sciences, basic medicine and clinical diagnostics. Since the 20th century, the field of optical microscopy imaging has achieved rapid development, and many new technologies and methods have emerged, including confocal microscopy, twoª²photon microscopy, light sheet illumination microscopy, and super resolution microscopy, and so on. Among these microscopic imaging technologies, twoª²photon microscopy imaging is one of the most landmark technologies, and fluorescence lifetime detection technology has opened up a new detection function for such an imaging.

Such technology was first implemented by Professor Webb£Û7£Ý in 1990. This technology uses twoª²photon excitation fluorescence signals for 3D microscopy imaging. The use of low scattering NIR light for local excitation makes it gain the advantages of low photobleaching and phototoxicity, super strong tissue penetration, subcellular level resolution, and inherent tomographic capabilities. In addition, this technology can also use endogenous optical markers to obtain contrast and achieve labelª²free imaging£Û3,46ª²47£Ý. In view of the above advantages, it is considered to be one of the most suitable technologies for in vivo optical microscopy imaging£Û47£Ý. It has gradually become a research method for the occurrence, development and potential treatment of diseases such as tumors and Alzheimer¡¯s disease. In addition, the method is nonª²invasive and can achieve labelª²free imaging characteristics, and its advantages in imaging depth 
and resolution make it one of the most promising clinical research tools. At present, it has been successfully used in clinical research on tumors, tissue lesions, controlled drug release, and in vivo drug screening£Û48£Ý.

Fluorescence lifetime detection refers to the use of time resolved technology to detect the dynamic process of fluorescence intensity attenuation. Under the excitation of high energy light, the fluorescent substance will transit to an unstable excited state,  and radiate fluorescent photons when it returns to a stable ground state. Therefore, the fluorescence lifetime reflects the average time that the fluorescent substance stays in the excited state£Û49£Ý. Similar to fluorescence spectroscopy, fluorescence lifetime is another important characteristic of fluorescent materials. Its detection breaks through the limitations of traditional steady state fluorescence detection and adds an independent dimension of new information to fluorescence imaging£Û50ª²52£Ý. Since the transition process of the fluorophore from the excited to the ground states is very easily affected by the local environment of the molecule, the lifetime can also sensitively reflect the pH, temperature, oxygen concentration, ion concentration, enzyme activity, and molecular configuration of the environment in which the molecule is located£Û50ª²53£Ý. For example, the freeª²stateª²reduced nicotinamide adenine dinucleotide (NADH) has a fluorescence lifetime of several hundred picoseconds, while proteinª²bound NADH has a fluorescence lifetime of several nanoseconds£Û54£Ý. In addition, the fluorescence lifetime measurement is usually independent of the concentration and quantum yield of fluorescent substances£Û52, 55£Ý. Therefore, the use of time resolved fluorescence detection technology to study biological systems has many unique advantages: 

(1) The fluorescence lifetime characteristics provide an additional contrast parameter for distinguishing fluorescent materials with overlapping emission spectra, so that biomolecules with overlapping spectra but different fluorescence decay times can be obtained. 

(2) The sensitivity of fluorescence lifetime measurement to various parameters of the microenvironment of biological tissues enables it to be used to detect local environmental parameters and study the interaction between proteins£Û51, 53£Ý. 

(3) The concentration of fluorescent substances in tissues is usually unknown and constantly changing, so the fluorescence lifetime is independent of fluorescent substance concentration and quantum yield characteristics, which makes it pos
sible to achieve more accurate in vivo quantitative measurements compared to 
steady state fluorescence detection technology£Û51, 53£Ý. 

Based on the above advantages, fluorescence lifetime imaging technology has received great attention from researchers, and has been widely used in biophysics and medical diagnostic research£Û56£Ý.

The combination of twoª²photon microscopy imaging and fluorescence lifetime detection technologies creates a winª²win situation with complementary advantages. On the one hand, the former technology can not only provide the pulsed excitation light source required for the later, but also benefit from its inherent tomographic ability. This technology can effectively avoid signals of different depths when performing fluorescence lifetime measurement on thick tissues. On the other hand, the combination with the latter enables the former to provide multiple dimensional fluorescence signal detection modes, and both the function and the application range have been expanded. Biomedicine and clinical diagnosis provide a new research method£Û52£Ý. Many leading scientific research teams in the world have been actively carrying out research on instruments and equipment, data analysis, biomedical and clinical applications related to twoª²photon fluorescence lifetime imaging, and have made many breakthroughs. But the research in our country is still in its infancy, and only a few scientific research teams are engaging in it. Among them, the team of Professor Qu Junle of Shenzhen University has long been committed to the research of former and its application in biomedicine. At present, the technology has been applied to the tumor mechanism and diagnostic methods and the molecular diagnoses£Û57£Ý. Our research group also has deep accumulation in this area, and is currently conducting on the diagnosis of benign and malignant diseases of the digestive 
tract and brain tumors based on the former. This book will briefly summarize the concept concerned, combined with the latest research results of this research group, summarize the research progress in tumor detection, and finally look forward to the future clinical applications, challenges and potential advantages.

The nonlinear optical spectrum signal is a new type of optical characterization technology due to the nonª²invasiveness and good biocompatibility. Since the Stokes Raman signal is often affected by the fluorescence effect of the detection object, in order to avoid the fluorescent signal, based on the characteristic that the frequency of the Stokes Raman signal is higher than the frequency of the fluorescent signal, the antiª²Stokes Raman measurement method was proposed, but the antiª²Stokes Raman signal was far smaller than the Stokes Raman signal. As a result, CARS and imaging technology came into being under the unremitting efforts of scientists, and successfully achieved signal measurement. A technique similar to CARS is SRS. At the same time, there are two other important nonlinear optical signals and their microscopic imaging methods, TPEF and SHG. The principles and experiments of these four types of nonlinear optical signals, especially 2D and 3D imaging, have great application potential in materials, chemistry and biomedicine. This book focuses on introducing the principles of SRS, CARS, TPEF and SHG signals, and appropriate calculation methods, as well as signal measurement, imaging methods, experimental results. The theoretical part starts from the nonlinear optics and the relationship between strong light, and gradually transits to specific calculation methods. The theoretical part contains the combination of classical and quantum theories, so that readers can understand the core of these technologies well. 
The experimental part mainly introduces the application of nonlinear optical spectroscopy and imaging technology in the fields of materials and biology. This book combines recent high quality scientific research results in the field of nonlinear optics at home and abroad as well as the authors¬ð years of research results and experience in this field. First of all, we will elaborate on the physical principles before the description of each nonlinear optical technology. The principle is roughly divided into two parts, and the theory is explained and discussed in depth. Secondly, we also review the experimental methods and excellent results of nonlinear optical spectroscopy and imaging in the world. Then, We introduce the latest experimental and theoretical results obtained by our group in the field of nonlinear optics and spectral imaging. In the end, the whole book has a unique way of explaining the theory and experimental methods in a small but precise manner, rather than a broad introduction to all nonlinear optics.






Chapter 2Basic theory of nonlinear optics


Nonlinear optics is a branch of modern optics that studies the nonlinear phenomena produced by strong media in the presence of strong coherent light and its applications. The study of nonlinear optics is of great significance to laser technology, spectroscopy development, and material structure analysis.The field of nonlinear optics is mainly concerned with various new optical phenomena and effects that occur during the interaction between intense laser radiation and matter, including in depth understanding and exploration of these new phenomena and the causes of the new process£Û58£Ý. Before the advent of lasers, some important formulas describing common optical phenomena were often linear. The polarization strength vector of the medium is such a very important physical quantity, which describes the important phenomena such as dispersion and scattering of light during the propagation of the medium. Before the appearance of a strong field laser, it is assumed to have a simple linear relationship with the incident 
electric field strength E :

P=¦Å0¦ÖE,(2.1)

where ¦Å0 is the vacuum dielectric constant; coefficient  ¦Ö is the dielectric 
polarization of the medium. From this perspective, in the theoretical framework of classical electrodynamics, the macroscopic Maxwell¡¯s equations describing the interaction between light and matter are also a set of linear partial differential equations. In other words, there is only a linear term in the  equation  that contains the field strength vector. Therefore, deductive reasoning can be seen that a bundle of monochromatic light is incident on the medium, and its frequency does not change; when light of different frequencies is incident at the same time, mutual coupling does not occur, and no new light is generated. But the above conclusions were fundamentally shaken in 1960. In this year, the world¡¯s first laser, the ruby laser, was born. The scientists used a 694.3 nm laser output from a pulsed ruby laser to enter the quartz crystal. For the first time, 347.2 nm multiplier coherent radiation was observed. After this incident, different abnormal optical phenomena have sprung up. In a short period of time, people have observed second harmonics, third harmonics, and optical and frequency in a series of media. Scientists pointed out that as long as the previous linear polarization theory is extended to higher order, the new effect can be perfectly explained. At this point, the polarization of the dielectric is no longer a simple linear relationship with the incident light field, but a higherª²order power series relationship, namely:





P=¦Å0£Û¦Ö(1): E+¦Ö(2): EE+¦Ö(3): EEE+¡­£Ý,(2.2)

where ¦Ö(1), ¦Ö(2) and ¦Ö(3) are the first order (linear), second order (nonlinear) and third order (nonlinear) polarization rates of the medium, respectively. In general, they all appear as tensor forms. By introducing the above representation of the polarization strength into Maxwell¡¯s equations, a set of nonlinear electromagnetic wave equations containing higher field strength terms can be derived. Thus, it is possible to explain the generation of frequency doubling radiation when a single frequency light is incident on a particular medium.

2.1Classical electromagnetic theory of 

nonlinear optics
2.1.1Measurement of nonlinear optical processes


If the relationship (2.1) is established in an isotropic or an anisotropic medium, when a monochromatic light wave is incident on the medium, the polarization intensity P is harmonically changed at the same frequency as E. And radiate electromagnetic waves of the same frequency, which is the secondary wave radiation.  The results obtained by the interaction of the secondary wave radiation with the incident light wave can explain the reflection, refraction and scattering of light. The linear wave equation in a homogeneous isotropic medium is

«ý2E-¦Ì¦Åªµ2Eªµt2=0.(2.3)

The above equation is a set of linear differential equations of three components of E, so that when light of different frequencies is simultaneously incident into 
the medium, no