5.7　Raman Imaging

Raman spectroscopy is a commonly used vibrational spectroscopy based on Raman scattering effect discovered by Indian physicist Raman in 1928.In Raman spectrum，wavenumber，intensity and width of the characteristic peaks effectively reveal fingerprint information of substances，and the intensity is concentration-dependent.Thus，Raman spectroscopy is a quantitative analysis method.Compared with other spectroscopy techniques，Raman spectroscopy has its unique advantages：①It can selectively excites the required chemical bonds of molecules by changing the excitation wavelength.②There is no requirement of any sample pretreatment.③The sample will not be destroyed during the measurement.④The Raman absorbance of water can be ignored，so it is easier to measure Raman spectra of samples in aqueous solution.⑤Raman spectroscopy has a wide measurement range，so that heavy atom vibrations can be detected.Based on the advantages of Raman spectroscopy，various new analytical techniques are derived，such as surface enhanced Raman spectroscopy（SERS），Fourier transform Raman spectroscopy（FTRaman），resonance Raman spectroscopy（RRS），confocal Raman microspectral imaging（CRMI）and coherent Raman scattering microscopy（CRSM）.

CRMI is a relatively novel technique for the construction of label-free images of biological entities，such as cells or tissue sections.This method utilizes thousands of spatially resolved Raman spectra，and sophisticated image analysis algorithms，to construct images which are strictly based on the inherent biochemical abundance contrast afforded by Raman microscopy.Since only the signal from the focal plane is collected through the confocal pinhole，the signal-to-noise ratio and the signal quality is greatly improved.CRMI can detect tiny samples or even single-cell level samples.Equipped with a high-magnification optical microscope and various types of excitation light sources，CRMI can scan and obtain a Raman microscopic image of the sample point by point，which exhibits high spatial resolution，high signal-to-noise ratio and high accuracy.In the past few years，CRMI has vigorously developed in the fields of tumor detection，cultural relics and archaeology，and public security law.When the composition of biological tissues is analyzed with Raman spectroscopy，some uncertain factors，such as the inhomogeneity of biological tissues and the complexity of chemical composition，will affect the results.CRMI with the advantages of Raman spectroscopy and confocal microscopic imaging，can effectively compensate for the cognitive limitations caused by these uncertain factors.It is a molecular level optical imaging method to study biochemical components and structure of biological tissues.Figure 5-16（a）shows a simplified schematic of CRMI system.Due to weak Raman scattering（Stokes and anti-Stokes shifts），the laser of CRMI system is about one millionth weaker than typical absorption spectroscopy.A beamsplitter splits the light，with the beam path focused onto the sample through an objective lens.The beam can be split between a camera and the spectrograph for observation or measurement，respectively，and after passing through a rejection filter to remove the Rayleigh scatter，the Stokes（and anti-Stokes）wavelengths are diffracted by a grating onto a detector，either a CCD or an InGaAs array.Thus，the spatial distribution of the chemical components within the sample are imaged.High-resolution confocal Raman microscopes acquire the information of a complete Raman spectrum at every image pixel and achieve a lateral resolution at the diffraction limit（circa A/2 of the excitation wavelength）.CRMI can perform analysis of cells or biological tissues at molecular level.It is valuable for researchers to observe the changes of the composition and distribution of biochemical components in the cell life cycle，cell damage or cancer.CRMI not only can monitor the changes of various cells or tissues at any time，but also can analyze the difference between healthy tissues and cancerous tissues.(https://www.daowen.com)

Skin is composed of proteins，nucleic acids，sugars，coenzymes and other components.It is the largest organ in human body，whose function is to protect internal organs and tissues from external physical and chemical stimuli and pathogens，etc.Skin consists of epidermis，dermis，and subcutaneous tissue，as well as some accessory organs（e.g.，sweat glands，sebaceous glands，and nails），blood vessels，lymphatic vessels，muscles，and others.Because different molecules contain chemical structures that have unique vibrational frequencies，vibrational spectroscopy and microscopy techniques can provide intrinsic and specific chemical contrast.Among these vibrational techniques，Raman spectroscopy has been extensively utilized in biomedical applications（e.g.，disease diagnosis）.In the experiment，Raman spectra of skin can provide the changes of the composition and distribution of biochemical components in different skin layers.For example，the difference of Raman spectra between stratum corneum and epidermis is the peaks located at 1062 cm-1，1126 cm-1，and 1296 cm-1，indicating that a large amount of ceramide is present in the stratum corneum.Compared with Raman spectroscopy，Raman spectral properties of isolated skin tissues are studied in CRMI.Raman spectra of chemical composition in skin tissues constitute a spectral image，exhibiting the changes of composition and distribution of biochemical components in different skin layers for visualized diagnosis.