Raman spectroscopy is dependent on scattering of light. When light interacts with matter, energy exchange can take place through absorption, simulated emission, Raman scattering or spontaneous emission. In Raman scattering, the emitted photons have less energy and lower frequency in comparison with the absorbed photons. Some energy of the emitted photons is transferred to the molecules. Raman spectroscopy is a method which collects the chemical fingerprints associated with molecules. Every molecule has different levels of vibrational energy and the emitted photons experience shift in their wavelengths. The wavelength shifts are collected,examined and analyzed for sample identification. Raman spectroscopy provides information on the state of cells and different stages of cancer present in the cells. It indicates whether the cells are infected by virus. A laser light is directed at the cell sample. The spectrum of light reflected from the cell sample is analyzed to obtain the required information about the cell. Raman scattering is a scarce phenomenon and approximately one photon in thirty million photons undergoes Raman scattering. The required signal is obtained after long duration and it is easily masked by fluorescence or other types of interferences(Scott,n.d.).
Confocal Raman microscopy refers to an imaging technique which produces high-resolution images and characterizes specimens and materials based on chemical composition. Sample preparation methods and specimen labeling and are not required necessary(Witec, n.d.).
Confocal Raman microscopy is highly challenging since a Raman image must be collected in a very short time and sensitivity, optical throughput must be optimized. A Raman image comprises of thousands of pixels/ spectra. The time of integration for each pixel/spectra should be less than one second per spectra. A system of confocal Raman microscope comprises of dual-output-port spectrometer which is combined with confocal microscope. Point-source illumination is provided by single-mode optical fiber. In a focal plane the confocal pinhole is provided by core diameter of multi-mode optical fiber. The other components present in the system include laser,sample chamber,couplers,holographic and removable beam splitters,notch filters,video camera,charge coupled device,computer,objective,photon-counting detector like Avalanche Photo Diode (APD) among others. In the system an excitation laser is coupled to microscope using a single-mode optical fiber cable. The laser beam which has a gaussian profile is focused to a spot size which is diffraction-limited. A holographic beam-splitter reflects the laser beam and it is then focused to the sample using a microscope objective. The sample is scanned using a piezoelectric scanner. It is accurate and fast. The same microscope objective is used for collecting the light which undergoes Raman-scattering. Rayleigh-scattered light and the reflected laser light is curbed using notch filter. Then the Raman scattered light is directed to multi-mode optical fiber cable and the core of fiber is the pinhole for the function of confocal microscopy.The multi-mode optical fiber is connected to a spectrometer which consists of two output ports. One port is connected to CCD camera which is used for spectral imaging. The second port is connected to APD for the purpose of Raman fast imaging.The multi-mode fiber core functions as entrance slit for the spectrometer. The computer generated multi-spectrum file is used for generation of images which are displayed on the monitor(Hollricher,2003).
Hot stage Raman spectroscopy(HSRS) is a useful method to study a mineral's thermal stability when extremely small quantity of mineral is available like in museum collection. In HSRS, the Raman spectra collected is a function of temperature and is obtained using a thermal stage. The changes in Raman spectra are collected corresponding to increasing temperature. The changes observed in the spectra are associated with changes present in the mineral's molecular structure. The thermal stability of mineral euchroite is studied using HSRS. The mineral disintegrated in the temperature range 125 -175 degree centigrade(Frost & Bahfenne,2009).
Scanning electron microscope(SEM) makes use of electrons to form the image. The advantages of SEM when compared to traditional microscopes include huge field depth,high resolution, clear images and the control of researcher over the degree of magnification. An electron gun at the apex of SEM produces beam of electrons. The beam travels vertically through a vacuum. The beam is focused by lenses and electromagnetic towards the sample. The sample ejects X-rays and electrons when the beam strikes it. The X-rays and electrons are collected by detectors and they are converted into signals. The signals are displayed as image on a monitor. SEM uses vacuum and electrons for producing the image. Hence the sample is treated before it is used. Water is removed from samples and non-metallic samples are made conductive by suitable method. Metallic samples being conductive require no prior preparation before they are used. There are concerns regarding radiation safety since backscattered electrons and X-rays are produced during the operation of SEM. Shielding is present in SEM and the radiation produced is with in the acceptable levels.(Purdue university,2014).
SEM is an integral component in all forensic investigations. It provides superior performance due to its various advantages and provides excellent results in the areas which include analysis of gunshot residues,comparison of bullet marks, jewellery and gemstones investigation has investigations of filament bulbs in traffic accidents, identification of forgery,detection of counterfeit currency among others(Azonetwork,2017).
The 3D images which are produced by SEM provide information regarding morphology,topography and composition of materials. Hence it is widely used in scientific research and industry. For proper operation of SEM, cooling system,vacuum system, steady power supply, vibration-free area is required. The SEM should be isolated from electric and magnetic fields which are present in the surroundings. The applications of SEM include examination of surface contamination, detection and analysis of surface fractures, identification of crystalline structures, micro-structure analysis, qualitative analyses of chemical compositions,detecting spatial variations in chemical formulations among others. SEMs are widely used in semiconductor inspection, assembly of IC chips for computing systems. The disadvantages associated with SEMs include their huge size,prohibitive cost, expensive maintenance,special training for operation of instrument and sample preparation among others. Sample preparation can result in generation of artifacts which negatively impact the final image. How ever certain artifacts can be identified and eliminated by experienced researchers. There are no methods presently available for identification and elimination of all artifacts. Only solid, inorganic samples which are small in size and which are easily positioned in the vacuum chamber can be analyzed by SEM(Anderson,2016).
References
Anderson , H. (2016). Scanning Electron Microscope. Retrieved January 19, 2017, from http://www.microscopemaster.com/scanning-electron-microscope.html
Azonetwork. (2017). Applications of Scanning Electron Microscopes in Forensic Investigations by Carl Zeiss. Retrieved January 21, 2017, from http://www.azom.com/article.aspx?ArticleID=5528
Frost,R. L., & Bahfenne,S. (2009). Thermal analysis and Hot-stage Raman spectroscopy of the basic copper arsenate mineral: Euchroite. Journal of Thermal Analysis and Calorimetry, 100(1), 89-94. doi: 10.1007/s10973-009-0599-x
Hollricher,O. (2003, November). Combine & Conquer. Retrieved January 20, 2017, from http://spie.org/newsroom/combine-and-conquer
Purdue. (2014). Scanning Electron Microscope. Retrieved January 21, 2017, from https://www.purdue.edu/ehps/rem/rs/sem.htm
Scott, K. (n.d.). Raman spectroscopy. Retrieved January 20, 2017, from https://www.st-andrews.ac.uk/seeinglife/science/research/Raman/Raman.html
Witec. (n.d.). RAMAN. Retrieved January 20, 2017, from http://www.witec.de/techniques/raman/
