Spectroscopy

1. Spectroscopy Mahsa Ranji (mahsa@drexel.edu) ECE-E641

2. Introduction • The history of spectroscopy goes back to the 17th century when Newton showed that the white light could be dispersed into a spectrum • The era of modern spectroscopy began with the invention of the laser • Spectroscopy could be: • Intrinsic fluorescence (IFS) • Diffuse reflectance (DRS) • Light scattering (LSS) • This science is concerned with the interaction of light and matter.The knowledge about the properties of atoms and molecules is extracted from the light properties and the changes light undergoes in the medium

3. Light Propagation • The scattering from very small scatteres (λ/10) is predominantly Rayleigh scattering.For particles that are comparable to the wavelength of light, Mie scattering predominates. Mie scattering produces a pattern like an antenna lobe, with a sharper and intense lobe for larger particles • When the light wavelength is similar to the particle diameter, light interacts with the particle over the scattering cross section, σs

4. Instrumentation • Fast excitation-emission matrix with 337nm nitrogen laser • White light(350-700nm) from Xe flash lamp • Delivery fiber of a 1mm diameter optical fiber probe • Photodetectors • Spectrum analyzer

5. Instrumentation(cont…) • For Fluorescence spectroscopy a fast excitation-emission matrix (FastEEM) consists of a 337 nm nitrogen laser as the light source and a 10 dye spinning wheel to obtain 11 different excitation wavelength (337-620nm) which coupled into the delivery fiber of a 1 mm diameter optical fiber probe • For the reflectance measurements, white light (350-700 nm) from the Xe flash lamp is coupled into the same probe. The probe is composed of six collection fibers surrounding the central light delivery fiber

6. Analysis • Mie scattering theory predicts that scattering cross section oscillates as a function of the wavelength. The frequency of these oscillations is directly proportional to the particle size and the amplitude proportional to the number of particles • The nuclear size distribution can be obtained from the amplitude of the Fourier transform of the oscillatory component of light reflected from the tissue

7. Reflectance • Since light penetrates only about ~1mm into tissue, most of the diffusely scattered return light is back-reflected due to photons that are singly back-scattered by the cell nuclei of the epithelium • Size distribution of normal and dysplastic nuclei which seems to be larger(12-20um) than normal nuclei ( 4-10um) and also crowding (increase in the total number of nuclei) can be obtained

8. Fluorescence • Fluorescence spectroscopy provide valuable information about changes taking place in cell biochemistry during the development of cancer • At 337-nm excitation the shape of the fluorescence spectrum broadens and shifts to the red region during the progression from normal to cancerous

9. Conclusion • Optical technologies offer the potential of detecting the very earliest cell changes in biochemical, microstructural levels • Light spectroscopy is a nonivasive, real time and high sensitive method for detecting cellular, molecular variations in their very early stage • Any cellular changes (dysplasia to invasive cancer) that can’t be detected by eye would be recognizable by light spectrum analysis • Spectroscopy is simple and contains detailed information about the sample and in medical applications offers a good diagnostic accuracy

10. Questions • What are the different regions of scattering due to scatteres size ? • What does Mie theory claim about scattering cross section s ? • What kind of light source is used for reflectance and fluorescence spectroscopy? • What change in fluorescence spectrum can be a signature of cancer? • What are some advantages of using light techniques such as spectroscopy?