Spectroscopy and its Application Chemical and Biological detection Professor: Nam Sun Wang Haimo Liu 12/04/2007

Spectrum and Spectroscopy  Spectrum: (a). Different colors observed when the white light was dispersed through the prism (b). The changing of light intensity as a function of frequency  Spectroscopy: Study of spectrum, to identify substances

Spectroscopy  Types of spectroscopy: (a) Continuous spectroscopy (b) Absorption spectroscopy (c) Emission spectroscopy

Emission Process Electrons ground level High energy level Absorb energy Energy emission

Equipment  Left: Equipment diagram  Right: Schematic diagram

Applications  Absorption spectrum: used in deducing the presence of elements in stars and other gaseous objects which cannot be measured directly.  Emission spectrum: provide a definition of the spectrum of each atom, used to be compared with absorption spectrum

Spectrum of planets  Compare the absorption spectrum with the element’s emission spectrum, people can build the spectrum of planets.

Fluorescence Spectroscopy  Light source, self-emission which means the electrons transferred to the lowest level spontaneously  Different fluorescence: (a) different meta-stable states (b) different various vibrational states of the ground state

Time-resolved fluorescence spectroscopy  It provides fluorescence intensity decay in terms of lifetimes  Advantages: enhance the discrimination among fluorophores (overlapping emission spectra ) sensitive to various parameters of the biological microenvironment

Time-resolved fluorescence spectroscopy  Time-resolved laser-induced fluorescence spectroscopy (tr-LIFS)

Mathematical method

If is an impulse, then the impulse response will be

Mathematical method  Based on the definition of convolution:

Mathematical method  For the tr-LIFS system, the impulse response function is what would be recorded as the observed fluorescence decay Estimation of the intrinsic fluorescence decay was carried out via deconvolution of the observed fluorescence

Spectral Imaging system  Imaging provides intensity at every pixel of the image I (x, y)  spectrometer provides the intensity of a single spectrum, I(λ)  spectral image provides a spectrum at each pixel, I (x, y, λ)

Observation of multiple activities  Trying to use 5 different kinds of fluorescent molecules to label each of the 24 chromosomes in human body  2 to 5 minus 1=31

Observation of multiple colors

Living cell spectral imaging  Compromise: only two kinds of cellular organ were labeled

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