Spectroscopy and its Application

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 Energy emission Absorb energy

High energy level 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 elements 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 y[n] T [ x[n]] T [ x[n] y[n]] T [ x[n]] T [ y[n]] T [ x[n]] T [ x[n]] x[n] x[k ] [n k ] k

T [ x[n]] T [ x[k ] [n k ] k Mathematical method T [ x[n]] x[k ]T [ [n k ]] k y[n] x[k ]T [ [n k ]] k

If [ x] is an impulse, then the impulse response will beh[ x ] T [ [ x]] h[n k ] T [ [n k ]] y[n] x[k ]h[n k ] k Mathematical method

Based on the definition of convolution: ( f g )(m) f (n) g (m n) n y[n] ( x h)[n] x[n] h[n] Mathematical method For the tr-LIFS system, the impulse (t )

responseIRF function is what would be recorded as the observed fluorescence F (t ) decay F (t ) I (t ) IRF (t ) 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() spectral image provides a spectrum at each pixel, I (x, y, )

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 Questions?