Spectroscopy and its Application

Chemical and Biological detectionProfessor: Nam Sun Wang

Haimo Liu12/04/2007

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 ProcessElectrons ground level

High energy level

Absorb energy

En

erg

y e

missio

n

EquipmentLeft: 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[ ] [ [ ]]y n T x n

[ [ ] [ ]] [ [ ]] [ [ ]]T x n y n T x n T y n

[ [ ]] [ [ ]]T x n T x n

[ ] [ ] [ ]k

x n x k n k [ [ ]] [ [ ] [ ]

k

T x n T x k n k

Mathematical method

[ [ ]] [ ] [ [ ]]k

T x n x k T n k

[ ] [ ] [ [ ]]k

y n x k T n k

[ ]x[ ] [ [ ]]h x T x

If is an impulse, then the impulse response will be

[ ] [ [ ]]h n k T n k

[ ] [ ] [ ]k

y n x k h n k

Mathematical method

Based on the definition of convolution:

( )( ) ( ) ( )n

f g m f n g m n

[ ] ( )[ ] [ ] [ ]y n x h n x n h n

Mathematical method

For the tr-LIFS system, the impulse response function is what would be recorded as the observed fluorescence decay

( )IRF t

( )F t

( ) ( ) ( )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(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

¿Questions?