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 be h[ 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 response
function IRF (t ) is what would be recorded as

the observed fluorescence decay 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()

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?