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

2. Spectrum and Spectroscopy
(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
In the past, the word spectrum was introduced into the area of optics at first, referring to the range of colors observed when white light was dispersed through a prism. Soon after that, a spectral density or spectrum is also known as the term referred to a plot of light intensity or power as a function of frequency or wavelength. Spectroscopy is the study of spectrum, study of the interaction between radiation , for identifying the substances through the spectrum emitted from or absorbed by them.

3. Spectroscopy Types of spectroscopy: (a) Continuous spectroscopy
(b) Absorption spectroscopy
(c) Emission spectroscopy
The absorption spectroscopy means that we observe the spectrum of the light right after it go through the substance, (look at the pic.) black line means the photons which have those specific kind of energy were absorbed by the substance; emission spectrum means the range of electromagnetic spectra in which a substance radiates (emits). The substance first must absorb energy. This energy can be from a variety of sources. We observe the emission light

4. Electrons ground level
Emission Process
Electrons ground level
High energy level
Absorb energy
Energy emission
when the electrons in the element are excited, they jump to higher energy levels, however, in both the atomic and molecular cases, the excited states do not persist: after some random amount of time, the atoms and molecules revert back to their original, lower energy state. In atoms, the excited electron returns to a lower orbital, emitting a photon. In molecules, the vibrational or rotational mode decays, also emitting a photon. As the electrons fall back down, and leave the excited state, energy is re-emitted, the wavelength of which refers to the discrete lines of the emission spectrum.

5. Equipment Left: Equipment diagram Right: Schematic diagram
One question is that they need to isolate the incident light and fluorescence light. The light from an excitation source passes through a filter or monochromator, and passes through the sample. Here some of it probably is absorbed, making some of the molecules in the sample fluoresce. The fluorescence is often measured at 90 degree relative to the excitation light, is to avoid the interference between the transmitted light and the emission light. When measuring at a 90 angle, only the light scattered by the sample causes stray light. This results in a better signal-to-noise ratio. (compare forward-transmitted light and backward reflection light)

6. 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
A material's absorption spectrum shows the fraction of incident electromagnetic radiation absorbed by the material over a range of frequencies. Every chemical element has absorption lines at several particular wavelengths corresponding to the differences between the energy levels of its atomic orbital. Absorption spectra can therefore be used to identify elements present in a gas or liquid.

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

8. 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
As mentioned above, the absorption and emission spectrums are the properties of the object itself performed with the incoming ray. The essential of the fluorescence technology is to label the object using the fluorescent molecules. the objects are the light source.
the photons will have different energies, (based on two factors )and thus frequencies. By using different kinds of dyes which have different fluorescent properties, we can label and track separate activities on the biological specimen.

9. 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-dependent measurements resolve fluorescence intensity decay in terms of lifetimes, and thus provide additional information about the underlying fluorescence dynamics. Therefore, fluorescence lifetime information could have distinct advantages in clinical research and practice as it: (a) may enhance the discrimination among fluorophores, especially for those with overlapping emission spectra but with different emission characteristics; (b) is sensitive to various parameters of the biological microenvironment (pH, ion concentration and binding, enzymatic activity, temperature), allowing these variables to be analyzed; and (c) is not, or only minimally affected by the variation of excitation or emission intensity due to intervening absorbers. [10]

10. Time-resolved fluorescence spectroscopy
Time-resolved laser-induced fluorescence spectroscopy (tr-LIFS)
The excitation light pulses are focused into the illumination channel of the fiber-optic probe. Following sample excitation, the emitted fluorescence light is captured and directed, via the collection channel of the probe, into the entrance slit of the dual- mode spectrometer. One of the two outputs (in scanning monochromator mode) is connected to a multichannel plate photomultiplier tube (MCP-PMT) while the other output (in spectrograph mode) is coupled to an intensified charge-coupled-device (ICCD) camera. Operation as a scanning monochromator allows for time-resolved measurements at discrete steps across the emission spectrum. Operation as a spectrograph allows for rapid single-shot acquisition of steady-state spectral emissions and time-gated detection of long lifetime decays (.100 ns) in a scan-free fashion. Both outputs are digitized and directed to the computer workstation, which controls all major subcomponents of the apparatus and serves as the user interface.

11. Mathematical method

12. Mathematical method is an impulse, then the impulse response will beIf
is an impulse, then the impulse response will be

13. Mathematical method
Based on the definition of convolution:

14. 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
It allows one to identify n types of stains and calculate the concentration of each. Moreover, it allows one to separate the complex color-image into a set of single color images where each one of them shows the sample as if it were stained with only a single stain.

15. 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)
In a spectral imaging system, imaging provides the intensity at every pixel of the image, I(x,y), and a typical spectrometer provides a single spectrum, I(), a spectral image provides a spectrum at each pixel, I(x, y, ). This is a 3D data set and can be viewed as a cube of information. The spectral information allows detecting and distinguishing among many different fluorochromes even if they have a similar color or overlapping spectra. This permits one to label different entities by different specific fluorescence in a sample simultaneously and to quantitatively analyze each entity.

16. 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
The basic idea of this technology is to label each of the 24 chromosomes in human body with a combination of 5 different kinds of fluorescent molecules. For each fluorescent molecule there are two possibilities: exist or doesn’t exist. Thus the number of the total possible different combinations is:
(Except the possibility of no any fluorescent molecules)

17. Observation of multiple colors

18. Living cell spectral imaging
Compromise: only two kinds of cellular organ were labeled
For the reason that the spectral imaging takes quite a long time to measure if we want to collect lots of data along the axle of the wavelength, the movement or the activity of the living cell will bring a huge influence to the imaging result. [8] When viewed from this aspect, it becomes clear that a sort of compromise will perform much better in the living cell spectral imaging measurement. For instance, if only two kinds of cellular organ were labeled, the signal-noise ratio will be much better than that we labeled all kinds of cellular organ.

19. ¿Questions ?