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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
Spectrum and Spectroscopy
 Types of spectroscopy:
(a) Continuous spectroscopy
(b) Absorption spectroscopy
(c) Emission spectroscopy
Spectroscopy
Emission Process
Electrons ground level
High energy level
Absorb energy
Energyemission
Left: Equipment diagram
Right: Schematic diagram
Equipment
 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
Applications
 Compare the
absorption
spectrum with the
element’s emission
spectrum, people
can build the
spectrum of
planets.
Spectrum of planets
 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
Fluorescence Spectroscopy
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)
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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Spectroscopy

  • 2.  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 Spectrum and Spectroscopy
  • 3.  Types of spectroscopy: (a) Continuous spectroscopy (b) Absorption spectroscopy (c) Emission spectroscopy Spectroscopy
  • 4. Emission Process Electrons ground level High energy level Absorb energy Energyemission
  • 5. Left: Equipment diagram Right: Schematic diagram Equipment
  • 6.  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 Applications
  • 7.  Compare the absorption spectrum with the element’s emission spectrum, people can build the spectrum of planets. Spectrum of planets
  • 8.  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 Fluorescence Spectroscopy
  • 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
  • 10. Time-resolved fluorescence spectroscopy  Time-resolved laser-induced fluorescence spectroscopy (tr- LIFS)
  • 11. 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)
  • 12. 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
  • 14. Living cell spectral imaging  Compromise: only two kinds of cellular organ were labeled

Editor's Notes

  1. 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.
  2. 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.
  3. 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.