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Spectroscopy
- 1. 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
- 13. Observation of multiple colors
- 14. Living cell spectral imaging Compromise: only two kinds of cellular organ were labeled
Editor's Notes
- 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.
- 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.
- 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.