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Chromogen Tracers

Chromogen Tracers. Use of colored compounds as tracers includes at least 4 primary approaches: Use of naturally colored molecules as endogenous indices of biological status Attachment of colored molecules to compounds of interest to make them visible

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Chromogen Tracers

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  1. Chromogen Tracers • Use of colored compounds as tracers includes at least 4 primary approaches: • Use of naturally colored molecules as endogenous indices of biological status • Attachment of colored molecules to compounds of interest to make them visible • Chemical reactions that produce dyes or change their colors • Conjugation of naturally colored molecules to binder- ligand components

  2. Naturally Colored Molecules Proteins: Heme proteins(hemoglobin, myoglobin, cytochrome C, hemocyanin) Redox & photoproteins(phytochromes, phycoerythrin, phycocyanin, chlorophylls, phycobiliproteins, rhodopsin, ferredoxins) Pigments: Melanins(black, brown) Xyanthophylls, flavinoids(yellow) Cyanins(blue),anthocyanins(red – blue) Carotinoids, lycopene(yellow, red) Minerals, metal ions, metallic chelates

  3. Synthetic Colored Molecules Metallic ions & chelates Aromatics & polyaromatics (especially heterocycles; quinoids, terpenes; extended π – bonded structures) Crystalline arrays

  4. Theory of Color The origins of color in minerals: http://www.minsocam.org/msa/collectors_corner/arc/color.htm Origins of color : http://www.chemistry.nmsu.edu/studntres/chem435/Lab5/ Color perception & mixing: http://www.handprint.com/HP/WCL/color14.html; http://www.csun.edu/~hchum001/bookcase/light/spectrum.html

  5. Measurement of Color Color indicates effective absorbance of electromagnetic radiation in or near the range of visual perception (~300 - 800 nm). If the endpoint is the presence of color, e.g., qualitative staining of a microscopic specimen, it can be directly observed or recorded on film or by digital means. If a quantitative test is done, color is usually measured with a spectrophotometer.

  6. Spectrometry The Joy of Visual Perception: http://www.yorku.ca/eye/spectru.htm

  7. Spectrometry http://www.irvingcrowley.com/cls/back.htm http://bouman.chem.georgetown.edu/S02/lect10/lect10.htm Single – beam Spectrophotometer Double – beam Spectrophotometer

  8. Atomic Absorption, Flame Photometry www.thebritishmuseum.ac.uk/.../ sr-tech-aas.html

  9. Spectrometry ULTRAVIOLET SPECTROSCOPY, ORIGINS OF COLOR: http://www.uwplatt.edu/~sundin/354-7/l547-38a.htm The Joy of Visual Perception: http://www.yorku.ca/eye/spectru.htm

  10. Spectrometry: Basic Equations %Transmittance = T = (I x100)/I0 where I = observed intensity & I0 = incident intensity T ranges from 0 – 100%; no radiation absorption = 100%; total radiation absorption = 0% Absorbance = A = - Log10(T) = Log10 (1/T) = Log10 (I0/I) A, or OD, optical density, ranges from 0 to ∞; no radiation absorption = 0; total radiation absorption =∞ A is specific for wavelength, compound, solvent, pH, & temperature

  11. Spectrometry: Beer/Lambert Law A = ελx b c where ελx = A for a 1 M/L solution of specific compound x in a specific solvent, pH, & temperature at wavelength λ; b is the pathlength of the observation cuvette or cell (usually in cm); & c is the concentration of the solution of x being measured (in M/L) If ελx& b are constant across a series of samples, A is proportional to c & may be used to estimate c in unknowns by comparison to standards or from published values of ελxif measurements are done on purified material in a known solvent. www.rx.uga.edu/.../phrm4120/ spectrophotometry.htm

  12. Impacts of pH on Absorbance Titration of phenol www.biomedcentral.com/1472-6793/ 2/5/figure/F3 wps.prenhall.com/.../724/ 741576/chapter_08.html

  13. Impacts of Temperature on Absorbance Melting DNA Thermosensing polymer wps.prenhall.com/.../724/ 741576/chapter_08.html reaktor.ch.pw.edu.pl/~dybko/ csrg/patents/thermo/

  14. Impacts of Solvent on Absorbance www.photobiology.com/photoiupac2000/ karapire/

  15. Complications for Spectrophotometry • Light scattering, A is not directly proportional to c • Can be used: nephelometry (scattered light) & turbidimetry (decrease in absorbance) • Photochemical reactions, the system is dynamic • Quenching, multiple compounds absorb light energy & alter spectral intensities acting on other compounds present • Nonlinearity or spectral impurity of light sources, illumination intensity varies by wavelength

  16. www.physics.montana.edu/.../ emissionspectra.html Light Source Intensity Variation by Wavelength www.pti-nj.com/ obb_spectra.html Other examples:www.firstrays.com/ spectra.htm

  17. Lasers: Coherent Light Sources Laser Theory: www.physics.ubc.ca/.../p420_95/ mark/htheory.html www1.union.edu/.../ laser_theory.htm webphysics.davidson.edu/.../ laserlab/theory.htm people.deas.harvard.edu/.../ ls2_unit_6.html www.people.fas.harvard.edu/.../ chemlaser.htm Infrared & Optical Masers:www.coseti.org/ schawlow.htm www.phy.davidson.edu/.../ diode_laser.htm

  18. Histochemical Staining www.udel.edu/.../illuspage/ lec1/lec1.htm Histochemical Society: www.histochem.org/ main.htm; www.lab.anhb.uwa.edu.au/mb140/ MoreAbout/stains.ht

  19. Histochemical Staining (cont.) www.udel.edu/.../illuspage/ lec1/lec1.htm

  20. Histochemical Staining (cont. 2) www.udel.edu/.../illuspage/ lec1/lec1.htm

  21. Chemistry of H&E Staining Eosin www.gspchem.com/ eosin.htm www.histology.to/ hemx.html Hematoxylin

  22. Link catalogs of histological microscopy images: turing.bear.uncw.edu/.../ vertebrate.asp www2.uerj.br/~micron/atlas/ atlasenglish/lymph/

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