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
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
Synthetic Colored Molecules
Metallic ions & chelates
Aromatics & polyaromatics (especially heterocycles; quinoids, terpenes; extended π – bonded structures)
Crystalline arrays
Origins of color :http://www.chemistry.nmsu.edu/studntres/chem435/Lab5/
The origins of color in minerals:http://www.minsocam.org/msa/collectors_corner/arc/color.htm
Color perception & mixing:http://www.handprint.com/HP/WCL/color14.html; http://www.cambridgeincolour.com/tutorials/color-perception.htm
Theory of Color
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.
Spectrometry
The Joy of Visual Perception: http://www.yorku.ca/eye/spectru.htm
http://www.irvingcrowley.com/cls/back.htm
Single – beam Spectrophotometer
Spectrometry
http://bouman.chem.georgetown.edu/S02/lect10/lect10.htm
Double – beam Spectrophotometer
http://www.britishmuseum.org/the_museum/departments/conservation_and_science/research/scientific_techniques/atomic_absorption_spectrophoto.aspx
Atomic Absorption, Flame Photometry
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
Spectrometry: Basic Equations
%Transmittance = T = (I x100)/I0
where I = observed intensity & I0 = incident intensityT 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
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 ελ
x if measurements are done on purified material in a known solvent.
www.rx.uga.edu/.../phrm4120/ spectrophotometry.htm
wps.prenhall.com/.../724/ 741576/chapter_08.html
Titration of phenol
Impacts of pH on Absorbance
www.biomedcentral.com/1472-6793/ 2/5/figure/F3
Impacts of Temperature on
Absorbance
wps.prenhall.com/.../724/ 741576/chapter_08.html
Melting DNA
reaktor.ch.pw.edu.pl/~dybko/ csrg/patents/thermo/
Thermosensing polymer
www.photobiology.com/photoiupac2000/ karapire/
Impacts of Solvent on
Absorbance
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
http://www.physics.montana.edu/demonstrations/video/7_modernphysics/demos/emissionspectra.html
Other examples: www.firstrays.com/ spectra.htm
http://www.obb1.com/LightSources/KiloArc.html#AboutLamps
Light Source Intensity
Variation by Wavelength
Infrared & Optical Masers: www.coseti.org/ schawlow.htm
webphysics.davidson.edu/.../ laserlab/theory.htm people.deas.harvard.edu/.../ ls2_unit_6.html
www1.union.edu/.../ laser_theory.htm www.physics.ubc.ca/.../p420_95/ mark/htheory.html
Laser Theory:
www.phy.davidson.edu/.../ diode_laser.htm
Lasers: Coherent Light Sources
Histochemical Society: www.histochem.org/ main.htm;
Histochemical Staining
www.udel.edu/.../illuspage/ lec1/lec1.htm
www.lab.anhb.uwa.edu.au/mb140/ MoreAbout/stains.ht
www.udel.edu/.../illuspage/ lec1/lec1.htm
Histochemical Staining (cont.)
www.udel.edu/.../illuspage/ lec1/lec1.htm
Histochemical Staining (cont. 2)
Hematoxylin
Chemistry of H&E Staining
Eosin
Link catalogs of histological microscopy images:
http://meded.ucsd.edu/hist-img-bank/
http://www.kumc.edu/instruction/medicine/anatomy/histoweb/
https://histo.life.uiuc.edu/histo/atlas/slides.php
http://www.itg.uiuc.edu/technology/atlas/
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