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/