Transcript Slide 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
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
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/chem
435/Lab5/
Color perception & mixing:
http://www.handprint.com/HP/WCL/color14.html;
http://www.csun.edu/~hchum001/bookcase/light/
spectrum.html
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
Spectrometry
http://www.
irvingcrowl
ey.com/cls/
back.htm
http://bouman.
chem.georgeto
wn.edu/S02/lec
t10/lect10.htm
Single – beam Spectrophotometer
Double – beam Spectrophotometer
Atomic Absorption, Flame Photometry
www.thebritishmuseum.ac.uk/.../ sr-tech-aas.html
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 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
Spectrometry: Beer/Lambert Law
A = ε λx 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 ελx if measurements are done
on purified material in a known solvent.
www.rx.uga.edu/.../phrm4120/ spectrophotometry.htm
Impacts of pH on
Absorbance
www.biomedcentral.com/
1472-6793/ 2/5/figure/F3
Titration of phenol
wps.prenhall.com/.../724/
741576/chapter_08.html
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/
Impacts of
Solvent on
Absorbance
www.photobiology.com/photoi
upac2000/ karapire/
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
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
Lasers: Coherent Light Sources
Infrared & Optical Masers: www.coseti.org/ schawlow.htm
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
www.phy.davidson.edu/.../ diode_laser.htm
Histochemical Staining
Histochemical Society: www.histochem.org/ main.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)
www.udel.edu/.../illuspage/ lec1/lec1.htm
Chemistry of H&E Staining
www.histology.to/ hemx.html
Eosin
Hematoxylin
www.gspchem.com/ eosin.htm
Link catalogs of histological microscopy images:
turing.bear.uncw.edu/.../ vertebrate.asp
www2.uerj.br/~micron/atlas/ atlasenglish/lymph/