Fluorescent Tracers
Fluorescence: “an effect in which a substance
releases electromagnetic radiation while absorbing
another form of energy, but ceases to emit the
radiation immediately upon cessation of the input
energy”; “the light emission of a given wavelength
by a substance that is activated by light of a
different wavelength.” Academic Press Dictionary of
Science and Technology, 1992.
“The emission of a longer wavelength radiation by a
substance as a consequence of absorption of
energy from a shorter wavelength radiation,
continuing only as long as the stimulus is present;”
“distinguished from phosphorescence in that, in
the latter, emission persists for a perceptible period
of time after the stimulus has been removed.”
Stedman’s Medical Dictionary, 24th ed., 1982.
http://chsfpc5.chem.ncsu.ed
u/Poznan/membrane_fuse/in
dex.htm
micro.magnet.fsu.edu/.../
jablonski/lightandcolor/
General Resources:
http://www.probes.com/handbook/
Fluorescent Probes:
www.sigmaaldrich.com/.../ Labels.html
Choice of Fluorescent Labels:
www.aecom.yu.edu/aif/instructions/ probes.htm
BioRad Fluorochrome Simulation Program:
microscopy.bio-rad.com/.../ fluorophoreDatab.htm
Fluorescence & Luminescence Standards:
www.cstl.nist.gov/nist839/ 839.04/luminescence.htm
Analyte Sensor:
biomed.tamu.edu/obsl/ analyte_fluor.htm
Fluorescence Polarization:
www.glycoforum.gr.jp/.../ GT-C06E.html
Fluorescence Calibration:
www.pti-nj.com/ tech_8.html
Validation of Comparative Fluorescence:
www.npl.co.uk/biotech/ validfluo.html
Extended Depth of Field Imaging:
www.colorado.edu/isl/intimages/ fluorescence1.html
Naturally Fluorescent Molecules
Proteins:
Heme proteins (hemoglobin, myoglobin,
cytochrome C, hemocyanin)
Redox & photoproteins (phytochromes,
phycoerythrin, phycocyanin, green fluorescent
protein (http://www.ucmb.ulb.ac.be/.../
GFP/Fluorophore.gif; http://wwwbioc.rice.edu/.../Phillips/ Papers/gfpbio.html;
dwb.unl.edu/.../projects/ gmocz/gfp.htm),
phycobiliproteins, rhodopsin, ferredoxins)
Pigments:
flavins, stilbenes, tryptophan, tyrosine, purines..
Minerals, metallic chelates
Synthetic Fluorescent Molecules
Metallic chelates
Aromatics & polyaromatics (extended
 – bonded structures)
Crystalline arrays (intersubband
semiconductors)
Theory of Fluorescence
Fluorescence Theory:
www.pharm.uky.edu/ASRG/ HPLC/fluorescence.html
The origins of fluorescence in minerals:
http://www.uhaul.com/supergraphics/rocks/fluorescence.
html
Measurement of Fluorescence
Fluorescence indicates effective absorbance
& re-emission of electromagnetic radiation
(often in the visible range, ~300 - 800 nm,
but may involve other ranges, e.g., x - ray.
If the endpoint is presence of fluorescence,
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 spectrofluorimeter.
Spectrofluorimetry
Fluorescence Spectrometer Simulation:
www.inform.umd.edu/.../ WWW/Fluorescence.html
http://www.ucd.ie/appphys/michael/ photosyn.html
http://www.shu.ac.uk/.../
tutorials/
Scanning Spectrofluorimeter:
www.laboratorytalk.com/.../ rs_5/rs_5_56.html
Microplate Spectrofluorometer
Http://www.moleculardevices.com/ media/gem_xs_optics.gif
Note the use of light pipes, E,J; the multiwell plate, G;
the flashlamp, C; & the photomultiplier tube, L.
Spectrofluorimetry: Basics
The Beer Lambert law applies to the initial absorption
of excitation energy but many other considerations
then enter that govern the efficiency & nature of the
re-emitted light observed.
Quality factors are associated with internal energy
conversion & transfer to nearby molecules.
Quantum efficiency governs the transition to the
meta-stable, pre-fluorescent excited state; this is a
measure of the number of photons emitted relative to
the number of photons exciting the fluorescing
structure.
Quench decreases efficiency.
Fluorescence, or fluorescent yield is specific for
wavelength, compound, solvent, pH, & temperature.
Spectrofluorimetry
Fluorescence scales are arbitrary & always
relative to a standard used to calibrate the
instrument. With no background, fluorescence
may reach sensitivity of single molecules.
Impacts of pH on
Fluorescence
Impacts of Temperature on Fluorescence
From 25-60ºC in 5ºC increments:
http://www.isainc.com/fluor/apandtc/tempcont.htm
Impacts of
Solvent on
Fluorescence
www.photobiology.com/photoi
upac2000/ karapire/
Complications for Spectrofluorimetry
Light scattering, F is not directly proportional to c
Photochemical reactions, the system is dynamic
Quenching, multiple compounds absorb light
energy & alter spectral intensities acting on other
compounds present
http://members.tripod.com/obsil/fluor.html
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
Other Applications of Fluorescence
Fluorescence Microscopy
Microscopy Primer:
micro.magnet.fsu.edu/.../ techniques/contrast.html
Confocal Microscopy Primers:
www.cas.muohio.edu/.../microscopes/ confocal.html
web.ncifcrf.gov/rtp/ ial/cml/details.asp
http://www.emsl.pnl.gov/homes/csd/sms/subhomes/r4.html
Confocal Microscopy & Confocal Simulator:
www.biosci.ohio-state.edu/.../ sackmicroscopy.htm
Imaging Techniques using Fluorescence:
www.bris.ac.uk/synaptic/info/ imaging/imaging_1.ht
www.imagingresearch.com/.../ Fluorescence.asp
Total Internal Reflectance Microscopy:
http://www.microscopyu.com/articles/fluor
escence/tirf/tirfintro.html
Chromosome Spreads with Different Stains:
amba.charite.de/cgh/ img/01/img01.html
Multi-photon Excitation Microscopy:
Http://www.loci.wisc.edu/images/ jpegs/amos.jpeg
Lens & Focus Formulae:
http://www.biodevice.com/optical_and_fluorescence_
formula.htm
Filter Selection Demo:
http://www.omegafilters.com/front/curvomatic/spectra.php
Dye Selection:
http://www.promega.com/geneticidproc/ussymp8proc/21.html
FISH:
http://www.devicelink.com/ivdt/archive/97/01/005.html
Fluorescence Image Gallery:
http://www.nobel.se/physics/educational/micro
scopes/fluorescence/gallery/8.html
Dividing CHO Cell
Zeiss
FISH, Human Cells
P. Björk, Stockholm University
C. elegans Brain
H. Hutter
Max Planck Institut, Heidelberg
Time Resolved Fluorescence (TRF):
www.utu.fi/research/residues/ methods.html
Fluorescence Activated Cell Sorting (FACS):
www.bioteach.ubc.ca/.../ FlowCytometry/
Fluorescence Resonance Energy Transfer (FRET):
www.neuro.mpg.de/research/ scn/celldyn/
laxmi.nuc.ucla.edu:8248/.../ part1/en_sens6.html
Real Time PCR &
Molecular Beacons
(NucliSens)
Fluorogenic response of differently colored molecular beacons to the
addition of target. A solution of each molecular beacon was placed in
a pair of test tubes. The molecular beacons contained (left to right)
coumarin (blue), EDANS (blue-green), fluorescein (green), Lucifer
yellow, tetramethylrhodamine (orange), and Texas red. All molecular
beacons contain DABCYL as a quencher. Complementary singlestranded oligonucleotides were added to the left tube of each pair,
and the tubes were illuminated with a broad-wavelength ultraviolet
lamp. (Tyagi et al., 1998). http://www.biolegio.com/beacons.htm
Chemistry of Fluorescent Staining
http://www.chemie.unimarburg.de/~butenuth/642/Lumi
Dateien/bilder/physik5.jpg
Link catalogs of fluorescent microscopy images:
turing.bear.uncw.edu/.../ vertebrate.asp
www2.uerj.br/~micron/atlas/ atlasenglish/lymph/
Types of Luminescence
Luminescence:
www.deakin.edu.au/.../ luminescence.htm
pcml.univ-lyon1.fr/Luminophores/ Luminophores.html
www.dcb.unibe.ch/.../guedel/ research/hug_lum.htm
www.chem.lsu.edu/.../spec/ spectroscopy.html
ice.chem.wisc.edu/.../light/ lightandcolor6.html
Luminescence Theory:
www.shu.ac.uk/.../tutorials/ molspec/lumin1.htm
www.tf.uni-kiel.de/.../kap_5/ backbone/r5_1_2.html
www.chemistry.nmsu.edu/.../ Lab6/intro.html
Chemiluminescence:
www.chm.bris.ac.uk/.../ fleming/intro.htm
www.lumigen.com/
www.turnerbiosystems.com/.../ 998_2620.html
www.scienceofspectroscopy.info/ theory/ADVANCE...
www.chm.bris.ac.uk/.../ fleming/mechanism.htm
Lanthanides:
www.cchem.berkeley.edu/ knrgrp/lantha.html
www.chem.ox.ac.uk/icl/heyes/ LanthAct/L9.html
web.chem.ufl.edu/~kschanze/ NIRLED.htm
lanthanides.tripod.com/ ln-cl.html
Testing Quantum Dots:
www.phy.hw.ac.uk/resrev/ EQUIS/WP5.html
www.ifm.liu.se/.../research/ nano/InAsQD.html
Luciferase Assays:
www.shpromega.com.cn/ 65-6921_09.html
Luminescent Organics:
www.ndt.net/.../wcndt00/papers/ idn105/idn105.htm
Acridinium Esters:
www.turnerbiosystems.com/ doc/appnotes/s_0017.html
www.metachem.co.uk/ Acridium_NHS_Ester.htm
Imidazopyrazinones:
www.ch.ic.ac.uk/.../echet96/ papers/055/ref/6.html
Dioxetanes:
www.michdiag.com/ app.html
Photoluminescence:
www.fz-rossendorf.de/FWI/ FWIM/lumi_photo_e.htm
Bioluminescence:
bioluminescence.free.fr/ dosatp.htm
Cathodoluminescence:
plaza.snu.ac.kr/~lee2602/ atlas/cath_intro.html
Scanning Tunneling Luminescence:
level2.phys.strath.ac.uk/ ssd/HTML/charhtml/stl.ht
Oxygen Electrodes:
www.nogi.ch/chemistry/ oxygensensor.html
chemistry.mtu.edu/.../thesis/ ch02/Chapter02.html
QuickTime Chemistry Movies:
www.shsu.edu/~chm_tgc/ sounds/sound.html
Chemiluminescence Detection in HPLC:
www.ftns.wau.nl/.../lunteren/ lunteren.htm
Multimode Plate Reader:
www.image1.com/resources/ insights/0306/analyst.cf
Materials Characterization:
www.ifm.liu.se/.../popular/ Chapter4.html
Biological Toxic Metal Assays:
www.aboatox.com/ environmental_analysis.html
Biospore Testing:
www.nasatech.com/Briefs/ Mar03/NPO30469.html
Use polycyclic arene esters to
produce luminescence in almost
any visible color. H. Bock & co-authors
report that they used the good chargetransport properties of columnar liquid crystals
of these compounds to produce light-emitting
diodes (LEDs) with color-tuned electroluminescence. [E.g.], they used perylene3,4,9,10-tetracarboxylic acid ester 1 to form an
LED with orange– red luminescence, while a
series of esters of the triphenylene derivative 2
produced luminescence in the violet–blue
range.
R is methyl through n-octyl. By varying the
arene core & the R group on the esters, the
authors demonstrated photoluminescence
wavelengths over the entire visible spectrum.
Particularly interesting was their use of multiple
layers of different esters to form LEDs with
almost white luminescence. (Angew. Chem.,
Int. Ed. 2001, 40, 2060–2063; WJP)
pubs.acs.org/.../ci/31/
i12/html/12hc3.html
www.biodevice.com/
FluorescenceChemil
uminescen...
Luminol Reaction:
www.newlisbon.k12.wi.us/
reactions/home.html%234
Bioluminescent Organisms:
www.mcb.harvard.edu/.../ bioluminescence.html
Railroad Worm
Bioluminescent
Chemistries:
http://www.turne
rbiosystems.co
m/
doc/appnotes/pr
o_bio.htm
Bacteria
Squid
Temperature Effects on Chemiluminescence:
dbweb.liv.ac.uk/ltsnpsc/ practs/djm3.htm
Bioaffinity Flow Analyzer:
www.analykem.lu.se/homepage/ res/res3.htm
Suppliers:
www.vectorlabs.com/ products.asp?catID=157&loc...