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Lecture 3
Fluorescence and Fluorescence Probes
BMS 524 - “Introduction to Confocal Microscopy and Image Analysis”
1 Credit course offered by Purdue University Department of Basic Medical Sciences, School of
Veterinary Medicine
J.Paul Robinson, Ph.D.
Professor of Immunopharmacology
Director, Purdue University Cytometry Laboratories
These slides are intended for use in a lecture series. Copies of the graphics are distributed and students
encouraged to take their notes on these graphics. The intent is to have the student NOT try to reproduce the
figures, but to LISTEN and UNDERSTAND the material. All material copyright J.Paul Robinson unless
otherwise stated, however, the material may be freely used for lectures, tutorials and workshops. It may not
be used for any commercial purpose.
The text for this course is Pawley “Introduction to Confocal Microscopy”, Plenum Press, 2nd Ed. A number of
the ideas and figures in these lecture notes are taken from this text.
Purdue University Cytometry Laboratories
UPDATED October 27, 1998
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Overview
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Fluorescence
Types of fluorescent probes
Problems with fluorochromes
General applications
Purdue University Cytometry Laboratories
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Excitation Sources
• Excitation Sources
Lamps
Xenon
Xenon/Mercury
Lasers
Argon Ion (Ar)
Krypton (Kr)
Helium Neon (He-Ne)
Helium Cadmium (He-Cd)
Krypton-Argon (Kr-Ar)
Purdue University Cytometry Laboratories
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Light Sources - Lasers
Laser
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Argon
Krypton-Ar
Helium-Neon
He-Cadmium
Abbrev.
Ar
Kr-Ar
He-Ne
He-Cd
Excitation Lines
353-461, 488, 514 nm
488, 568, 647 nm
633 nm
325 - 441 nm
(He-Cd light difficult to get 325 nm band through some optical systems)
Purdue University Cytometry Laboratories
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Arc Lamp Excitation Spectra
Xe Lamp
Irradiance at 0.5 m (mW m-2 nm-1)



Hg Lamp



Purdue University Cytometry Laboratories
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Fluorescence
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What is it?
Where does it come from?
Advantages
Disadvantages
Purdue University Cytometry Laboratories
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Fluorescence
Jablonski Diagram
Singlet States
Triplet States
Vibrational energy levels
Rotational energy levels
Electronic energy levels
S2
ENERGY
T2
S1
IsC
T1
ABS
FL
I.C.
PH
IsC
S0
[Vibrational sublevels]
ABS - Absorbance
S 0.1.2 - Singlet Electronic Energy Levels
FL - Fluorescence
T 1,2 - Corresponding Triplet States
I.C.- Nonradiative Internal Conversion IsC
- Intersystem Crossing
PH - Phosphorescence
Purdue University Cytometry Laboratories
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Parameters
• Extinction Coefficient
– 
refers to a single wavelength (usually the absorption maximum)
• Quantum Yield
– Qf
is a measure of the integrated photon emission over the
fluorophore spectral band
• At sub-saturation excitation rates,
fluorescence intensity is proportional to the
product of  and Qf
Purdue University Cytometry Laboratories
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Excitation Saturation
• The rate of emission is dependent upon the time the molecule remains
within the excitation state (the excited state lifetime f)
• Optical saturation occurs when the rate of excitation exceeds the
reciprocal of f
• In a scanned image of 512 x 768 pixels (400,000 pixels) if scanned in
1 second requires a dwell time per pixel of 2 x 10-6 sec.
• Molecules that remain in the excitation beam for extended periods
have higher probability of interstate crossings and thus
phosphorescence
• Usually, increasing dye concentration can be the most effective means
of increasing signal when energy is not the limiting factor (ie laser
based confocal systems)
Purdue University Cytometry Laboratories
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How many Photons?
• Consider 1 mW of power at 488 nm focused to a Gaussian
spot whose radius at 1/e2 intensity is 0.25m via a 1.25
NA objective
• The peak intensity at the center will be 10-3W [.(0.25 x
10-4 cm)2]= 5.1 x 105 W/cm2 or 1.25 x 1024 photons/(cm2
sec-1)
• At this power, FITC would have 63% of its molecules in
an excited state and 37% in ground state at any one time
Purdue University Cytometry Laboratories
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Raman Scatter
• A molecule may undergo a vibrational transition (not
an electronic shift) at exactly the same time as
scattering occurs
• This results in a photon emission of a photon differing
in energy from the energy of the incident photon by
the amount of the above energy - this is Raman
scattering.
• The dominant effect in flow cytometry is the stretch
of the O-H bonds of water. At 488 nm excitation this
would give emission at 592 nm
Purdue University Cytometry Laboratories
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Rayleigh Scatter
• Molecules and very small particles do not absorb,
but scatter light in the visible region
• Rayleigh scattering is directly proportional to the
electric dipole and inversely proportional to the 4th
power of the wavelength of the incident light
• e.g. the sky looks blue because the gas molecules
scatter more light at shorter (blue) rather than longer
wavelengths
Purdue University Cytometry Laboratories
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Photobleaching
• Defined as the irreversible destruction of an
excited fluorophore (discussed in later lecture)
• Methods for countering photobleaching
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–
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Scan for shorter times
Use high magnification, high NA objective
Use wide emission filters
Reduce excitation intensity
Use “antifade” reagents (not compatible with viable
cells)
Purdue University Cytometry Laboratories
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Photobleaching example
• FITC - at 4.4 x 1023 photons cm-2 sec-1 FITC
bleaches with a quantum efficiency Qb of 3 x 10-5
• Therefore FITC would be bleaching with a rate
constant of 4.2 x 103 sec-1 so 37% of the molecules
would remain after 240 sec of irradiation.
• In a single plane, 16 scans would cause 6-50%
bleaching
Purdue University Cytometry Laboratories
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Antifade Agents
• Many quenchers act by reducing oxygen concentration to
prevent formation of singlet oxygen
• Satisfactory for fixed samples but not live cells!
• Antioxidents such as propyl gallate, hydroquinone, pphenylenediamine are used
• Reduce O2 concentration or use singlet oxygen quenchers such
as carotenoids (50 mM crocetin or etretinate in cell cultures);
ascorbate, imidazole, histidine, cysteamine, reduced
glutathione, uric acid, trolox (vitamin E analogue)
Purdue University Cytometry Laboratories
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Excitation - Emission Peaks
Fluorophore
FITC
Bodipy
Tetra-M-Rho
L-Rhodamine
Texas Red
CY5
EXpeak EM peak
496
503
554
572
592
649
518
511
576
590
610
666
% Max Excitation at
488
568 647 nm
87
58
10
5
3
1
0
1
61
92
45
11
0
1
0
0
1
98
Note: You will not be able to see CY5 fluorescence
under the regular fluorescent microscope because
the wavelength is too high.
Purdue University Cytometry Laboratories
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Probes for Proteins
Probe
Excitation
Emission
FITC
PE
APC
PerCP™
Cascade Blue
Coumerin-phalloidin
Texas Red™
488
488
630
488
360
350
610
550
540
640
525
525
650
680
450
450
630
575
575
670
Tetramethylrhodamine-amines
CY3 (indotrimethinecyanines)
CY5 (indopentamethinecyanines)
Purdue University Cytometry Laboratories
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Probes for Nucleic Acids
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Hoechst 33342 (AT rich) (uv)
Dapi (uv)
PI (uv/vis)
Acridine Orange (vis)
TOTO-1, YOYO-3, BOBO (vis)
Pyrine Y (vis)
Thiazole Orange (vis)
Purdue University Cytometry Laboratories
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Probes for Ions
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INDO-1
QUIN-2
Fluo-3
Fura -2
Purdue University Cytometry Laboratories
Ex350
Ex350
Ex488
Ex330/360
Em405/480
Em490
Em525
Em510
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pH Sensitive Indicators
Probe
Excitation
Emission
• SNARF-1
488
575
• BCECF
488
440/488
525/620
525
[2’,7’-bis-(carboxyethyl)-5,6-carboxyfluorescein]
Purdue University Cytometry Laboratories
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Probes for Oxidation States
Probe
• DCFH-DA
• HE
• DHR 123
DCFH-DA
HE
DHR-123
Purdue University Cytometry Laboratories
Oxidant
Excitation
(H2O2)
(O2-)
(H2O2)
488
488
488
Emission
525
590
525
- dichlorofluorescin diacetate
- hydroethidine
- dihydrorhodamine 123
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Specific Organelle Probes
Probe
BODIPY
NBD
DPH
TMA-DPH
Rhodamine 123
DiO
diI-Cn-(5)
diO-Cn-(3)
Purdue University Cytometry Laboratories
Site
Golgi
Golgi
Lipid
Lipid
Excitation
505
488
350
350
Mitochondria 488
Lipid
488
Lipid
550
Lipid
488
Emission
511
525
420
420
525
500
565
500
BODIPY - borate-dipyrromethene complexes
NBD - nitrobenzoxadiazole
DPH - diphenylhexatriene
TMA - trimethylammonium
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Other Probes of Interest
• GFP - Green Fluorescent Protein
– GFP is from the chemiluminescent jellyfish Aequorea victoria
– excitation maxima at 395 and 470 nm (quantum efficiency is 0.8)
Peak emission at 509 nm
– contains a p-hydroxybenzylidene-imidazolone chromophore
generated by oxidation of the Ser-Tyr-Gly at positions 65-67 of the
primary sequence
– Major application is as a reporter gene for assay of promoter
activity
– requires no added substrates
Purdue University Cytometry Laboratories
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Multiple Emissions
• Many possibilities for using multiple probes
with a single excitation
• Multiple excitation lines are possible
• Combination of multiple excitation lines or
probes that have same excitation and quite
different emissions
– e.g. Calcein AM and Ethidium (ex 488)
– emissions 530 nm and 617 nm
Purdue University Cytometry Laboratories
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Energy Transfer
• Effective between 10-100 Å only
• Emission and excitation spectrum must
significantly overlap
• Donor transfers non-radiatively to the
acceptor
• PE-Texas Red™
• Carboxyfluorescein-Sulforhodamine B
Purdue University Cytometry Laboratories
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Conclusions
• Confocal Microscopes are designed to use fluorescence
• Dye molecules must be close to, but below saturation
levels for optimum emission
• Fluorescence emission is longer than the exciting
wavelength
• The energy of the light increases with reduction of
wavelength
• Fluorescence probes must be appropriate for the
excitation source and the sample of interest
• Correct optical filters must be used for multiple color
fluorescence emission
Purdue University Cytometry Laboratories
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