Slide 1 t:/powerpnt/course/lect4.ppt Purdue University Cytometry Laboratories 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 UPDATED October 27, 1998 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.
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Slide 1 t:/powerpnt/course/lect4.pptPurdue University Cytometry Laboratories
Lecture 3Fluorescence 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
UPDATED October 27, 1998
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.
Slide 2 t:/powerpnt/course/lect4.pptPurdue University Cytometry Laboratories
Overview
• Fluorescence
• Types of fluorescent probes
• Problems with fluorochromes
• General applications
Slide 3 t:/powerpnt/course/lect4.pptPurdue University Cytometry Laboratories
Excitation Sources
• Excitation SourcesLamps
XenonXenon/Mercury
LasersArgon Ion (Ar)Krypton (Kr)Helium Neon (He-Ne)Helium Cadmium (He-Cd)Krypton-Argon (Kr-Ar)
Slide 4 t:/powerpnt/course/lect4.pptPurdue University Cytometry Laboratories
Light Sources - Lasers
• Argon Ar 353-461, 488, 514 nm
• Krypton-Ar Kr-Ar 488, 568, 647 nm
• Helium-Neon He-Ne 633 nm
• He-Cadmium He-Cd 325 - 441 nm(He-Cd light difficult to get 325 nm band through some optical systems)
Laser Abbrev. Excitation Lines
Slide 5 t:/powerpnt/course/lect4.pptPurdue University Cytometry Laboratories
Arc Lamp Excitation SpectraIr
rad
ian
ce a
t 0.
5 m
(m
W m
-2 n
m-1)
Xe Lamp
Hg Lamp
Slide 6 t:/powerpnt/course/lect4.pptPurdue University Cytometry Laboratories
Fluorescence
• What is it?
• Where does it come from?
• Advantages
• Disadvantages
Slide 7 t:/powerpnt/course/lect4.pptPurdue University Cytometry Laboratories
FluorescenceE
NE
RG
Y
S0
S1
S2
T2
T1ABS FL I.C.
ABS - Absorbance S 0.1.2 - Singlet Electronic Energy LevelsFL - Fluorescence T 1,2 - Corresponding Triplet StatesI.C.- Nonradiative Internal Conversion IsC - Intersystem Crossing PH - Phosphorescence
IsC
IsC
PH
[Vibrational sublevels]
Jablonski Diagram
Vibrational energy levelsRotational energy levelsElectronic energy levels
Singlet States Triplet States
Slide 8 t:/powerpnt/course/lect4.pptPurdue University Cytometry Laboratories
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
Slide 9 t:/powerpnt/course/lect4.pptPurdue University Cytometry Laboratories
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)
Slide 10 t:/powerpnt/course/lect4.pptPurdue University Cytometry Laboratories
How many Photons?
• Consider 1 mW of power at 488 nm focused to a Gaussian spot whose radius at 1/e2 intensity is 0.25m 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
Slide 11 t:/powerpnt/course/lect4.pptPurdue University Cytometry Laboratories
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
Slide 12 t:/powerpnt/course/lect4.pptPurdue University Cytometry Laboratories
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
Slide 13 t:/powerpnt/course/lect4.pptPurdue University Cytometry Laboratories
Photobleaching• Defined as the irreversible destruction of an
excited fluorophore (discussed in later lecture)• Methods for countering photobleaching
– 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)
Slide 14 t:/powerpnt/course/lect4.pptPurdue University Cytometry Laboratories
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
Slide 15 t:/powerpnt/course/lect4.pptPurdue University Cytometry Laboratories
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, p-phenylenediamine 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)
Slide 16 t:/powerpnt/course/lect4.pptPurdue University Cytometry Laboratories
Slide 23 t:/powerpnt/course/lect4.pptPurdue University Cytometry Laboratories
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
Slide 24 t:/powerpnt/course/lect4.pptPurdue University Cytometry Laboratories
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
Slide 25 t:/powerpnt/course/lect4.pptPurdue University Cytometry Laboratories
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
Slide 26 t:/powerpnt/course/lect4.pptPurdue University Cytometry Laboratories
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