SlideShare ist ein Scribd-Unternehmen logo
1 von 46
Downloaden Sie, um offline zu lesen
Fundamentals of Fluorescence
Sam Wells, 9/15/03
Wells, 9/15/03
What is fluorescence?
Optical and Physical Properties
Detection and Measurement
Chemical properties and Biological
applications to be discussed later
Wells, 9/15/03
Definition of Fluorescence
Light emitted by singlet excited states of molecules following
absorption of photons from an external source
Requirements for Fluorescence
Fluorescent Dye (Fluorophore). A molecule with a rigid conjugated
structure (usually a polyaromatic hydrocarbon or heterocycle).
Excitation. Creation of dye excited state by absorption of a photon.
Wells, 9/15/03
Molecular Structure Features and Fluorescence
High ring density of π electrons increase fluorescence. Aromatic
hydrocarbons (π to π * absorption) are frequently fluorescent.
Increased hydrocarbon conjugation shifts π το π* absorption to
the red and increases the probability of fluorescence.
(C3H3) n, n = 3,5,7 Cy3, Cy5, Cy7
Protonation (affected by e- withdrawing/donating groups). OH
produces blue shift and decreased Qf in fluorescein. Halogens
(e- withdrawing) lower pKa and alkyl groups (e- donating) raise the
pKa.
Planarity and rigidity affect fluorescence. Increased viscosity
may slow free internal rotations and increase fluorescence.
C6H5 CH CH C6H5
Wells, 9/15/03
Fluorescent PROBES: Tags, Tracers, Sensors, etc.
Fluorescent tags generate contrast between an analyte and background, e.g., to identify
receptors, or decorate cellular anatomy.
Fluorescent sensors or indicators change spectral properties in response to dynamic
equilibria of chemical reactions, intracellular ions e.g., Ca2+, H+, and membrane potential.
Wells, 9/15/03
Fluorescence Combines Chemical
and Optical Recognition
Analyte
isolated or complex
dynamic mixture
Probe
targeting group
+ organic dye
chemical
connection
optical
connection
Detection
Wells, 9/15/03
ANALYTES can evaluated by fluorescence in a wide range of
environments, including single molecules in solutions, gels or
solids, cultured cells, thick tissue sections, and live animals.
?
Wells, 9/15/03
Optical Properties
Spectral properties of fluorescent probes define
their utility in biological applications.
Fluorescence spectra convey information
regarding molecular identity.
Variations in fluorescence spectra characterize
chemical and physical behavior.
Wells, 9/15/03
1 micron spheres viewed under transmitted incandescent “white” light
The light passing through the sample is diffracted, partially
absorbed and transmitted to the detector.
Wells, 9/15/03
1 micron spheres emitting fluorescence
The light passing through the sample is diffracted and absorbed, then
FILTERED to detect light originating from molecules within the sample.
Wells, 9/15/03
Fluorescence-related Terminology
Excitation – process of absorbing optical energy.
Extinction coefficient – efficiency of absorbing optical energy; this
value is wavelength dependent.
Emission – process of releasing optical energy.
Quantum yield – efficiency of releasing energy (0-1), generally in the
form of fluorescent light; not wavelength dependent.
Stokes shift – difference in energy between excitation and emission
wavelength maxima – the key to contrast generation.
Spectrum – distribution of excitation and emission wavelengths
Wells, 9/15/03
Spectral Features of Fluorescence
λ2 - λ1 = Stokes shift
F = I0ЄλC0LQf
I0
(Єλ)
a
Absorption and Fluorescence Excitation Spectra:
Same or Different?
Incident intensity (Io) Transmitted intensity (It)
Fluorescence intensity (IF)
Absorption spectrum: Scan Io(λ), detect It
Fluorescence excitation spectrum: Scan Io(λ), detect IF
Fluorescenceexcitation
For pure dye molecules in solution, the
absorption and fluorescence excitation spectra
are usually identical and can be used
interchangeably. When more than one
absorbing or fluorescent species is present in
the sample, they are typically different, as in
the case of protein-conjugated
tetramethylrhodamine dyes, shown right.
Absorption
500 550 600
Wavelength (nm)
Iain Johnson [Molecular Probes]
Wells, 9/15/03
DETECTION involves only a single observable
parameter = INTENSITY.
The spectral properties [COLOR] are
discriminated by optical filtering and sometimes
temporal filtering prior to measuring the intensity.
Therefore, it is extremely important to configure
the filtering and optical collection conditions
properly to avoid artifacts and faulty
observations!
Wells, 9/15/03
EX = excitation filter
DB = dichroic beamsplitter
EM = emission filter
Contrast by Epi-illumination (reflection mode)
and Optical Filtering
Wells, 9/15/03
Chemical Identification and Physical
Properties by Excitation and Emission
(A) 1 dye:1 excitation, 1 emission
(B) 1 dye:2 excitation, 1 emission or
(C) 1 excitation, 2 emission
(D) 2 dyes:1 excitation, 2 emission or
(E) 2 excitation, 2 emission
(F) >1 dye: >1 excitation, >1 emission
wavelength= emission,= excitation,
Wells, 9/15/03
detector
DETECTION / Microscopy, Imaging
Basic components for reflected light
fluorescence microscopy, a.k.a.
incident light fluorescence, or epi-
fluorescence.
1. Excitation light source
2. Filter set
3. Objective lens (the microscope)
4. Fluorescent specimen (analyte + probe)
5. Detector
Wells, 9/15/03
Wide-field,
inverted
fluorescence
microscope
http://micro.magnet.fsu.edu/primer/techniques/fluorescence/fluorhome.html
Wells, 9/15/03
Spectral Optimization
• Absorption wavelength Optimize excitation by matching
dye absorption to source output (Note: total excitation is
the product of the source power and dye absorption)
• Emission wavelength Impacts the ability to discriminate
fluorescence signals from probe A versus probe B and
background autofluorescence
• Emission bandwidth Narrow bandwidths minimize
overspill in multicolor label detection
Wells, 9/15/03
Limitations to Sensitivity (S/N)
Noise Sources
Instrument
Stray light, detector noise
Sample
Sample–related autofluorescence, scattered
excitation light (particle size and wavelength
dependent)
Reagent
Unbound or nonspecifically bound probes
Signal Losses
Inner filter effects, Emission scattering, Photobleaching
Wells, 9/15/03
Single T2 genomic DNA
macromolecule stained with
YOYO-1 (10 nM loading in
very low concentration DNA
in agarose). Wide-field
image using a 1.4NA
objective and CCD camera.
Unfortunately, YOYO-1
bleaches very fast.
Wells, 9/15/03
Viability
2 dyes, 1excitation, 2 emissions, 1 LP filter set
calcien AM (green, live cells), ethidium homodimer (red, dead cells)
Wells, 9/15/03
Organelles
1 dye, 2 excitation, 2 emissions,
switch between 2 filter sets
BODIPY FL C5-ceramide accumulation in
the trans-Golgi is sufficient for excimer
formation (top panel).
Images by Richard Pagano, Mayo Foundation.
Wells, 9/15/03
Cytoskeleton
3 dyes, 3 excitation, 3 emissions
BPAE cells: microtubules (green, Bodipy-antibody), F-actin (red, Texas Red-X
phalloidin) and nuclei (blue, DAPI). Triple exposure on 35mm film. This can be
done with a triple-band filter set or by 3 filter sets.
Wells, 9/15/03
1 (UV) excitation “band,” 8 (visible) emission “bands”
detected simultaneously on 35mm color film
Macro-image
Wells, 9/15/03
Physical Origins of Fluorescence
1 Dye excited state created by absorption of a photon
from an external light source
2 Fluorescence photon emitted has lower energy
(longer wavelength) than excitation photon. Ratio of
emitted photons/absorbed photons (fluorescence
quantum yield) is usually less than 1.0
3 Unexcited dye is regenerated for repeat
absorption/emission cycles. Cycle can be broken by
photobleaching (irreversible photochemical
destruction of excited dye)
Wells, 9/15/03
Energy Flow: Photons→Electrons→Photons
Different dye molecules
exhibit different excitation
and emission “profiles.”
Energy is
proportional
to 1/λ
1 = absorbance of light (excitation)
2 = vibronic relaxation, (Stokes shift)
3 = emission of light
Wells, 9/15/03
Fluorescence Excitation-Emission Cycle
Relaxation
Phosphorescence
hνPH
S1'
S1
Excitation
hνEX
Energy(hν)
T1
Emission
hνFL
photoproducts
S0
S0 = ground electronic state
S1 = first singlet excited state
T1= triplet excited state
Radiative transition
Nonradiative transition
Iain Johnson [Molecular Probes]
Wells, 9/15/03
Molecular Electronic State Transitions
Jablonski
Diagram
Iain Johnson [Molecular Probes]
Wells, 9/15/03
• Intermolecular Energy Transfer - characterized by donor-acceptor pairing
short range exchange interactions within interatomic collision diameter
long range interactions by sequential short range exchange
optical interactions between transition dipoles - decays as 1/r6
exciton migration = electron-hole pair migration - decays as 1/r3
eximer = complex between excited and ground state of same type molecules
exiplex = complex between excited and ground state of different type molecules
• Intramolecular Energy Transfer
intersystem crossing, e.g., E-type delayed fluorescence, phosphorescence
internal conversion, e.g., vibronic transitions
• Polarized Transitions and Rotational Diffusion
angular orientation between excitation and emitted light polarization changes
when a fluorophore rotates during the excited-state lifetime (τf)
Molecular Interactions and Fluorescence
excitation
emission
D < 100 Α
emission dipole
excitation
time
excitation dipole
Wells, 9/15/03
Two Photon Excitation
(hνEX)/2
hνEM
Energy
S1'
S1
(hνEX)/2
S0
1
2
Radiative transition Nonradiative transition
Wells, 9/15/03
One-photon ≠ Two-photon Excitation Spectra
Xu, Zipfel, Shear, Williams,
Webb. Proc Natl Acad Sci
USA 93:10763 (1996)
Wells, 9/15/03
Quantitative Fluorometry
• Measured fluorescence intensities are products of dye-
dependent (ε, QY), sample-dependent (c, L) and instrument-
dependent (I0
, k) factors
• Sample-to-sample and instrument-to-instrument comparisons
should ideally be made with reference to standard materials such
as fluorescent microspheres
• Attempts to increase signal levels by using higher concentrations
of dye may have the opposite effect due to dye–dye interactions
(self-quenching) or optical artifacts (e.g. inner filter effects, self-
absorption of fluorescence)
Wells, 9/15/03
Photometric Output Factors: Absorption
• Molar extinction coefficient (aka molar absorptivity).
Symbol: ε Units: cm-1 M-1
Defined by the Beer-Lambert law log I0/It = ε·c·l
• Quantifies efficiency of light absorption at a specific
wavelength (εmax = peak value)
• Typically 10,000 – 200,000 cm-1 M-1
• Not strongly environment dependent
Wells, 9/15/03
Photometric Output Factors: Emission
• Fluorescence quantum yield
– Symbol: QY or φF or QF Units: none
• Number of fluorescence photons emitted per photon
absorbed
• Typically 0.05 – 1.0
• Strongly environment dependent
• Other emission parameters: lifetime, polarization
Wells, 9/15/03
Quantifying Fluorescence Intensity
I
F
= I
0
·QY·(1-e
–2.303 ε.c.L
)·k
For (ε·c·L) < 0.05
I
F
= I
0
·QY·(2.303·ε·c·L)·k = Fluorescence emission intensity at λEM
QY = Fluorophore quantum yield
ε = Molar extinction coefficient of fluorophore at λEX
c = Fluorophore concentration
L = Optical pathlength for excitation
I0
= Excitation source intensity at λEX
k = Fluorescence collection efficiency*
*In a typical epifluorescence microscope, about 3% of the total available emitted
photons are collected (k =0.03) [Webb et al, PNAS 94, 11753 (1997)]
Wells, 9/15/03
IF will decrease when (ε·c·L) > 0.05
Inner Filter Effect Simulation
0.0
0.2
0.4
0.6
0.8
1.0
0 0.5 1 1.5 2
ε .c.L
Intensity
1-e-2.303.ε.c.L
IF = Io.(1-e-2.303.ε.c.L)
Io
Iain Johnson [Molecular Probes]
Wells, 9/15/03
Total Output Limit: Photobleaching
• Irreversible destruction of excited fluorophore
• Proportional to time-integrated excitation intensity
• Avoidance: minimize excitation, maximize detection
efficiency, antifade reagents
• QB (photobleaching quantum yield). QF/QB = number
of fluorescence cycles before bleaching. About
30,000 for fluorescein.
Photobleaching Reactions
O
O
1O2
3Dye* + 3O2
1Dye +
1O2
Photosensitized generation of singlet oxygen
Anthracene Nonfluorescent endoperoxide
The lifetime of 1O2 in cells is <0.5 µs versus 4 µs in H2O.
Why? Iain Johnson [Molecular Probes]
Wells, 9/15/03
• Electronic Excitation-Relaxation Rate R = I0 OD
• Excitation-Relaxation Rate Limit Rmax α (ελ τf )-1
• Fluorescence Rate (zero bleaching) Kf = Qf R
• Bleaching Rate KB = QB R
• Total Photons Emitted / Molecule Qf / QB
• Bleaching reduces the dye concentration c(t) = c0exp(-KBt)
Definitions
I0 = incident excitation intensity (W/cm2)
c0 = dye concentration prior to bleaching (mole/L)
ελ = molar extinction coefficient (M-1 -cm-1)
OD = optical density at fixed λ and pathlength = ελc0
Kf,B = fluorescence (f) or bleaching (B) rate (seconds-1)
Qf,B = fluorescence (f) or bleaching (B) quantum yield (environmentally dependent)
τf = fluorescence lifetime (seconds)
Dynamic Intensity Parameters
Fluorescence Rate (+ bleaching)
F(t) = Kf exp(-KBt)
Wells, 9/15/03
Photobleaching [QB] Varies between
dyes and chemical environments
Alexa Fluor 488
phalloidin
Fluorescein
phalloidin
Fluorescein dextran photobleaching under
one- and two-photon excitation
Biophys J 78:2159 (2000)
One-photon Two-photon
Fluorescence intensity Photobleaching rate
Wells, 9/15/03
Photochemistry: everything you didn’t want to know.
Cells containing ONLY DAPI may turn green!
1st exposure final exposureDIC, t0
DAPI
channel
fluorescein
channel
Wells, 9/15/03
Avoid dyes when there not essential to the experiment
DIC
fluorescence
DIC + fluorescence
Wells, 9/15/03
Antifade Reagents
Fixed Specimens
P-phenylene diamine
N-propyl gallate
DABCO
Hydroquinone
Live Specimens
Mitochondrial particles
Bacterial membrane “Oxyrase”
Vit. C, E
Trolox (water soluble E analogue)
O
CH
3
HO
H
3
C
CH
3
CH
3
C OH
O
Trolox® is a registered trademark of Hoffman – La Roche
Wells, 9/15/03
Guiding Principles for Imaging Fluorescence
Select the proper dye for the job – not always obvious
Optimize light collection conditions – use the proper
optics, filters, detectors, etc.
Minimize excitation exposure – minimize bleaching,
reduce sample perturbations, artifacts
Maximize the dynamic range – contrast depends on this!
Minimize background signal – maximize sensitivity

Weitere ähnliche Inhalte

Was ist angesagt?

Atomic Fluorescence Spectroscopy (AFS)
Atomic Fluorescence Spectroscopy (AFS)Atomic Fluorescence Spectroscopy (AFS)
Atomic Fluorescence Spectroscopy (AFS)Sajjad Ullah
 
fluroscence spectroscopy
fluroscence spectroscopyfluroscence spectroscopy
fluroscence spectroscopyPooja Dhurjad
 
Fluorescence spectroscopy, by kk sahu
Fluorescence spectroscopy, by kk sahuFluorescence spectroscopy, by kk sahu
Fluorescence spectroscopy, by kk sahuKAUSHAL SAHU
 
Atomic absorption spectrophotometry
Atomic absorption spectrophotometryAtomic absorption spectrophotometry
Atomic absorption spectrophotometryBasil "Lexi" Bruno
 
factors affecting fluorescence & phosphorescence
 factors affecting fluorescence & phosphorescence factors affecting fluorescence & phosphorescence
factors affecting fluorescence & phosphorescenceRamsha Afzal
 
Atomic Absorption Spectroscopy (AAS)
Atomic Absorption Spectroscopy (AAS)Atomic Absorption Spectroscopy (AAS)
Atomic Absorption Spectroscopy (AAS)Sajjad Ullah
 
Flourescence spectroscopy- instrumentation and applications
Flourescence spectroscopy-  instrumentation and applicationsFlourescence spectroscopy-  instrumentation and applications
Flourescence spectroscopy- instrumentation and applicationssinghsnehi01
 
Atomic absorption spectroscopy
Atomic absorption spectroscopyAtomic absorption spectroscopy
Atomic absorption spectroscopySaptarshi Das
 
Fluorescence and phosphorescence
Fluorescence and phosphorescenceFluorescence and phosphorescence
Fluorescence and phosphorescenceSamawiaIqbal
 
Fluorescence spectroscopy
Fluorescence spectroscopyFluorescence spectroscopy
Fluorescence spectroscopyNimisha Dutta
 
Light sources for atomic absorption spectroscopy (aas)
Light sources for atomic absorption spectroscopy (aas)Light sources for atomic absorption spectroscopy (aas)
Light sources for atomic absorption spectroscopy (aas)Dr. Mallikarjunaswamy C
 

Was ist angesagt? (20)

Fluorescence
FluorescenceFluorescence
Fluorescence
 
Flourescence & Phosphorescence
Flourescence & PhosphorescenceFlourescence & Phosphorescence
Flourescence & Phosphorescence
 
Atomic Fluorescence Spectroscopy (AFS)
Atomic Fluorescence Spectroscopy (AFS)Atomic Fluorescence Spectroscopy (AFS)
Atomic Fluorescence Spectroscopy (AFS)
 
fluroscence spectroscopy
fluroscence spectroscopyfluroscence spectroscopy
fluroscence spectroscopy
 
Phosphorimetry
PhosphorimetryPhosphorimetry
Phosphorimetry
 
Fluorescence spectroscopy, by kk sahu
Fluorescence spectroscopy, by kk sahuFluorescence spectroscopy, by kk sahu
Fluorescence spectroscopy, by kk sahu
 
Atomic absorption spectrophotometry
Atomic absorption spectrophotometryAtomic absorption spectrophotometry
Atomic absorption spectrophotometry
 
factors affecting fluorescence & phosphorescence
 factors affecting fluorescence & phosphorescence factors affecting fluorescence & phosphorescence
factors affecting fluorescence & phosphorescence
 
Atomic Absorption Spectroscopy (AAS)
Atomic Absorption Spectroscopy (AAS)Atomic Absorption Spectroscopy (AAS)
Atomic Absorption Spectroscopy (AAS)
 
Fluorescence spectroscopy
Fluorescence spectroscopyFluorescence spectroscopy
Fluorescence spectroscopy
 
Flourescence spectroscopy- instrumentation and applications
Flourescence spectroscopy-  instrumentation and applicationsFlourescence spectroscopy-  instrumentation and applications
Flourescence spectroscopy- instrumentation and applications
 
Atomic absorption spectroscopy
Atomic absorption spectroscopyAtomic absorption spectroscopy
Atomic absorption spectroscopy
 
Laser spectroscopy
Laser spectroscopyLaser spectroscopy
Laser spectroscopy
 
Fluorescence and phosphorescence
Fluorescence and phosphorescenceFluorescence and phosphorescence
Fluorescence and phosphorescence
 
Atomic absorption spectrophotometer
Atomic absorption spectrophotometerAtomic absorption spectrophotometer
Atomic absorption spectrophotometer
 
Fluorescence spectroscopy
Fluorescence spectroscopyFluorescence spectroscopy
Fluorescence spectroscopy
 
Dispersive & FTIR
Dispersive & FTIRDispersive & FTIR
Dispersive & FTIR
 
Light sources for atomic absorption spectroscopy (aas)
Light sources for atomic absorption spectroscopy (aas)Light sources for atomic absorption spectroscopy (aas)
Light sources for atomic absorption spectroscopy (aas)
 
Chemiluminescence
ChemiluminescenceChemiluminescence
Chemiluminescence
 
Flourescence
FlourescenceFlourescence
Flourescence
 

Andere mochten auch

Andere mochten auch (20)

Fluorescence spectroscopy
Fluorescence spectroscopyFluorescence spectroscopy
Fluorescence spectroscopy
 
Fluorimetry
FluorimetryFluorimetry
Fluorimetry
 
Fluorescence Microscopy
Fluorescence MicroscopyFluorescence Microscopy
Fluorescence Microscopy
 
Synthesis of thienyl appended porphyrins for cancer treatment by edith amuhaya
Synthesis of thienyl appended porphyrins for cancer treatment by edith amuhayaSynthesis of thienyl appended porphyrins for cancer treatment by edith amuhaya
Synthesis of thienyl appended porphyrins for cancer treatment by edith amuhaya
 
Photochemistry
PhotochemistryPhotochemistry
Photochemistry
 
Trans effect
Trans effectTrans effect
Trans effect
 
LIGHT SOURCE IN PHOTO CHEMISTRY
LIGHT SOURCE IN PHOTO CHEMISTRYLIGHT SOURCE IN PHOTO CHEMISTRY
LIGHT SOURCE IN PHOTO CHEMISTRY
 
Photochemistry
PhotochemistryPhotochemistry
Photochemistry
 
Photochemistry
PhotochemistryPhotochemistry
Photochemistry
 
Zappos final
Zappos finalZappos final
Zappos final
 
Dinkars presentation on potochemistry.
Dinkars presentation on potochemistry.Dinkars presentation on potochemistry.
Dinkars presentation on potochemistry.
 
Photochemistry
PhotochemistryPhotochemistry
Photochemistry
 
Dr.wael elhelece photochemistry 431chem
Dr.wael elhelece photochemistry 431chemDr.wael elhelece photochemistry 431chem
Dr.wael elhelece photochemistry 431chem
 
Photochemistry
PhotochemistryPhotochemistry
Photochemistry
 
Phosphorescence
PhosphorescencePhosphorescence
Phosphorescence
 
Photochemistry
PhotochemistryPhotochemistry
Photochemistry
 
Infrared spectroscopy
Infrared spectroscopyInfrared spectroscopy
Infrared spectroscopy
 
Application of fluorescence quenching
Application of fluorescence quenchingApplication of fluorescence quenching
Application of fluorescence quenching
 
Photochemistry
PhotochemistryPhotochemistry
Photochemistry
 
Nmr spectroscopy.
Nmr spectroscopy.Nmr spectroscopy.
Nmr spectroscopy.
 

Ähnlich wie Principles of fluorescence

Spectrofluorimetry
SpectrofluorimetrySpectrofluorimetry
SpectrofluorimetryMaharshi9
 
Fluorescence microscopy Likhith K
Fluorescence microscopy Likhith KFluorescence microscopy Likhith K
Fluorescence microscopy Likhith KLIKHITHK1
 
UV VISIBLE SPECTROSCOPY.pptx
UV VISIBLE SPECTROSCOPY.pptxUV VISIBLE SPECTROSCOPY.pptx
UV VISIBLE SPECTROSCOPY.pptxianjalliyadav
 
Flourimetry 140618015916-phpapp01 - copy
Flourimetry 140618015916-phpapp01 - copyFlourimetry 140618015916-phpapp01 - copy
Flourimetry 140618015916-phpapp01 - copyMANISH KUMAR
 
UV-visible spectroscopy - 2021
UV-visible spectroscopy - 2021UV-visible spectroscopy - 2021
UV-visible spectroscopy - 2021Ahmed Metwaly
 
Chemical fluorescent prode
Chemical fluorescent prodeChemical fluorescent prode
Chemical fluorescent prodealfachemistry
 
Flourimetry
FlourimetryFlourimetry
Flourimetrynehla313
 
1250706_634570128835168750.ppt
1250706_634570128835168750.ppt1250706_634570128835168750.ppt
1250706_634570128835168750.pptArun Nt
 
Fluorimetry/ Fluoroscences
Fluorimetry/ FluoroscencesFluorimetry/ Fluoroscences
Fluorimetry/ FluoroscencesROHIT
 
Payalfluorimetryupload
PayalfluorimetryuploadPayalfluorimetryupload
PayalfluorimetryuploadKhannapayal
 
SEMINAR ON UV SPECTROSCOPY
SEMINAR ON UV SPECTROSCOPYSEMINAR ON UV SPECTROSCOPY
SEMINAR ON UV SPECTROSCOPYNITIN KANWALE
 

Ähnlich wie Principles of fluorescence (20)

Spectrofluorimetry
SpectrofluorimetrySpectrofluorimetry
Spectrofluorimetry
 
Fluorometry Manik
Fluorometry Manik Fluorometry Manik
Fluorometry Manik
 
Spectrofluorimetry
SpectrofluorimetrySpectrofluorimetry
Spectrofluorimetry
 
Fluorescence microscopy Likhith K
Fluorescence microscopy Likhith KFluorescence microscopy Likhith K
Fluorescence microscopy Likhith K
 
Photoluminescence.pptx
Photoluminescence.pptxPhotoluminescence.pptx
Photoluminescence.pptx
 
UV VISIBLE SPECTROSCOPY.pptx
UV VISIBLE SPECTROSCOPY.pptxUV VISIBLE SPECTROSCOPY.pptx
UV VISIBLE SPECTROSCOPY.pptx
 
Flourimetry 140618015916-phpapp01 - copy
Flourimetry 140618015916-phpapp01 - copyFlourimetry 140618015916-phpapp01 - copy
Flourimetry 140618015916-phpapp01 - copy
 
Photoluminescence -chapter-6.pdf
Photoluminescence -chapter-6.pdfPhotoluminescence -chapter-6.pdf
Photoluminescence -chapter-6.pdf
 
UV-visible spectroscopy - 2021
UV-visible spectroscopy - 2021UV-visible spectroscopy - 2021
UV-visible spectroscopy - 2021
 
Fluorimetry
FluorimetryFluorimetry
Fluorimetry
 
Chemical fluorescent prode
Chemical fluorescent prodeChemical fluorescent prode
Chemical fluorescent prode
 
Flourimetry
FlourimetryFlourimetry
Flourimetry
 
1250706_634570128835168750.ppt
1250706_634570128835168750.ppt1250706_634570128835168750.ppt
1250706_634570128835168750.ppt
 
Radio isotopes
Radio isotopesRadio isotopes
Radio isotopes
 
Spectroflurometer
SpectroflurometerSpectroflurometer
Spectroflurometer
 
Fluorimetry/ Fluoroscences
Fluorimetry/ FluoroscencesFluorimetry/ Fluoroscences
Fluorimetry/ Fluoroscences
 
Payalfluorimetryupload
PayalfluorimetryuploadPayalfluorimetryupload
Payalfluorimetryupload
 
Specrtofluorometer
SpecrtofluorometerSpecrtofluorometer
Specrtofluorometer
 
FLUORIMETRY
FLUORIMETRYFLUORIMETRY
FLUORIMETRY
 
SEMINAR ON UV SPECTROSCOPY
SEMINAR ON UV SPECTROSCOPYSEMINAR ON UV SPECTROSCOPY
SEMINAR ON UV SPECTROSCOPY
 

Kürzlich hochgeladen

P4C x ELT = P4ELT: Its Theoretical Background (Kanazawa, 2024 March).pdf
P4C x ELT = P4ELT: Its Theoretical Background (Kanazawa, 2024 March).pdfP4C x ELT = P4ELT: Its Theoretical Background (Kanazawa, 2024 March).pdf
P4C x ELT = P4ELT: Its Theoretical Background (Kanazawa, 2024 March).pdfYu Kanazawa / Osaka University
 
HED Office Sohayok Exam Question Solution 2023.pdf
HED Office Sohayok Exam Question Solution 2023.pdfHED Office Sohayok Exam Question Solution 2023.pdf
HED Office Sohayok Exam Question Solution 2023.pdfMohonDas
 
Patterns of Written Texts Across Disciplines.pptx
Patterns of Written Texts Across Disciplines.pptxPatterns of Written Texts Across Disciplines.pptx
Patterns of Written Texts Across Disciplines.pptxMYDA ANGELICA SUAN
 
CHUYÊN ĐỀ DẠY THÊM TIẾNG ANH LỚP 11 - GLOBAL SUCCESS - NĂM HỌC 2023-2024 - HK...
CHUYÊN ĐỀ DẠY THÊM TIẾNG ANH LỚP 11 - GLOBAL SUCCESS - NĂM HỌC 2023-2024 - HK...CHUYÊN ĐỀ DẠY THÊM TIẾNG ANH LỚP 11 - GLOBAL SUCCESS - NĂM HỌC 2023-2024 - HK...
CHUYÊN ĐỀ DẠY THÊM TIẾNG ANH LỚP 11 - GLOBAL SUCCESS - NĂM HỌC 2023-2024 - HK...Nguyen Thanh Tu Collection
 
How to Add a New Field in Existing Kanban View in Odoo 17
How to Add a New Field in Existing Kanban View in Odoo 17How to Add a New Field in Existing Kanban View in Odoo 17
How to Add a New Field in Existing Kanban View in Odoo 17Celine George
 
Easter in the USA presentation by Chloe.
Easter in the USA presentation by Chloe.Easter in the USA presentation by Chloe.
Easter in the USA presentation by Chloe.EnglishCEIPdeSigeiro
 
How to Use api.constrains ( ) in Odoo 17
How to Use api.constrains ( ) in Odoo 17How to Use api.constrains ( ) in Odoo 17
How to Use api.constrains ( ) in Odoo 17Celine George
 
The basics of sentences session 10pptx.pptx
The basics of sentences session 10pptx.pptxThe basics of sentences session 10pptx.pptx
The basics of sentences session 10pptx.pptxheathfieldcps1
 
How to Make a Field read-only in Odoo 17
How to Make a Field read-only in Odoo 17How to Make a Field read-only in Odoo 17
How to Make a Field read-only in Odoo 17Celine George
 
Maximizing Impact_ Nonprofit Website Planning, Budgeting, and Design.pdf
Maximizing Impact_ Nonprofit Website Planning, Budgeting, and Design.pdfMaximizing Impact_ Nonprofit Website Planning, Budgeting, and Design.pdf
Maximizing Impact_ Nonprofit Website Planning, Budgeting, and Design.pdfTechSoup
 
Human-AI Co-Creation of Worked Examples for Programming Classes
Human-AI Co-Creation of Worked Examples for Programming ClassesHuman-AI Co-Creation of Worked Examples for Programming Classes
Human-AI Co-Creation of Worked Examples for Programming ClassesMohammad Hassany
 
How to Add Existing Field in One2Many Tree View in Odoo 17
How to Add Existing Field in One2Many Tree View in Odoo 17How to Add Existing Field in One2Many Tree View in Odoo 17
How to Add Existing Field in One2Many Tree View in Odoo 17Celine George
 
Ultra structure and life cycle of Plasmodium.pptx
Ultra structure and life cycle of Plasmodium.pptxUltra structure and life cycle of Plasmodium.pptx
Ultra structure and life cycle of Plasmodium.pptxDr. Asif Anas
 
The Stolen Bacillus by Herbert George Wells
The Stolen Bacillus by Herbert George WellsThe Stolen Bacillus by Herbert George Wells
The Stolen Bacillus by Herbert George WellsEugene Lysak
 
The Singapore Teaching Practice document
The Singapore Teaching Practice documentThe Singapore Teaching Practice document
The Singapore Teaching Practice documentXsasf Sfdfasd
 
Diploma in Nursing Admission Test Question Solution 2023.pdf
Diploma in Nursing Admission Test Question Solution 2023.pdfDiploma in Nursing Admission Test Question Solution 2023.pdf
Diploma in Nursing Admission Test Question Solution 2023.pdfMohonDas
 
AUDIENCE THEORY -- FANDOM -- JENKINS.pptx
AUDIENCE THEORY -- FANDOM -- JENKINS.pptxAUDIENCE THEORY -- FANDOM -- JENKINS.pptx
AUDIENCE THEORY -- FANDOM -- JENKINS.pptxiammrhaywood
 
Education and training program in the hospital APR.pptx
Education and training program in the hospital APR.pptxEducation and training program in the hospital APR.pptx
Education and training program in the hospital APR.pptxraviapr7
 
How to Add a many2many Relational Field in Odoo 17
How to Add a many2many Relational Field in Odoo 17How to Add a many2many Relational Field in Odoo 17
How to Add a many2many Relational Field in Odoo 17Celine George
 

Kürzlich hochgeladen (20)

P4C x ELT = P4ELT: Its Theoretical Background (Kanazawa, 2024 March).pdf
P4C x ELT = P4ELT: Its Theoretical Background (Kanazawa, 2024 March).pdfP4C x ELT = P4ELT: Its Theoretical Background (Kanazawa, 2024 March).pdf
P4C x ELT = P4ELT: Its Theoretical Background (Kanazawa, 2024 March).pdf
 
HED Office Sohayok Exam Question Solution 2023.pdf
HED Office Sohayok Exam Question Solution 2023.pdfHED Office Sohayok Exam Question Solution 2023.pdf
HED Office Sohayok Exam Question Solution 2023.pdf
 
Patterns of Written Texts Across Disciplines.pptx
Patterns of Written Texts Across Disciplines.pptxPatterns of Written Texts Across Disciplines.pptx
Patterns of Written Texts Across Disciplines.pptx
 
CHUYÊN ĐỀ DẠY THÊM TIẾNG ANH LỚP 11 - GLOBAL SUCCESS - NĂM HỌC 2023-2024 - HK...
CHUYÊN ĐỀ DẠY THÊM TIẾNG ANH LỚP 11 - GLOBAL SUCCESS - NĂM HỌC 2023-2024 - HK...CHUYÊN ĐỀ DẠY THÊM TIẾNG ANH LỚP 11 - GLOBAL SUCCESS - NĂM HỌC 2023-2024 - HK...
CHUYÊN ĐỀ DẠY THÊM TIẾNG ANH LỚP 11 - GLOBAL SUCCESS - NĂM HỌC 2023-2024 - HK...
 
How to Add a New Field in Existing Kanban View in Odoo 17
How to Add a New Field in Existing Kanban View in Odoo 17How to Add a New Field in Existing Kanban View in Odoo 17
How to Add a New Field in Existing Kanban View in Odoo 17
 
Easter in the USA presentation by Chloe.
Easter in the USA presentation by Chloe.Easter in the USA presentation by Chloe.
Easter in the USA presentation by Chloe.
 
How to Use api.constrains ( ) in Odoo 17
How to Use api.constrains ( ) in Odoo 17How to Use api.constrains ( ) in Odoo 17
How to Use api.constrains ( ) in Odoo 17
 
The basics of sentences session 10pptx.pptx
The basics of sentences session 10pptx.pptxThe basics of sentences session 10pptx.pptx
The basics of sentences session 10pptx.pptx
 
How to Make a Field read-only in Odoo 17
How to Make a Field read-only in Odoo 17How to Make a Field read-only in Odoo 17
How to Make a Field read-only in Odoo 17
 
Finals of Kant get Marx 2.0 : a general politics quiz
Finals of Kant get Marx 2.0 : a general politics quizFinals of Kant get Marx 2.0 : a general politics quiz
Finals of Kant get Marx 2.0 : a general politics quiz
 
Maximizing Impact_ Nonprofit Website Planning, Budgeting, and Design.pdf
Maximizing Impact_ Nonprofit Website Planning, Budgeting, and Design.pdfMaximizing Impact_ Nonprofit Website Planning, Budgeting, and Design.pdf
Maximizing Impact_ Nonprofit Website Planning, Budgeting, and Design.pdf
 
Human-AI Co-Creation of Worked Examples for Programming Classes
Human-AI Co-Creation of Worked Examples for Programming ClassesHuman-AI Co-Creation of Worked Examples for Programming Classes
Human-AI Co-Creation of Worked Examples for Programming Classes
 
How to Add Existing Field in One2Many Tree View in Odoo 17
How to Add Existing Field in One2Many Tree View in Odoo 17How to Add Existing Field in One2Many Tree View in Odoo 17
How to Add Existing Field in One2Many Tree View in Odoo 17
 
Ultra structure and life cycle of Plasmodium.pptx
Ultra structure and life cycle of Plasmodium.pptxUltra structure and life cycle of Plasmodium.pptx
Ultra structure and life cycle of Plasmodium.pptx
 
The Stolen Bacillus by Herbert George Wells
The Stolen Bacillus by Herbert George WellsThe Stolen Bacillus by Herbert George Wells
The Stolen Bacillus by Herbert George Wells
 
The Singapore Teaching Practice document
The Singapore Teaching Practice documentThe Singapore Teaching Practice document
The Singapore Teaching Practice document
 
Diploma in Nursing Admission Test Question Solution 2023.pdf
Diploma in Nursing Admission Test Question Solution 2023.pdfDiploma in Nursing Admission Test Question Solution 2023.pdf
Diploma in Nursing Admission Test Question Solution 2023.pdf
 
AUDIENCE THEORY -- FANDOM -- JENKINS.pptx
AUDIENCE THEORY -- FANDOM -- JENKINS.pptxAUDIENCE THEORY -- FANDOM -- JENKINS.pptx
AUDIENCE THEORY -- FANDOM -- JENKINS.pptx
 
Education and training program in the hospital APR.pptx
Education and training program in the hospital APR.pptxEducation and training program in the hospital APR.pptx
Education and training program in the hospital APR.pptx
 
How to Add a many2many Relational Field in Odoo 17
How to Add a many2many Relational Field in Odoo 17How to Add a many2many Relational Field in Odoo 17
How to Add a many2many Relational Field in Odoo 17
 

Principles of fluorescence

  • 2. Wells, 9/15/03 What is fluorescence? Optical and Physical Properties Detection and Measurement Chemical properties and Biological applications to be discussed later
  • 3. Wells, 9/15/03 Definition of Fluorescence Light emitted by singlet excited states of molecules following absorption of photons from an external source Requirements for Fluorescence Fluorescent Dye (Fluorophore). A molecule with a rigid conjugated structure (usually a polyaromatic hydrocarbon or heterocycle). Excitation. Creation of dye excited state by absorption of a photon.
  • 4. Wells, 9/15/03 Molecular Structure Features and Fluorescence High ring density of π electrons increase fluorescence. Aromatic hydrocarbons (π to π * absorption) are frequently fluorescent. Increased hydrocarbon conjugation shifts π το π* absorption to the red and increases the probability of fluorescence. (C3H3) n, n = 3,5,7 Cy3, Cy5, Cy7 Protonation (affected by e- withdrawing/donating groups). OH produces blue shift and decreased Qf in fluorescein. Halogens (e- withdrawing) lower pKa and alkyl groups (e- donating) raise the pKa. Planarity and rigidity affect fluorescence. Increased viscosity may slow free internal rotations and increase fluorescence. C6H5 CH CH C6H5
  • 5. Wells, 9/15/03 Fluorescent PROBES: Tags, Tracers, Sensors, etc. Fluorescent tags generate contrast between an analyte and background, e.g., to identify receptors, or decorate cellular anatomy. Fluorescent sensors or indicators change spectral properties in response to dynamic equilibria of chemical reactions, intracellular ions e.g., Ca2+, H+, and membrane potential.
  • 6. Wells, 9/15/03 Fluorescence Combines Chemical and Optical Recognition Analyte isolated or complex dynamic mixture Probe targeting group + organic dye chemical connection optical connection Detection
  • 7. Wells, 9/15/03 ANALYTES can evaluated by fluorescence in a wide range of environments, including single molecules in solutions, gels or solids, cultured cells, thick tissue sections, and live animals. ?
  • 8. Wells, 9/15/03 Optical Properties Spectral properties of fluorescent probes define their utility in biological applications. Fluorescence spectra convey information regarding molecular identity. Variations in fluorescence spectra characterize chemical and physical behavior.
  • 9. Wells, 9/15/03 1 micron spheres viewed under transmitted incandescent “white” light The light passing through the sample is diffracted, partially absorbed and transmitted to the detector.
  • 10. Wells, 9/15/03 1 micron spheres emitting fluorescence The light passing through the sample is diffracted and absorbed, then FILTERED to detect light originating from molecules within the sample.
  • 11. Wells, 9/15/03 Fluorescence-related Terminology Excitation – process of absorbing optical energy. Extinction coefficient – efficiency of absorbing optical energy; this value is wavelength dependent. Emission – process of releasing optical energy. Quantum yield – efficiency of releasing energy (0-1), generally in the form of fluorescent light; not wavelength dependent. Stokes shift – difference in energy between excitation and emission wavelength maxima – the key to contrast generation. Spectrum – distribution of excitation and emission wavelengths
  • 12. Wells, 9/15/03 Spectral Features of Fluorescence λ2 - λ1 = Stokes shift F = I0ЄλC0LQf I0 (Єλ) a
  • 13. Absorption and Fluorescence Excitation Spectra: Same or Different? Incident intensity (Io) Transmitted intensity (It) Fluorescence intensity (IF) Absorption spectrum: Scan Io(λ), detect It Fluorescence excitation spectrum: Scan Io(λ), detect IF Fluorescenceexcitation For pure dye molecules in solution, the absorption and fluorescence excitation spectra are usually identical and can be used interchangeably. When more than one absorbing or fluorescent species is present in the sample, they are typically different, as in the case of protein-conjugated tetramethylrhodamine dyes, shown right. Absorption 500 550 600 Wavelength (nm) Iain Johnson [Molecular Probes]
  • 14. Wells, 9/15/03 DETECTION involves only a single observable parameter = INTENSITY. The spectral properties [COLOR] are discriminated by optical filtering and sometimes temporal filtering prior to measuring the intensity. Therefore, it is extremely important to configure the filtering and optical collection conditions properly to avoid artifacts and faulty observations!
  • 15. Wells, 9/15/03 EX = excitation filter DB = dichroic beamsplitter EM = emission filter Contrast by Epi-illumination (reflection mode) and Optical Filtering
  • 16. Wells, 9/15/03 Chemical Identification and Physical Properties by Excitation and Emission (A) 1 dye:1 excitation, 1 emission (B) 1 dye:2 excitation, 1 emission or (C) 1 excitation, 2 emission (D) 2 dyes:1 excitation, 2 emission or (E) 2 excitation, 2 emission (F) >1 dye: >1 excitation, >1 emission wavelength= emission,= excitation,
  • 17. Wells, 9/15/03 detector DETECTION / Microscopy, Imaging Basic components for reflected light fluorescence microscopy, a.k.a. incident light fluorescence, or epi- fluorescence. 1. Excitation light source 2. Filter set 3. Objective lens (the microscope) 4. Fluorescent specimen (analyte + probe) 5. Detector
  • 19. Wells, 9/15/03 Spectral Optimization • Absorption wavelength Optimize excitation by matching dye absorption to source output (Note: total excitation is the product of the source power and dye absorption) • Emission wavelength Impacts the ability to discriminate fluorescence signals from probe A versus probe B and background autofluorescence • Emission bandwidth Narrow bandwidths minimize overspill in multicolor label detection
  • 20. Wells, 9/15/03 Limitations to Sensitivity (S/N) Noise Sources Instrument Stray light, detector noise Sample Sample–related autofluorescence, scattered excitation light (particle size and wavelength dependent) Reagent Unbound or nonspecifically bound probes Signal Losses Inner filter effects, Emission scattering, Photobleaching
  • 21. Wells, 9/15/03 Single T2 genomic DNA macromolecule stained with YOYO-1 (10 nM loading in very low concentration DNA in agarose). Wide-field image using a 1.4NA objective and CCD camera. Unfortunately, YOYO-1 bleaches very fast.
  • 22. Wells, 9/15/03 Viability 2 dyes, 1excitation, 2 emissions, 1 LP filter set calcien AM (green, live cells), ethidium homodimer (red, dead cells)
  • 23. Wells, 9/15/03 Organelles 1 dye, 2 excitation, 2 emissions, switch between 2 filter sets BODIPY FL C5-ceramide accumulation in the trans-Golgi is sufficient for excimer formation (top panel). Images by Richard Pagano, Mayo Foundation.
  • 24. Wells, 9/15/03 Cytoskeleton 3 dyes, 3 excitation, 3 emissions BPAE cells: microtubules (green, Bodipy-antibody), F-actin (red, Texas Red-X phalloidin) and nuclei (blue, DAPI). Triple exposure on 35mm film. This can be done with a triple-band filter set or by 3 filter sets.
  • 25. Wells, 9/15/03 1 (UV) excitation “band,” 8 (visible) emission “bands” detected simultaneously on 35mm color film Macro-image
  • 26. Wells, 9/15/03 Physical Origins of Fluorescence 1 Dye excited state created by absorption of a photon from an external light source 2 Fluorescence photon emitted has lower energy (longer wavelength) than excitation photon. Ratio of emitted photons/absorbed photons (fluorescence quantum yield) is usually less than 1.0 3 Unexcited dye is regenerated for repeat absorption/emission cycles. Cycle can be broken by photobleaching (irreversible photochemical destruction of excited dye)
  • 27. Wells, 9/15/03 Energy Flow: Photons→Electrons→Photons Different dye molecules exhibit different excitation and emission “profiles.” Energy is proportional to 1/λ 1 = absorbance of light (excitation) 2 = vibronic relaxation, (Stokes shift) 3 = emission of light
  • 28. Wells, 9/15/03 Fluorescence Excitation-Emission Cycle Relaxation Phosphorescence hνPH S1' S1 Excitation hνEX Energy(hν) T1 Emission hνFL photoproducts S0 S0 = ground electronic state S1 = first singlet excited state T1= triplet excited state Radiative transition Nonradiative transition Iain Johnson [Molecular Probes]
  • 29. Wells, 9/15/03 Molecular Electronic State Transitions Jablonski Diagram Iain Johnson [Molecular Probes]
  • 30. Wells, 9/15/03 • Intermolecular Energy Transfer - characterized by donor-acceptor pairing short range exchange interactions within interatomic collision diameter long range interactions by sequential short range exchange optical interactions between transition dipoles - decays as 1/r6 exciton migration = electron-hole pair migration - decays as 1/r3 eximer = complex between excited and ground state of same type molecules exiplex = complex between excited and ground state of different type molecules • Intramolecular Energy Transfer intersystem crossing, e.g., E-type delayed fluorescence, phosphorescence internal conversion, e.g., vibronic transitions • Polarized Transitions and Rotational Diffusion angular orientation between excitation and emitted light polarization changes when a fluorophore rotates during the excited-state lifetime (τf) Molecular Interactions and Fluorescence excitation emission D < 100 Α emission dipole excitation time excitation dipole
  • 31. Wells, 9/15/03 Two Photon Excitation (hνEX)/2 hνEM Energy S1' S1 (hνEX)/2 S0 1 2 Radiative transition Nonradiative transition
  • 32. Wells, 9/15/03 One-photon ≠ Two-photon Excitation Spectra Xu, Zipfel, Shear, Williams, Webb. Proc Natl Acad Sci USA 93:10763 (1996)
  • 33. Wells, 9/15/03 Quantitative Fluorometry • Measured fluorescence intensities are products of dye- dependent (ε, QY), sample-dependent (c, L) and instrument- dependent (I0 , k) factors • Sample-to-sample and instrument-to-instrument comparisons should ideally be made with reference to standard materials such as fluorescent microspheres • Attempts to increase signal levels by using higher concentrations of dye may have the opposite effect due to dye–dye interactions (self-quenching) or optical artifacts (e.g. inner filter effects, self- absorption of fluorescence)
  • 34. Wells, 9/15/03 Photometric Output Factors: Absorption • Molar extinction coefficient (aka molar absorptivity). Symbol: ε Units: cm-1 M-1 Defined by the Beer-Lambert law log I0/It = ε·c·l • Quantifies efficiency of light absorption at a specific wavelength (εmax = peak value) • Typically 10,000 – 200,000 cm-1 M-1 • Not strongly environment dependent
  • 35. Wells, 9/15/03 Photometric Output Factors: Emission • Fluorescence quantum yield – Symbol: QY or φF or QF Units: none • Number of fluorescence photons emitted per photon absorbed • Typically 0.05 – 1.0 • Strongly environment dependent • Other emission parameters: lifetime, polarization
  • 36. Wells, 9/15/03 Quantifying Fluorescence Intensity I F = I 0 ·QY·(1-e –2.303 ε.c.L )·k For (ε·c·L) < 0.05 I F = I 0 ·QY·(2.303·ε·c·L)·k = Fluorescence emission intensity at λEM QY = Fluorophore quantum yield ε = Molar extinction coefficient of fluorophore at λEX c = Fluorophore concentration L = Optical pathlength for excitation I0 = Excitation source intensity at λEX k = Fluorescence collection efficiency* *In a typical epifluorescence microscope, about 3% of the total available emitted photons are collected (k =0.03) [Webb et al, PNAS 94, 11753 (1997)]
  • 37. Wells, 9/15/03 IF will decrease when (ε·c·L) > 0.05 Inner Filter Effect Simulation 0.0 0.2 0.4 0.6 0.8 1.0 0 0.5 1 1.5 2 ε .c.L Intensity 1-e-2.303.ε.c.L IF = Io.(1-e-2.303.ε.c.L) Io Iain Johnson [Molecular Probes]
  • 38. Wells, 9/15/03 Total Output Limit: Photobleaching • Irreversible destruction of excited fluorophore • Proportional to time-integrated excitation intensity • Avoidance: minimize excitation, maximize detection efficiency, antifade reagents • QB (photobleaching quantum yield). QF/QB = number of fluorescence cycles before bleaching. About 30,000 for fluorescein.
  • 39. Photobleaching Reactions O O 1O2 3Dye* + 3O2 1Dye + 1O2 Photosensitized generation of singlet oxygen Anthracene Nonfluorescent endoperoxide The lifetime of 1O2 in cells is <0.5 µs versus 4 µs in H2O. Why? Iain Johnson [Molecular Probes]
  • 40. Wells, 9/15/03 • Electronic Excitation-Relaxation Rate R = I0 OD • Excitation-Relaxation Rate Limit Rmax α (ελ τf )-1 • Fluorescence Rate (zero bleaching) Kf = Qf R • Bleaching Rate KB = QB R • Total Photons Emitted / Molecule Qf / QB • Bleaching reduces the dye concentration c(t) = c0exp(-KBt) Definitions I0 = incident excitation intensity (W/cm2) c0 = dye concentration prior to bleaching (mole/L) ελ = molar extinction coefficient (M-1 -cm-1) OD = optical density at fixed λ and pathlength = ελc0 Kf,B = fluorescence (f) or bleaching (B) rate (seconds-1) Qf,B = fluorescence (f) or bleaching (B) quantum yield (environmentally dependent) τf = fluorescence lifetime (seconds) Dynamic Intensity Parameters Fluorescence Rate (+ bleaching) F(t) = Kf exp(-KBt)
  • 41. Wells, 9/15/03 Photobleaching [QB] Varies between dyes and chemical environments Alexa Fluor 488 phalloidin Fluorescein phalloidin
  • 42. Fluorescein dextran photobleaching under one- and two-photon excitation Biophys J 78:2159 (2000) One-photon Two-photon Fluorescence intensity Photobleaching rate
  • 43. Wells, 9/15/03 Photochemistry: everything you didn’t want to know. Cells containing ONLY DAPI may turn green! 1st exposure final exposureDIC, t0 DAPI channel fluorescein channel
  • 44. Wells, 9/15/03 Avoid dyes when there not essential to the experiment DIC fluorescence DIC + fluorescence
  • 45. Wells, 9/15/03 Antifade Reagents Fixed Specimens P-phenylene diamine N-propyl gallate DABCO Hydroquinone Live Specimens Mitochondrial particles Bacterial membrane “Oxyrase” Vit. C, E Trolox (water soluble E analogue) O CH 3 HO H 3 C CH 3 CH 3 C OH O Trolox® is a registered trademark of Hoffman – La Roche
  • 46. Wells, 9/15/03 Guiding Principles for Imaging Fluorescence Select the proper dye for the job – not always obvious Optimize light collection conditions – use the proper optics, filters, detectors, etc. Minimize excitation exposure – minimize bleaching, reduce sample perturbations, artifacts Maximize the dynamic range – contrast depends on this! Minimize background signal – maximize sensitivity