The document provides an overview of fluorescence, including:
- Definitions of key fluorescence concepts like excitation, emission spectra, and quantum yield
- How molecular structure influences fluorescence properties
- Common fluorescent probes and their applications in tagging molecules and sensing analytes
- Techniques for detection and measurement of fluorescence, including microscopy and filtering of excitation and emission wavelengths
- Factors that influence fluorescence intensity and limit sensitivity, such as photobleaching
Fluorescence spectroscopy becomes a widely used tool at the interface of biology, chemistry and physics, because of its precise sensitivity and recent technical advancements. The measurements can provide information on a wide range of molecular processes including the interactions of solvent molecules with fluorophores, rotational diffraction of biomolecules, distance between sites of biomolecules, conformational changes and binding interactions. These advances in fluorescence technology are decreasing the cost and complexity of previously complex processes. Fluorescence spectroscopy is a highly developed and non-invasive technique that enables the on-line measurements of substrate and product concentrations or the identification of characteristic process states.
Fluorescence spectroscopy becomes a widely used tool at the interface of biology, chemistry and physics, because of its precise sensitivity and recent technical advancements. The measurements can provide information on a wide range of molecular processes including the interactions of solvent molecules with fluorophores, rotational diffraction of biomolecules, distance between sites of biomolecules, conformational changes and binding interactions. These advances in fluorescence technology are decreasing the cost and complexity of previously complex processes. Fluorescence spectroscopy is a highly developed and non-invasive technique that enables the on-line measurements of substrate and product concentrations or the identification of characteristic process states.
Introduction to Spectroscopy,
Introduction to UV, electronic transitions, terminology, chromophore, Auxochrome, Examples and Applications.
Introduction to IR, Fundamental vibrations, Types of Vibrations, Factors affecting the vibrational freaquencies, Group frequencies, examples and applications.
Photoluminescence Spectroscopy for studying Electron-Hole pair recombination ...RunjhunDutta
Description of Photoluminescence Spectroscopy: Principle, Instrumentation & Application.
Three research papers have been summarized which lay stress on Photoluminescence Study for Electron-Hole Pair Recombination for characterizing the properties of semiconductors used in Photoelectrochemical Splitting of Water.
describes the complete history, mechanisms, instrumentation(jablonski diagram), types, comparision and factors affecting, applications of fluorescence and phosphorescence and describes about quenching and stokes shift.
Basic operating principle and instrumentation of photo-luminescence technique. Brief description about interpretation of a photo-luminescence spectrum. Applications, advantages and disadvantages of photo-luminescence.
Introduction to Spectroscopy,
Introduction to UV, electronic transitions, terminology, chromophore, Auxochrome, Examples and Applications.
Introduction to IR, Fundamental vibrations, Types of Vibrations, Factors affecting the vibrational freaquencies, Group frequencies, examples and applications.
Photoluminescence Spectroscopy for studying Electron-Hole pair recombination ...RunjhunDutta
Description of Photoluminescence Spectroscopy: Principle, Instrumentation & Application.
Three research papers have been summarized which lay stress on Photoluminescence Study for Electron-Hole Pair Recombination for characterizing the properties of semiconductors used in Photoelectrochemical Splitting of Water.
describes the complete history, mechanisms, instrumentation(jablonski diagram), types, comparision and factors affecting, applications of fluorescence and phosphorescence and describes about quenching and stokes shift.
Basic operating principle and instrumentation of photo-luminescence technique. Brief description about interpretation of a photo-luminescence spectrum. Applications, advantages and disadvantages of photo-luminescence.
431chem course Aljouf university, college of science, chemistry department.
. Fates of Excited State Molecules.
• Absorption and emission of electromagnetic radiation.
• Einstein coefficients, absorption probabilities.
• Fluorescence and phosphorescence.
• Internal conversion and intersystem crossing.
• Photodissociation and predissociation.
• Jablonski diagram.
. Lasers.
• Requirements for laser action.
• Population inversions.
• Properties of laser radiation.
• Examples of lasers.
• Applications in spectroscopy and photochemistry.
Dr Wael A. Elhelece.
Introduction, theoretical principle, quantum efficiency of fluorescence, molecular structure of
fluorescence, instrumentation, factors influencing the intensity of fluorescence, comparison of
fluorometry with spectrophotometry, application of fluorometry in pharmaceutical analysis
Fluorescence as a phenomenon is part of a larger family of related luminescent processes in which a susceptible substance absorbs light, only to reemit light (photons) from electronically excited states after a given time.
Photo luminescent processes that are generated through excitation, whether this is via physical, mechanical, or chemical mechanisms, can generally be subdivided into fluorescence and phosphorescence. Absorption of a light quantum (blue) causes an electron to move to a higher energy orbit. After residing in this “excited state” for a particular time, the fluorescence lifetime, the electron falls back to its original orbit and the fluorochrome dissipates the excess energy by emitting a photon (green).
Compounds that display fluorescent properties are generally termed fluorescent probes or dyes. Often ‘fluorochrome’ and ‘fluorophore’ are used interchangeably. The term ‘fluorophore’ refers to fluorochromes that are conjugated covalently or through adsorption to biological macromolecules, such as nucleic acids, lipids, or proteins. Fluorochromes come in different flavors and include organic molecules (dyes), inorganic ions (e.g., lanthanide ions such as Eu, Tb, Yb, etc.)fluorescent proteins (e.g., green fluorescent protein) atoms (such as gaseous mercury in glass light tubes).
Recently, inorganic luminescent semiconducting nanoparticles, quantum dots, have been introduced as labels for biological assays, bio-imaging applications, and theragnostic purposes (the combination of diagnostic and therapeutic modalities in one and the same particle).
Fluorescence microscopy provides an efficient and unique approach to study fixed and living cells because of its versatility, specificity, and high sensitivity.
Fluorescence microscopes can both detect the fluorescence emitted from labeled molecules in biological samples as images or photometric data from which intensities and emission spectra can be deduced. By exploiting the characteristics of fluorescence, various techniques have been developed that enable the visualization and analysis of complex dynamic events in cells, organelles, and sub-organelle components within the biological specimen.
The most common techniques are
Fluorescence recovery after photo bleaching (FRAP)
Fluorescence loss in photo bleaching (FLIP)
Fluorescence localization after photo bleaching (FLAP)
Fluorescence resonance energy transfer (FRET)
Alfa Chemistry offers an extensive catalog of building blocks, reagents, catalysts, reference materials, and research chemicals in a wide range of applications. We also provide analytical services and laboratory services to our customers. We provide Chemical Fluorescence Probe. Visit https://www.alfa-chemistry.com/products/chemical-fluorescence-probe-173.htm for more information.
PRINCIPLE
1-Collisional deactivation:
2-Flourescence:
Phosphorescence:
JABLONSKI DIAGRAM
TYPES OF FLOURESCENCE
FACTORS INFLUENCING FLOURESCENCE INTENSITY
EFFECT OF CONCENTRATION ON FLOURESCENCE INTENSITY
QUENCHING OF FLOURESCENCE AND TYPES
INSTRUMENTATION OF FLOURIMETRY
a. Single beam filter flourimeter
b. Double beam (filter) fiourimeter
c. Spectroflourimeter (double beam)
ADVANTAGES AND LIMITATIONS OF FLOURIMETRY
Fluorescence is the phenomenon whereby a molecule, after absorption radiation, emits radiation of a longer wavelength.
A compound absorbs radiation in the UV-rgion and emits visible light.
Absorption of uv/visible radiation causes transition of electrons from ground state (low energy) to excited state (high energy).
This increase in wavelength is known as the Stokes shift.
Students, digital devices and success - Andreas Schleicher - 27 May 2024..pptxEduSkills OECD
Andreas Schleicher presents at the OECD webinar ‘Digital devices in schools: detrimental distraction or secret to success?’ on 27 May 2024. The presentation was based on findings from PISA 2022 results and the webinar helped launch the PISA in Focus ‘Managing screen time: How to protect and equip students against distraction’ https://www.oecd-ilibrary.org/education/managing-screen-time_7c225af4-en and the OECD Education Policy Perspective ‘Students, digital devices and success’ can be found here - https://oe.cd/il/5yV
Instructions for Submissions thorugh G- Classroom.pptxJheel Barad
This presentation provides a briefing on how to upload submissions and documents in Google Classroom. It was prepared as part of an orientation for new Sainik School in-service teacher trainees. As a training officer, my goal is to ensure that you are comfortable and proficient with this essential tool for managing assignments and fostering student engagement.
Synthetic Fiber Construction in lab .pptxPavel ( NSTU)
Synthetic fiber production is a fascinating and complex field that blends chemistry, engineering, and environmental science. By understanding these aspects, students can gain a comprehensive view of synthetic fiber production, its impact on society and the environment, and the potential for future innovations. Synthetic fibers play a crucial role in modern society, impacting various aspects of daily life, industry, and the environment. ynthetic fibers are integral to modern life, offering a range of benefits from cost-effectiveness and versatility to innovative applications and performance characteristics. While they pose environmental challenges, ongoing research and development aim to create more sustainable and eco-friendly alternatives. Understanding the importance of synthetic fibers helps in appreciating their role in the economy, industry, and daily life, while also emphasizing the need for sustainable practices and innovation.
We all have good and bad thoughts from time to time and situation to situation. We are bombarded daily with spiraling thoughts(both negative and positive) creating all-consuming feel , making us difficult to manage with associated suffering. Good thoughts are like our Mob Signal (Positive thought) amidst noise(negative thought) in the atmosphere. Negative thoughts like noise outweigh positive thoughts. These thoughts often create unwanted confusion, trouble, stress and frustration in our mind as well as chaos in our physical world. Negative thoughts are also known as “distorted thinking”.
Extraction Of Natural Dye From Beetroot (Beta Vulgaris) And Preparation Of He...SachinKumar945617
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The Roman Empire A Historical Colossus.pdfkaushalkr1407
The Roman Empire, a vast and enduring power, stands as one of history's most remarkable civilizations, leaving an indelible imprint on the world. It emerged from the Roman Republic, transitioning into an imperial powerhouse under the leadership of Augustus Caesar in 27 BCE. This transformation marked the beginning of an era defined by unprecedented territorial expansion, architectural marvels, and profound cultural influence.
The empire's roots lie in the city of Rome, founded, according to legend, by Romulus in 753 BCE. Over centuries, Rome evolved from a small settlement to a formidable republic, characterized by a complex political system with elected officials and checks on power. However, internal strife, class conflicts, and military ambitions paved the way for the end of the Republic. Julius Caesar’s dictatorship and subsequent assassination in 44 BCE created a power vacuum, leading to a civil war. Octavian, later Augustus, emerged victorious, heralding the Roman Empire’s birth.
Under Augustus, the empire experienced the Pax Romana, a 200-year period of relative peace and stability. Augustus reformed the military, established efficient administrative systems, and initiated grand construction projects. The empire's borders expanded, encompassing territories from Britain to Egypt and from Spain to the Euphrates. Roman legions, renowned for their discipline and engineering prowess, secured and maintained these vast territories, building roads, fortifications, and cities that facilitated control and integration.
The Roman Empire’s society was hierarchical, with a rigid class system. At the top were the patricians, wealthy elites who held significant political power. Below them were the plebeians, free citizens with limited political influence, and the vast numbers of slaves who formed the backbone of the economy. The family unit was central, governed by the paterfamilias, the male head who held absolute authority.
Culturally, the Romans were eclectic, absorbing and adapting elements from the civilizations they encountered, particularly the Greeks. Roman art, literature, and philosophy reflected this synthesis, creating a rich cultural tapestry. Latin, the Roman language, became the lingua franca of the Western world, influencing numerous modern languages.
Roman architecture and engineering achievements were monumental. They perfected the arch, vault, and dome, constructing enduring structures like the Colosseum, Pantheon, and aqueducts. These engineering marvels not only showcased Roman ingenuity but also served practical purposes, from public entertainment to water supply.
2024.06.01 Introducing a competency framework for languag learning materials ...Sandy Millin
http://sandymillin.wordpress.com/iateflwebinar2024
Published classroom materials form the basis of syllabuses, drive teacher professional development, and have a potentially huge influence on learners, teachers and education systems. All teachers also create their own materials, whether a few sentences on a blackboard, a highly-structured fully-realised online course, or anything in between. Despite this, the knowledge and skills needed to create effective language learning materials are rarely part of teacher training, and are mostly learnt by trial and error.
Knowledge and skills frameworks, generally called competency frameworks, for ELT teachers, trainers and managers have existed for a few years now. However, until I created one for my MA dissertation, there wasn’t one drawing together what we need to know and do to be able to effectively produce language learning materials.
This webinar will introduce you to my framework, highlighting the key competencies I identified from my research. It will also show how anybody involved in language teaching (any language, not just English!), teacher training, managing schools or developing language learning materials can benefit from using the framework.
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
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,
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]
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
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)
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