supervisor: dr. kartikeya murari kathryn simone, phd student...

ENEL 606Fluorescence: Theory and Applications

Kathryn Simone, PhD StudentSupervisor: Dr. Kartikeya Murari

source : www2.warwick.ac.uk

FluorescenceEmission of electromagnetic radiation by a material that has absorbed electromagnetic radiation

Detection of fluorescence is possible due to difference in wavelength of emitted photons from that of the absorbed photons

Endogenous and engineered fluorophores

Life sciences, industrial, and commercial applications

Lecture Outline❏ Principles of Fluorescence

❏ Detection of Fluorescence

❏ Applications

❏ Related Concepts

Principles of Fluorescence ● Molecule in the ground state is moved to an excited singlet state when a photon is absorbed

○ Conformational changes○ Several vibrational levels

● Excitation and emission light● Delay: fluorescence lifetime a few

nanoseconds● (mostly) Reversible process● Energy is conserved

source : www.thermofisher.com

Principles of Fluorescence

source : www.thermofisher.com

v : frequency (1/s)h: planck’s constant (6.626x10^-34 J/s)c: speed of light (3.0x10^8 m/s): wavelength (m)

Principles of Fluorescence● Polyatomic species: Broad

energy distributions due to multiple vibrational and ground states

● Excitation and emission spectra● Ability to emit or excite/absorb● Difference in peaks is Stokes’

shift: 10-20 nm● “Absorption” ~ “Excitation”● One species ~ one peak

Source: quora.com

QUESTIONIs this fluorescence?

Principles of Fluorescence

● How does the wavelength of excitation affect the emission?

Source: quora.com

Principles of Fluorescence● Diffuse sources

○ Isotropic (all directions) radiation○ EXCEPTION: crystal/lattice structures >

● Polarization-dependent○ Excitation depends on alignment of electric

field with molecular axis

● Inefficient Process!○ Concentration of fluorophore in solution

○ Absorption cross section: = incident photons/absorbed photons

○ Quantum Efficiency (QE) of fluorophore = emitted photons/absorbed photons



❏ Applications


Detection of Fluorescence

SPECIMENEXCITATION EMISSIONLIGHT SOURCE DETECTOR

Transmitted light fluorescence:

SPECIMEN

EXCITATION

EMISSION

LIGHT SOURCE

DETECTOR

Epifluorescence:


● Fluorescence is an inefficient process○ In life sciences, a few fW emission for a few μW excitation

■ 1 billion : 1○ Excitation light leaks through to detector

■ transmission and epifluorescence setups○ Large background: reduced sensitivity to fluorescence, detector may

saturate● Ideally, only emission light will reach the detector● Good design exploit both the optical and electrical nature of the signals

Detection of Fluorescence● Optical filters separate

excitation and emission light● Excitation filter: allow for use

with a broad spectrum source (easy switching)

● Dichroic mirror: reflects or transmits light depending on wavelength

● Collimation optics: Angle of Incidence

● Interference filters have

3 key selection criteria:1. Stop band rejection (%T or OD)2. Sharpness3. Passband efficiency

Dichroic reflects Dichroic transmits

Detection of Fluorescence● Sources of excitation may be narrow-spectrum

○ Lasers (specialized applications)○ LEDs

● Or broad-spectrum○ Xe, Hg (UV/Vis)○ Nd:YAG Laser

■ (neodymium-doped yttrium aluminum garnet)

Source: http://www.lumileds.com/uploads/265/DS68-pdf

Source: http://zeiss-campus.magnet.fsu.edu/

Detection of Fluorescence● Selection of a detector should involve consideration of:

○ Strength of emission (concentration, QE, etc of fluorophore)○ Temporal nature of emission○ Spectral nature of the emission○ Spatial characteristics of beam (eg. imaging vs. single point)

Detection of FluorescenceSome Practical Considerations

1. Photobleaching: Irreversible destruction of fluorophore from exposure to light. Causes dimming over time.

2. Background fluorescence: Emitted photons from endogenous fluorophores in sample/system compete with the fluorophore of interest for detector dynamic range, and add shot noise to the measurement.

3. Quenching: Reduced fluorescence due to chemical interactions with fluorophore

Detection of Fluorescence - Photobleaching● Photobleaching follows an

exponential decay

● Effect is removed in signal post-processing

Detection of Fluorescence - Coherent Detection● Improve sensitivity by exploiting the fact that excitation and emission are near

simultaneous.○ “Low-level detection”, “lock-in amplification”

Signal: Acos(⍵t)

Reference: Bcos(⍵t)

0.5[AB+cos(2⍵t)]

LOW PASS FILTER

MULTIPLIER

0.5*AB


1/f noise

a) Detector output b) After Multiplication with reference

c) Output of low-pass filter



❏ Applications


Applications - Labeling● Chemical attachment of a fluorophore to a target molecule● Visualize biological structure and function with high contrast dyes● Extensive use in the life sciences

○ Monitor behaviour of antibodies, enzymes and other proteins or peptide chains;

○ Target fluorescent probes to chromosomal sites, identify deletions/duplications of genes

● Fluorescent proteins and dyes○ Size matters for interactions at the nanoscale○ Dyes preferred as it doesn’t affect the interaction

Applications - Monitor Physiological Dynamics● Fluorescent sensors in biology and

neuroscience○ Conformational changes, due to

physiology, affect brightness○ Monitor neuronal dynamics with light:

Calcium, membrane potential, neurotransmitters

○ Genetic targeting to specific cell types● GCaMP (popular Calcium Sensor) >

○ Engineered fusion of GFP and calmodulin proteins

Source: Badura, A. et al 2014

Applications - Monitoring Physiological Dynamics

● Spontaneous behaviour in individual cells

● Synchronization

● Events can be correlated with behaviour to establish causality (not shown here)

http://www.youtube.com/watch?v=t3TaMU_qXMc

Applications - Labelling● Transgenic animals enable

targeting of fluorophores in vivo >

● Imaging with multiple labels is possible with spectral differentiation

● How was this image created? 50 μm

Source: Fuzesi, T. et al. 2016

Applications - 2 Photon Excitation● Absorption of two photons,

each carrying half the excitation energy

○ Twice the excitation wavelength

○ Emission follows single-photon case

● Unlikely Process○ Arrive at the same molecule,

at the same time○ Low probability

Source: http://www.bates.edu/durst-research-group

Applications - 2 Photon Excitation

● Increase intensity temporally and spatially

● Femtosecond lasers○ Photons arrive simultaneously

○ Higher achievable peak-power with pulsed lasers

● High NA lens for focusing

Source: http://www.bates.edu/durst-research-group

Applications - 2 Photon Excitation

● Advantages○ Used in the life sciences for imaging tissue○ “Redder is better”: scattering of light by tissue is strongest in visible wavelengths, the region

many engineered probes are excited○ Out of plane autofluorescence is eliminated

● Nonlinear process○ The probability of excitation increases with I2

QUESTIONIf the excitation intensity for 2PE is doubled, how

would you expect the emission intensity to change?

Applications - Laser-induced Fluorescence● Non-intrusive study of fluid dynamic flowfields● Fluorescent particles are added to the flow and excited with laser light● The flow is then imaged in real time with sensitive detectors (CCD cameras)● Thermodynamic variations may cause fluorescence quenching

Applications - Laser-Induced Fluorescence

http://www.youtube.com/watch?v=JWvJLs47EKg

Applications - Commercial Applications● Fluorescent Lighting

○ Mercury vapor excited with electric current produces UV light○ Interior phosphor coating glows in response

● White LEDs○ Progress in high efficiency blue LEDs enabled white LEDs○ Phosphor coating absorbs high-energy blue light and emits yellow light○ “Mix” is perceived as white by the human eye

Applications - Commercial Applications

Cool White

Warm White



❏ Applications


Related Concepts● Chemiluminescence - energy from chemical reaction● Phosphorescence - transition to a “forbidden” triplet state

supervisor: dr. kartikeya murari kathryn simone, phd student...

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