Fluorescence

1
Fluorescence

• Yin Yeh
• Department of Applied Science
• Center for Biophotonics

2
Fluorescence

• Two photons (at the very minimum) are involved in
  this process.
• Absorption
• Emission
• Electric Dipole Process
• Polarization sensitive
• Lifetime in the nsec regime

3
Principles of fluorescence

• Quantum mechanical process
• Quantum efficiency of process
• Lifetime of the excited state (Fluorescence
  lifetime)
• Lifetime dynamics as a measurement of molecular
  activity (reaction, rotation)
• Lifetime sampling and imaging

4
Fluorescence Spectroscopy Basics
s1
kisc
T1
Fluorescence
Excitation
Phosphorescence
s0
5
Fluorescence

• Stokes Shift
• Due to broad energy levels, the absorption and
• emission are in spectral bands lem gt lab

Stokes Shift is 25 nm
Fluorescein molecule
520 nm
495 nm
Fluorescnece Intensity
Wavelength
6
Quantum Efficiency
Lifetime of Fluorescence
7
Controlling Qu. Efficiency

• Radiationless transitions compete
• Collisional transfer
• Dipole-dipole transfer - FRET, self-quenching
• Intersystem crossover - Phosphorescence
• Environment as a mode of dissipation
• Taking advantage of these effects
• Eliminating factors leading to effects
• Singlet O2 control

8
Utilizing Photobleaching

• Fluorescence recovery after photobleaching
• Translational dynamics
• Rotational dynamics
• Description of the processes
• The fast bleaching pulse
• The subsequent monitoring of recovery

9
Fluorescence Depolarization

• Dynamic monitoring of fast movements
• Rotational motion
• Reactions
• Triplet state dynamics
• Transient absorption dynamics
• Phosphorescence dynamics

10
Pulse-Probe Spectroscopy

• Excitation pulse is considered a d-function
• Probing fluorescence decay via
• Sampling rotational motion of fluorophore

11
Triplet state spectroscopy

• Phosphorescence measurement
• Slow dynamics (rotational movement of larger
  molecules
• Slow reactions
• Triplet state absorption spectroscopy
• Transient Dichroic Absorption

12
Phase modulation fluorescence

• CW excitation of the fluorophore
• Comparing phase shifts upon interaction
• Excitation amplitude Aex
• Fluorescence amplitude AF
• Tan f -wt

13
Two types of fluorophores

• Intrinsic
• Trp, Tyr, Phe are intrinsic emitters h 0.2
• Some defects in nucleotides - Benzl-(a)-pyrene
• Extrinsic
• Dye molecules (covalently, intercalated)
• Attachment via antibodies
• Introduction via transfection (GFP, phytochromes)

14
Fluorescence Microscopy

• Microscope configurations
• Epi-fluorescence
• Confocal scanning microscopy
• Total internal reflection (TIR) fluorescence
• Lifetime imaging
• Spectroscopy
• Structural
• Dynamical

15
Fluorescent Microscope
Arc Lamp
EPI-Illumination
Excitation Diaphragm
Excitation Filter
Ocular
Dichroic Filter
Objective
Emission Filter
16
Experimental Strategy
Flow
Y-shaped Flow-cell
Bead-DNA-RecBCD (Sample side inlet port)
Mg-ATP (Reaction side inlet port)
17
c
16
-ATP
Helicase-DNA complex
enters
ATP channel
ATP
Processive
unwinding
occurs
(rate
443 bp/sec)
corr
Unwinding
ceases due to
helicase
dissociation
18
Fluorescence in Cell Sorting

• Flow cytometry diagnostics
• Scattering
• Fluorescence
• Sorting according to the level of hybridization
  (Cell cycle monitoring)
• Sorting according to specific labels
• Intrinsically produced
• Labeling methods

19
Fluorescence Detectors
Laser
20
Fluorescence Activated Cell Sorting
488 nm laser
FALS Sensor
Fluorescence detector
-
Charged Plates
Single cells sorted into test tubes
21
Normal Cell Cycle
M
G0
G2
DNA Analysis
G1
s
Count
s
0
200
400
600
800
1000
DNA content
22
A typical DNA Histogram
G0-G1
G2-M
S
of Events
Fluorescence Intensity
23
RPE FACS tBH and C6
A
B
C
Necrotic Quadrant
Late Apoptotic
Live
Early Apoptotic
Early Apoptotic 2.04 Late Apoptotic
11.64 Total Apoptotic 13.68
Early Apoptotic 1.29 Late Apoptotic
12.25 Total Apoptotic 13.54
Early Apoptotic 1.66 Late Apoptotic
2.43 Total Apoptotic 4.09
RPE cells die by apoptosis in response to
inducers. A is untreated cells B is treated
with 300uM tBH for 3 hours C is treated with
50uM C6-Ceramide for 24 hours. Flow cytometry
and quadrant analysis performed as described in
methods section.
24
Confocal Microscopy

• Enhanced resolution by 2X
• Background rejection substantially improved
• Optical sectioning capability created
• Scanning sample or the light source needed
• Laser use is the way most instruments are made

25
Confocal Principle
Laser
Excitation Pinhole
Excitation Filter
PMT
Objective
Emission Filter
Emission Pinhole
26
Images of RPE cells at different levels of cell
death
C
D
E
Viability analysis by flow cytometric analysis of
hRPE cells treated with unlabelled CTB.
Treatment with unlabelled Cholera toxin subunit B
does not induce apoptosis or death. C-E are
confocal images of the stained cells sorted by
flow cytometry representing each of the three
dying subpopulations used in quadrant analysis.
C is early apoptotic quadrant D is late
apoptotic quadrant and E is necrotic quadrant
cell type.
27
TPE or MPE Fluorescence

• Nonlinear optical phenomena used for excitation
  of the state
• Due to weaker nonlinear interaction, can achieve
  optical sectioning at the focal spot
• Achieve depth excitation in thicker samples
• Use of mode-locked laser for high intensity

28
MPE Fluorescence apparatus
29
TPEF of cellsblue - Nucleusred - Actingreen -
Sphingo-myelin
30
Fluorescence resonance energy transfer

• We plan to further develop this technique to
  perform antibunching on the fluorescence
  resonance energy transfer (FRET) process.

The rate of energy transfer, kT is as predicted
by Förster theory
where R0, the Förster distance is given by
31
Fluorescence

• Resonance Energy Transfer

Fluorescence
Fluorescence
ACCEPTOR
DONOR
Intensity
Absorbency
Absorbency
Wavelength
32
Intensity FRET
NBD (Donor)
TR (Acceptor)

• Limitations
• Bleed-through or crosstalk.
• Photobleach.
• Correction Algorithms
• Fc Ff - Df (Fd / Dd) - Af (Fa / Aa).
• (7 images, 3 channels, 3 samples).

Df
Af
Ff
33
Lipid-lipid Interaction
A half bilayer (3 NBD DMPC) was erased by UV
light, then backfilled with TR POPC (2 mg/ml)
bilayer. Capture three channel (Af, Df, Ff)
images every 5 minutes until 105 minutes.
Corrected FRET Image
Intensity Distribution
34
FRET advantages

• Localized distance measurement of FRET pairs -
  Molecular Ruler
• Can examine biomolecular reactions via FRET
  blinking
• Leads to time-based sampling

35
Fluorescence Correlation Spectroscopy (FCS)

• Sampling local fluctuations of species
• Coupled with confocal or FRET methods to increase
  the extent of fluctuation
• Time correlation can be obtained using hardware
  correlator systems or computer software sampling

36
Finite domain sampling

• Confocal
• Multi-photon excitation
• Maiti, Haupts, Webb,
• PNAS 9411753,1997

37
Fluorescence Correlation Spectroscopy
38
Imaging FCS

• Sampling of two-dimensional domains most readily
  accessible
• Software method developed
• With high sensitivity CCD camera, useful for very
  minute quantity of samples
• Readily adaptable to tissue samples

39
I-FCS Autocorrelation function

• Cluster Density (CD)

2. Degree of Aggregation (DA)
40
Fluorescent Beads Diffusing
Diffusion Characteristic Time td 0.8 s.
41
Anti-bunching Spectroscopy

• FCS applied to non-classical photon system
• Equivalent to a pure number state
• Expect the FCS response at zero-time to give
  g2(0) 0
• Allows for a measure of single fluorophore
  lifetime

42
Microscope Set-up
Scanning Confocal Microscope
avalanche photo-diodes
bandpass filters
50/50 beamsplitter
Long pass filter
confocal aperture

• 100X, 1.4 NA microscope objective is used.
• Spot size is 350 nm
• Appropriate interference filters are placed in
  signal path to separate out the excitation light
  as well as other sources of background.

dichroic filter
Collimated source
high NA objective
Sample scanning stage
43
Results

• Results for antibunching measurements carried out
  on the three different DNA samples and for an
  ensemble of Atto 655 dye molecules (bottom
  right).
• Frequency histogram of the distribution of m
  values will be constructed for each of the three
  samples after carrying out the measurement on
  several individual molecules separately.

m1.1
m1.9
m3.2
mgt100
44
Experiments are now conducted in flowcells
35mer template
12mer primer
Cy5
strepavidin
biotin
glass coverslip

• Carboxl groups on PAA couple to amino group on
  biotin derivative.
• Negatively charged PAA limits nonspecific binding
  of nucleotides.

Bravlovsky, et. al., PNAS
45
We have designed a photon antibunching experiment
to test the outcome of DNA synthesis on short
segments of DNA

• DNA template and primer strands annealed
  before attached to surface

• Polymerase and nucleotides are introduced. If
  all nucleotides are incorporated, correlation
  measurement gives three dyes present.

Cy5
3-GCT ATC GCC TAC ATC TTC GTC TTT CTC TTC
TTA CG-5
Biotin-5-CGA TAG CGG ATG UAG AAG CAG AAA GAG AAG
AAU GC-3
46
Total Internal Reflection

• Evanescent wave excitation within a limited layer
  of sample
• Useful for depth rejection
• Increases S/N
• Applied to surface or near-surface reactions
• Has polarization sensitivity
• Prism or objective configurations available

47
Total Internal Reflection Polarization Features
48
TIRF Microscopy
49
Using TIR microscopy to examine the binding of
RNA Polymerase to DNA
50
TIRF (Total Internal Reflectance Fluorescence )
Epi-fluorescence
TIRF
TIRF using high N.A. objective (Nikon USA )
51
A Scanning Protein
52
GFP Molecule

• Strip model Ball Stick model

53
The GFP Structure

• GFP is a beta-can.
• On the outside, 11 antiparallel beta strands
  (green) form a very compact cylinder.
• Inside this beta-structure there is an
  alpha-helix (dark blue), in the middle of which
  is the chromophore (red).
• There are also short helical segments (light
  blue) on the end of the can.
• The cylinder has a diameter of about 30 Å and a
  length of about 40 Å

54
Detail of Mechanism

• GFP has a single tryptophan, Trp 57 (blue), which
  is located 13 to 15 Å away from the chromophore
  (red). Trp 57 is seperated from the chromophore
  by Phe 64 and Phe 46 (yellow).
• Long axis of its ring system is nearly parallel
  to the long axis of the chromophore. Therefore,
  an efficient energy transfer from Trp to the
  chromophore should be possible, hence no separate
  tryptophan emission can be observed.

55
Absorption and Emission Bands

• The excitation spectrum of native GFP from A.
  victoria (blue) has two excitation maxima at 395
  nm and at 470 nm.
• The fluorescence emission spectrum (green) has a
  peak at 509 nm and a shoulder at 540 nm.

56
The Four-State Model of GFPLight Proton Driven
57
Foerster Mechanism in GFP

• Upper left Tyr66 is in its hydroxyl form
  Absorbs at 395 nm
• Lower left Tyr66 is in its phenolate form
  Absorbs at 470 nm.
• Phenols are known to be more acidic in their
  excited state than in their ground state.
• Protonated excited form of the fluorophore (upper
  right) converts to
• Excited phenolate (lower right), the only
  fluorescent species and emits light at 509 nm.
• A cycle is formed the fluorophore absorbs a
  photon, then loses a proton, emits a photon and
  finally takes up a proton, returning to its
  original state.