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Quick Reference for Confocal Time-Resolved Microscopy (FLIM, FRET, FCS)
Point Spread Function (PSF) - Confocal Fluorescence Microscopy
Point Spread Function (PSF)
out of focus plane
Simplified Perrin - Jablonski Energy Diagram
PICOQUANT
Time Scales and Techniques
3D Gaussian approximation
(Ideal imaging system)
1 ns
1 µs
1 ms
Time
scale
Confocal Principle
1s
focal plane
S1
26 %
from laser
from laser
0.74 0
0.606
0.5 0
s
FWHM
kisc
S1
T1
translational diffusion
kFRET
0
kexc
kfluorescence
kNonRadiative
kphosphorescence
kT,NonRadiative
big conformation changes, docking,..
coupled
transitions
2
(1/e )
Radial co-ordinate
-1 -0.5 0 0.5 1
Distance (airy units)
rotational diffusion, triplett
vibrational relaxation
0
R*
0
Radial co-ordinate
kbleaching
pinhole
lexc
lem
s
to detector
Reference:
Fluorescence Microscopy: From
Principles to Biological Applications,
Edited by Ulrich Kubitschek
to detector
0
S0
excitation wavelength
emission wavelength
standard deviation
numerical aperture
pinhole size
amplitude of the Gaussian
shape parameter
2
1/e radius along the axial direction
excitation
10
-15
vibrational
relaxation
10
fluorescence
-12
10
phosphorescence
-9
10
-6
10
Fitting Fluorescence Decays
Time-Correlated Single Photon Counting (TCSPC)
Measurement principle
vibrational relaxation
S0
Fluorescence decay histogram
Excited state
fluorescence signal
IRF
model
time [s]
-3
[ns]
[µs]
[s]
fluorescence decay
correlation (FCS)
blinking analysis
Fluorescence Correlation Spectroscopy (FCS) and Diffusion
Exponential decay fit:
Single molecule transit
through confocal volume
Autocorrelation curve
Intensity fluctuations
2,5
104
2,0
1,5
“Start”
Laser
PMT
Optics
Electr.
G( )
Detector
Counts
hn
hn
Light source
Instrument Response Function (IRF):
1000
Fluorophore
slower
diffusion
higher
concentration
1,0
< >
IRF
100
0,5
Ä
“Stop”
Ä
Ä
average number
of particles in
detection volume
diffusion time
0,0
10
Ground state
-0,5
10
100
[ms]
1000
10000
1
Time
5
10
15
20
25
30
Examples of fitting models:
N-exponential decay
2
Decay distribution models
0
5
10
15
20
25
283.15
13
30
Time [ns]
Average lifetime:
[10-4 Pa×s]
-4
lifetime distribution i.e.
the total counts corresponding
to lifetime component i
288.15
Photon
filtering
Laser pulse
Photon
Synchronization
marker (M)
303.15
13
11
11
9
9
7
7
20
30
Temperature, [°C]
1.0
Marker time 1
Photon time 2
Time tag
T
E
Norm. values
exc. donor
em. donor
exc. acceptor
em. acceptor
spectral overlap
Norm. values
t
Photon
correlation
Correlation
Photon time 1
Start
}
}
Start-stop-time 2
exc. ATTO 425
exc. Rh6G
em. ATTO 425
em. Rh6G
E=1
E=0.5
0
1
RDAIR0
0.4
0.2
E=0
Wavelength [nm]
0.6
0.0
T
t
5 ns
2.5 µs
CH Detector 1
T
T
3.1 µs
M
Marker signal (e.g. from scanner)
1
6
Scanner
integration
7 ns
3.8 µs
Förster radius ~ 4-7 nm
FRET efficiency
spectral overlap integral
refractive index of the medium between the
donor and acceptor
quantum yield of the donor
orientation factor
Avogadro number
donor emission
normalised acceptor absorption
wavelength
CH Detector 2
Spectral overlap integral
TTTR data file
400
500
Wavelength [nm]
40
20
400
600
Wavelength [nm]
800
Type
Timing resolution (detector FWHM)
Max. detection efficiency
Spectral range
Diameter of active area
1000
PMA Series
PMT
< 180 ps
40 % at 400 nm*
185 - 920 nm
8 mm
* model dependent, see detection efficiency curves above
PMA 192
PMA 182
PMA 175
40
exc. ATTO 488
exc. ATTO 655
em. ATTO 488
em. ATTO 655
0.6
0.4
Wavelength [nm]
FA I FA
E=
FD I FD + FA I FA
30
20
Counts (log scale)
no FRET
medium FRET
high FRET
tDA
tD (no FRET)
500
600
700
Wavelength [nm]
800
1.0
exc. TSBeads - blue
exc. TSBeads - orange
em. TSBeads - blue
em. TSBeads - orange
0.8
Time [ns]
Wavelength [nm]
FA FD
FA FD
0.0
Donor lifetime
Norm. values
SPCM-AQRH SPAD
PDM SPAD
PMA Hybrid - 40 (mod)
PMA Hybrid - 06
PMA Hybrid - 50
Detection efficiency [%]
Detection efficiency [%]
ã PicoQuant GmbH, April 2015
Intensity
50
60
0
200
Intensity
Single Photon Sensitive Detectors
Acceptor emission
quantum yields
fluorescence counts corrected for background and
detection efficiencies
lifetime of the donor in the presence of the acceptor
lifetime of the donor in the absence of the acceptor
[nm]
436
501
663
530
484
523
684
555
45000
90000
125000
116000
Fluorophore
0.2
Donor emission
80
600
0.8
Lifetime
Recorded TTTR data stream
ATTO 425
ATTO 485
ATTO 655
Rh6G
[nm]
Molar absorption
coefficient [cm M]
Max. excitation
Max. emission
-1
200
PMA Hybrid Series
Hybrid PMT
< 50 to < 140 ps
45 % at 500 nm*
220 - 890 nm
3, 5 or 6 mm
400
600
Wavelength [nm]
800
E=1-
0.6
0.4
0.2
tDA
tD (no FRET)
0.0
SPCM-AQRH
SPAD
< 200 to < 600 ps
70 % at 700 nm
400 - 1100 nm
180 µm
PDM Series
SPAD
< 50 to < 250 ps
49 % at 550 nm
400 - 1100 nm
20, 50 or 100 µm
Fraction of
FRET-active donors
log
Fit of n-exponential decay
t1
FRET inactive
in this case n = 2
Time [ns]
Time [ns]
3.6
4.1
1.8
4.1
[nm]
in water at 25°C (298.15 K)
-6
2 -1
[10 cm s ]
in water
[ns]
400
500
Wavelength [nm]
600
ATTO 425 - carboxylic acid
ATTO 655 - maleimid
ATTO 655 - carboxylic acid
ATTO 655 - NHS esther
Cy5
Alexa 647
Alexa 633
Rhodamine 6G
Rhodamine B
Rhodamine 123
Rhodamine 110
Fluorescein
Oregon Green 488
Atto488-carboxylic acid
TetraSpeck Beads,
0.1 µm diameter
484
686
685
685
670
665
647
550
560
530
535
520
550
523
430, 515, 580, 680
4.07
4.26
4.25
3.6
3.3
3.4
4.14
4.5
4.6
4.7
4.25
4.11
4.10
4.0
0.044
3.6
1.8
1.8
1.8
0.9
1.0
3.2
3.9
1.7
4.0 (in PBS)
4.0
4.3
4.1
4.1
--
For more details please see our Technical Note on “Absolute Diffusion Coefficients: Compilation of Reference Data for FCS Calibration” available for download
on our website.
Want to Learn More? Visit our Workshop and Courses!
FRET
t2
0.9
0.8
0.3
0.95
[ns]
Lifetime
Determination of donor species
log
Lifetime
Diffusion coefficient
10
0
Quantum
yield
Max. emission
1.0
Norm. values
Förster radius
Fluorophore
2
Time
t
Typical values for the effective
FCS volume are 0.5-2 fl
depending on the objective and
the wavelength and the pinhole.
Properties of Standard Samples
Time
Start-stop-time 1
Boltzmann constant
temperature
hydration radius of
diffusing particle
viscosity of solvent
mass of diffusing
particle
40
0.8
TCSPC
time
-5
A = 2.414·10 Pa·s
B = 247.8 K
C = 140 K
T temperature in Kelvin
temperature in °C
FRET efficiency
RDA
Counts
Photon
298.15
Förster Resonance Energy Transfer (FRET)
Time Tagged Time-Resolved (TTTR) Data Acquisition
Laser pulse
293.15
(Pa·s) = A · 10B / (T-C)
10
Laser pulse
Stokes-Einstein (Spherical particles)
Absolute temperature, T [K]
-2
Viscosity,
! Repeat this time measurement many
times and count “how many photons have
arrived after what time”
! Sort the photons into a histogram
according to their arrival times
! Exponential fit of fluorescence decay to
obtain fluorescence lifetime
For TCSPC we need:
! A defined “start” of the experiment: pulsed excitation
! A defined “stop” of the experiment: photon counting detector
! A fast “stopwatch” to measure time difference between “start”
and “stop”
Residuals
4
Fraction of free
(FRET-inactive) donors
!
!
!
!
International Workshop on “Single Molecule Spectroscopy and Super-resolution Microscopy in the Life Sciences, Berlin, Germany
Short course on “Principles and Applications of Time-resolved Fluorescence Spectroscopy”, Berlin, Germany
Short Course on “Time-resolved Microscopy and Correlation Spectroscopy”, Berlin, Germany
Science in your labs - workshops or courses organized by PicoQuant along with a local research institute
www.picoquant.com
objective
lifetime
Acceptor
Confocal volume
Dye label/sample
properties
Rayleigh criterion
Diffraction pattern
Typical values for the FWHM of the PSF depending on the
type of objective and the wavelength: ~ 250 to 450 nm
Data recording and
analysis scheme
Point-like
object