HORIBA JobinYvon Inc., Leading
the 21st Century in Time-Resolved
Fluorescence Instrumentation
Dr. Adam M. Gilmore
Applications Scientist
Fluorescence Division
HORIBA Jobin Yvon Inc.
Edison, NJ USA
1923 Logo
Jobin Yvon (JY)
• JY is a World leader in
Optical Spectroscopy
founded in 1819 in Paris
• Supplier of Scientific
Instrumentation and Custom
Diffraction Gratings used in
the detection, measurement,
and analysis of light around
the Globe
1848 - Introduction of the Saccharimeter
1882 - Introduction of the Polarimeter
JY-Horiba Divisions
Gratings / OEM
Emission Spectrometry
Optical Spectroscopy
Fluorescence
Raman Spectroscopy
Thin Films
Forensic
Fluorescence Group
“The World’s Most Sensitive Instruments”
• Specializing in research grade fluorescence
detection
• Steady state and time-resolved (ps to hrs)
• All reflective optics
• No chromatic aberration
• High throughput (S/N>5000)
• Modular and self-contained instruments
• Thousands Operating Worldwide
Fluorescence: a type of light emission
• First observed from quinine by Sir J. F. W. Herschel
in 1845
Yellow glass of wine
Em filter > 400 nm
1853 G.G. Stoke
coined term
“fluorescence”
Blue glass
Filter
Church Window!
<400nm
Quinine
Solution
Light Absorption and Fluorescence
Absorbance energy
Absorption=10-15 s
S2 excited state
S1 excited state
Fluorescence =10-9 s
Ground State
Electrons
Nonradiative
dissipation
What is a Fluorescence Lifetime?
Random Decay Back to Ground State:
Each Molecule Emits 1 Photon
1
I(t)
0.8
0.6
t=1/e=37%
0.4
0.2
0
0
Population of Molecules Excited
With Instantaneous Flash
500
time, ps
1000
Why measure lifetimes?
 Absolute quantities- not merely ratios or timeaveraged intensities
 A snapshot of the excited state behavior
 Largely independent of sample concentration and
absorbance cross-section-in contrast to- steady
state
 Dynamic information-rotation-correlation times,
collisional quenching rates and energy transfer
processes
 Additional dimensions for fluorescence data –
increased specificity, sensitivity and selectivity
 Lifetime senses local molecular environment (e.g.
polarity, pH, temperature, electrostatics etc)
Fluorolog-Tau3 Picosecond Lifetime System
Classical Ruled Diffraction Grating
Diffracted
light cone
Slit
Diffracted
light
Normal
to groove face
Blaze
angle
Reflex
angle
Grating
normal
Incident
light
Groove
spacing
Grating Information
• Groove Density  Higher Resolution
• Blaze Wavelength (Angle) Peak
Efficiency
– Rule Of Thumb: 2/3 l to 2 l
• Slit (Bandpass) Determines Resolution
– 1200g/mm
– Reciprocal Linear Dispersion 4 nm/mm
Stray Light Reduction:
Front Face Accessory
Avoid Specular Reflectance at 45°:
Collect at 22.5°
Ref
diode
Grating
1
Swing away
Mirror: 22.5°
Grating
2
Front Face
Solid Sample
Excitation
Stray Light Reduction II:
Front Face Accessory
Ref
diode
Avoid Specular Reflectance off
solid samples: Collect at 22.5°
Grating
1
Grating
2
Front Face
Solid Sample
Excitation
1.5
1.5
M
(
)
(
)
Frequency Domain
Transform Principle
0.5
0.5
00
tan f  t f
-0.5
-0.5
100
1010
100
10
100
10
100
10
100
Frequency,
MHz
Frequency,
MHz
Frequency,
MHz
Frequency,
MHz
Frequency, MHz
1 11
0.9
0.9
0.9
0.8
0.8
0.8
0.7
0.7
0.7
0.6
0.6
0.6
0.5
0.5
0.5
0.4
0.4
0.4
0.3
0.3
0.3
0.3
0.2
0.2
0.2
0.2
0.2
0.1
0.1
0.1
0.1
0.1
00
00
0
1000
1000
1000
1000
1000
M
M
M
M
M
f
f
f
TIME
TIME
1   2t M2
f
1
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
1000
f
M
909090
808080
707070
606060
505050
404040
303030
2020
20
2020
10
10
10
1010
00
00 0
11
11 1
M 
10
100
Frequency, MHz
-1.5
-1.5
1
90
80
70
60
50
40
30
20
10
0
-1
-1
1
AMPLITUDE
AMPLITUDE
11
=
M
f
Spectracq
MHz
FFT:
Fast
Fourier
Transform
amp
SLAVE Rf + Df
Rf+Df
X
Rf
Df=cross correlation frequency
R928P
PMT
MASTER Rf
450W
cw xenon
amp
filter
Pockels Cell
sample
reference
sample turret
Fluorolog-Tau3:
Multifrequency Fluorometer
450 W Xe
300 nm blaze
1200 g/mm
exit slit
iris
pockels
polarizer
NIR:
9170-75=950-1700 nm
1000 nm blaze
600 g/mm grating
slit
r
V
V
V
UV-VIS:
R928 = 250-850nm
500 nm blaze
1200 g/mm grating
Mirror
Mirror
Tau-3-Fluoromap
Olympus BX51
Lens
Pinhole,
d=0.1-3 mm
Microscope
lens (f = 180mm)
<15 mm
Dichroic
mirror
Mirror
Lens
Objective
s
1 mm mapping
Ex-Mono
XYZ-Scanning Stage
Mirror
Mirror
Em-Mono
PMT
Triax 320
CCD
Pockels
Cell
Hallmarks of Frequency Domain

Rapid, robust data collection, no worry about pulse
pileup as single-photon techniques

True differential technique, no deconvolution of IRF

Economical 10 ps resolution with common cw sources
Xenon lamps and lasers (strong, affordable UV
source!)

Intuitive numerical data interpretation

Compatible with global analysis (separate complex decays)
Data Analysis in Frequency Domain
• If the time domain response expression is given by
I(t), it will have sine and cosine transform
expressions:

N 
 I (t ) sin (t ) dt
0

 I (t ) dt
0

D 
 I (t ) cos (t ) dt
0

 I (t ) dt
0
in which N and D are the numerator and denominator terms
Data Analysis in Frequency Domain
• For the sum of exponentials model, the sine transform is:
 it i2
N  
2 2
(
1


t )
i
 t
i i
i
and the cosine transform is:
 it i2
D  
2 2
(
1


t )
i
 t
i i
i
Data Analysis in Frequency Domain
• From the sine and cosine transforms, one calculates,
at each frequency, the expected phase and
magnitude terms:
 N 

fc,  tan 
 D 
1
mc,  N  D
2
2
Data Analysis in Frequency Domain
• Compare the calculated values to actual
data
• Calculate the reduced c2 value and
residuals
• Interpret physical significance of the
results
Data Analysis in Frequency Domain
• Calculation of c2 and the residuals of the fit:
c  
2

1  (fc ,  f )  (mc ,  m ) 


 
df
dm

2
where df and dm are the errors of the measurement, and
 is the number of degrees of freedom.
2
Multiexponential decay in Frequency Domain
Mixtures and multicomponent decays on the SPEX Tau3
This data is clearly not single exponential, we need to increment the model
Mixtures and multicomponent decays
Mixtures and multicomponent decays on SPEX Tau3
Adding a second component greatly improves the fit – and is justified
statistically
Fluorescence Polarization
SPEX Fluorescence
Anisotropy Measurements in Steady State
• Anisotropy - measure polarized emission
• Uses polarizers in excitation and emission
paths
• Measure vertical (V) and horizontal (H)
intensities
• Calculate <r> from these intensity
measurements
Anisotropy and Polarization
Polarized emission
with polarized
excited light
P=
I║ - I ┴
y
I║ + I ┴
x
I║ - I ┴
r = I + 2I
║
┴
P=
z
3r
2+r
; r=
Photoselection
2P
3- P
Anisotropy
r=
-
x
IVV
IHH
+2
x
IVH
IHV
H
V
Grating Factor
G=
G
H
zx
x
V
yy
Steady State Anisotropy: the Perrin equation
r
1

r0


t
1  
 f
r0 is the fundamental anisotropy (at zero time),
t is the (average) fluorescence lifetime, and
f is the (average) rotational correlation time
0.5
Dimers:
Larger
Slow rotation
Hindered by Viscosity
High anisotropy
Polarized
0.4
Anisotropy, r
Monomers:
Small
Rapid rotation
Unhindered
Low anisotropy
Depolarized
0.3
0.2
0.1
0
0
20
40
60
[Protein], mM
80
100
120
Fluorescence Emission Dipole
Long wavelength absorption (430nm)
TREA of perylene in oil
Short wavelength absorption (256nm)
Excitation Anisotropy Spectrum of Perylene in Oil
TREA of perylene in oil
• Fun questions:
– does perylene rotate like a sphere?
– or like a disk?
– how can we tell?
– We know perylene is a D2h rotor, can we see different modes?
– what can the Fluorolog-Tau3 show us is
happening?
– (Assume isotropic solvent - oil in this case)
TREA of perylene - using anisotropic rotor model
Correct Model!
Anisotropy Decay - TREA Modeling
Current Hot Topics and Applications
for JY-IBH Instruments
• Nanoparticles: quantum dots, nanotubes for physical and
molecular research
• Semiconductor PL: QC and applied LED and LD researchdevelopment
• FRET-resonance energy transfer (FRET): distance and orientation
of donors-acceptors
• Green, red and yellow fluorescent proteins (FPs): in vivo
molecular markers
• Fluorescence Lifetime Microscopy: ultrastructural and
biochemical characterization at 1 micron x-y-z resolution
• Photosynthesis: natural, engineered and artificial systems
• Drug- and Protein-design: ligand binding, anisotropy, molecular
beacons
• Etc…
Our Line of Jobin Yvon-Spex
Spectrofluorometers
FMAX3
World’s Most Sensitive
Self-Contained
Fluorometer
5000U
TCSPC
Flagship
Tau3
World’s Most Reliable
Frequency
Fluorometer
SkinSkan
World’s Most Sensitive
Skin-Surface
FLLOG3
Fluorometer
World’s Most Sensitive
Modular
Fluoromap
Fluorometer
1 micron Spatial
Resolution Lifetime
Microscope
Thanks for Your Attention!
Visit Our Websites:
www.ibh.co.uk
www.jobinyvon.com