1. Fluorescence Lifetime Imaging Microscopy FLIM
Inga Sliskovic
November 18, 2003

2. Fluorescence Lifetime
General Background
Fluorescence
Fluorescence Lifetime
Development of Fluorescence Lifetime Imaging Microscopy
Applications
Monitoring structural changes in proteins
FLIM-FRET tandem for conformational changes
Monitoring intracellular pH by FLIM
Using FLIM in vivo for unstained tissues
Advantages
Limitations
Conclusions and Future Work
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3. Pioneering work
Stoke (1852): observed a wavelength shift during fluorescence phenomena (abs< fl)
A pure substance in a solution has invariant fluorescence spectrum, independent of ex
The fluorescence spectrum appears as a mirror image of the absorption band of least frequency
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4. Figure 1: Jablonski energy diagram
Different pathways for an excited molecule to leave the excited state:
The intrinsic rate of fluorescence, internal conversion, static quenching, dynamic quenching, FRET, charge transfer/molecular isomerization/bimolecular reactions, intersystem crossing…
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5. Methods for Measuring Fluorescence
Pulse fluorimetry: measurements performed in the time domain
Phase and Modulation fluorimetry: measurements performed in the frequency domain
Confocal scanning light microscope: measurements made on point-by-point bases (single-pixel) and scanned across the 2D image plane.
Conventional fluorescence microscope: measurements based on the whole 2D image field
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6. Figure 2: Examples of Microscopes
Then…
Now…
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Figure 2: Examples of Microscopes

7. Figure 3: Problems with Fluorescence microscopy
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8. Development of FLIM Fluorescence microscopy measurements:
Fluorescence intensity
Intensity ratios
First images obtained in (Lakowicz JR. et al. Cell Calcium 1994; 15: 7-27.)
FLIM: measuring fluorescence lifetimes at each point in the image
FLIM: sensitive to the changes in the microenvironment, ion concentration, pH, etc., BUT not to the concentration of the fluorophore
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9. Figure 4: Fluorescence Lifetime
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Paul J. Tadrous. J Pathol 2000; 191:

10. FLIM Parameters  = fluorescence lifetime
0= fluorescence quantum yield
Kr = radiative deactivation parameter
 = 0/ Kr
Radioactive decay time:
r = 1/ Kr
 = 0 X r
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Szmacinski H., Lakowicz J. R., Johnson M. L. Methods in Enzymology, 240,

11. Figure 5: Time and frequency domain
= (m+ p )/ 2
p= phase of fluorescence
m=modulation depth of fluorescence
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Philippe I. H. Bastiaens and Anthony Squire. trends in CELL BIOLOGY (Vol. 9) February 1999

12. General Features of FLIM
Idea: measuring fluorescence emission decay rates of intrinsic or applied fluorescent molecules, influenced by biochemical environment.
Contrast: differentiate between different tissue types, metabolic status, structural conformation, presence of ions, etc…
Other: chemically specific in vivo histology and possible tomographic modality
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Paul J. Tadrous. J Pathol 2000; 191:

13. FLIM for Structural Information
Combining fluorophores with biomolecules to study their microenvironment.
Monitoring conformational changes of proteins
Real time diagnostic imaging (detection of cancerous cells)
FRET and FLIM for studying structural changes
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14. Flourescence Resonance Energy Transfer
Based on the energy transfer between two fluorophores
Distance dependant physical process
Upon bond cleavage, energy transfer is interrupted
FRET combined with FLIM provides a direct evidence for the physical interactions between two or more proteins.
Also enables high spatial and temporal resolution.
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15. Monitoring conformational changes in proteins
Calleja et al. (Biochem. J. (2003) 372, 33–40) used FRET/FLIM approach to study a conformational changes of proteins in cells
Target protein: protein kinase B (PKB/Akt)
Two different fluorophores:
Green fluorescent protein (GFP)
Yellow fluorescent protein (YFP)
Also, dark yellow fluorescent protein (YFPdark) as a control for enhanced acceptor fluorescence (EAF)
Goal: to study conformational changes of a full-length PKB in response to growth factor
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16. Figure 6:
PKB/AKT: FUNCTIONAL INSIGHTS FROM GENETIC MODELS
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17. Experimental
Zeiss laser scanning confocal microscope; Argon 458/488 laser
FRET by FLIM in the frequency domain; ex=488 nm; Ar/Kr laser
Time domain FLIM: verification of FRET in GFP-Akt-YFP
GFP fused at the N-terminal and YFP at the C-terminal
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18. p= phase of fluorescence m=modulation depth of fluorescence
Figure 7: Assessment of FRET in GFP-Akt-YFP by time-domain FLIM and YFPdark
GFP-Akt-YFPdark: Tyr67 to Leu
= (m+ p )/ 2
p= phase of fluorescence m=modulation depth of fluorescence
Blue= GFP-Akt-YFPdark
Green= GFP-Akt
Red= GFP-Akt-YFP
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19. Figure 8: Time domain FLIM
Logarithmic plot of the flourescence decay of the GFP-Akt-YFP
Bi-exponential decay
Residual component
GFP-Akt-YFP construct undergoes FRET and the changes in FLIM are due to the energy transfer
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20. Figure 9: Change in GFP-Akt-YFP conformation upon cell stimulation in NIH3T3
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21. Figure 10: GFP-Akt-YFP conformational change is phosphoinositide 3-kinase dependent
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22. FLIM & structural changes
FLIM can be used as a tool to study molecular state and structural information
Conformational changes in proteins based on the variations in energy resonance transfer can be measured by FLIM
FLIM can be applied to physiological situations
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23. Monitoring intracellular pH
Lakowicz et al. (Cytometry Part A. 52A: 77-89, 2003) used FLIM as a tool to study intracellular pH changes.
Frequency-domain measurements
Neutral and acidic pH lifetime-probes to study pH environment in cytosole and lysosomes of living cells
C-SNAFL2 was used to study cytosolic pH
DND-160, DM-NERF and OG-514 were used for lysosomes study
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24. Figure 11: Schematic diagram of the FLIM instrumentations
CCD: charged coupled device;
MCP: microchannel plate photomultiplier;
ND: neutral density.
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25. Figure 12: Monitoring Cytosolic pH by C-SNALF2
Table 1: Comparison of Resting Cytosolic pH levels of Adherant cells
Cytosolic pH
Adherant Cells FLIM Reference
3T3 Fibroblasts ± ; 6.83±0.38
CHO cells ± ; 7.3±0.2
MCF-7 cells ± ; 6.65±0.4 a) b) c)
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26. Figure 13: Time Course of cytosolic pH variations in CHO cells after different manipulations
Time (min)
Time (min)
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27. Figure 14: Treatement of ionophores and weak bases perturb cytosolic pH of CHO cells
Nigericin incubation
NMG+
BA added
NH3/NH4+ incubation 2min
NH3/NH4+ incubation 10min
Hank’s solutions washing
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28. Figure 15: In vitro pH-dependant spectral shift and lifetime responsive curves of DND-160 in 3T3 fibroblasts
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29. Table 2: Comparison of lysosomal pH levels derived from FLIM measurements
Low pH indicator From FLIM From literature
DND ±
DM-NERF dextrans 4.3±
OG-514 carboxylic acid dextran 4.9±0.1 N/A
Fluorescein dextrans N/A 5.14,
C-fluorescein dextrans N/A
OG-488 dextrans N/A
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30. Points of interest
FLIM as a method to measure intracellular pH values of different cell types
FLIM measurements differentiated between the appropriate fluorescent pH indicators in cells
DND-160 is not a suitable probe for monitoring lysosomal pH
Lysosomal pH images showed greater degrees of heterogeneity than cytosolic
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31. Wrapping up the FLIM
FLIM is a potential tool to study biochemical processes in living cells
Tissue component contrasting and biochemically specific imaging in unstained and unfixed tissue
Monitoring conformational changes of proteins in physiological environment through FRET by FLIM
Studying intracellular pH changes with respect to different stimuli
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32. Advantages Tracking down changes in chemical microenvironment.
Simultaneous measurement of multiple FLIM from a single measurement resolved by frequency domain.
Ability to suppress the emission signal for any desired lifetime.
Can be used on autofluorescent natural cell constituents.
Parallel readout
Non-invasiveness of the fluorophore
Probe concentration independent fluorescence lifetime
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33. Limitations Several images need to be collected for each FL image
To reduce noise generated by the sensitive imaging system
For correction of background
Large image processing requirement to calculate FL image from the phase data
Photochemistry of the probe
$$$$$ equipment such as lasers, linear CCDs and modulated photomultipliers.
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34. Future work
For pH variations: more fluorescent pH indicators with substantial lifetime sensitivity at the desired pH ranges
Development of multiphotone multiconfocal microscope
Multiple harmonic modulation frequencies FLIM: simultaneous detection of several tagged biomolecules and resolution of their interactions
Improvement of temporal and spatial resolutions
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