Fluorescence Lifetime Imaging Microscopy FLIM Inga Sliskovic November 18, 2003

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 11/13/2018

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 11/13/2018

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… 11/13/2018

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 11/13/2018

Figure 2: Examples of Microscopes Then… Now… 11/13/2018 Figure 2: Examples of Microscopes

Figure 3: Problems with Fluorescence microscopy 11/13/2018

Development of FLIM Fluorescence microscopy measurements: Fluorescence intensity Intensity ratios First images obtained in 1994. (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 11/13/2018

Figure 4: Fluorescence Lifetime 11/13/2018 Paul J. Tadrous. J Pathol 2000; 191: 229-234.

FLIM Parameters  = fluorescence lifetime 0= fluorescence quantum yield Kr = radiative deactivation parameter  = 0/ Kr Radioactive decay time: r = 1/ Kr  = 0 X r 11/13/2018 Szmacinski H., Lakowicz J. R., Johnson M. L. Methods in Enzymology, 240, 723-748

Figure 5: Time and frequency domain = (m+ p )/ 2 p= phase of fluorescence m=modulation depth of fluorescence 11/13/2018 Philippe I. H. Bastiaens and Anthony Squire. trends in CELL BIOLOGY (Vol. 9) February 1999

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 11/13/2018 Paul J. Tadrous. J Pathol 2000; 191: 229-234.

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 11/13/2018

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. 11/13/2018

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 11/13/2018

Figure 6: PKB/AKT: FUNCTIONAL INSIGHTS FROM GENETIC MODELS 11/13/2018

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 11/13/2018

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 11/13/2018

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 11/13/2018

Figure 9: Change in GFP-Akt-YFP conformation upon cell stimulation in NIH3T3 11/13/2018

Figure 10: GFP-Akt-YFP conformational change is phosphoinositide 3-kinase dependent 11/13/2018

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 11/13/2018

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 11/13/2018

Figure 11: Schematic diagram of the FLIM instrumentations CCD: charged coupled device; MCP: microchannel plate photomultiplier; ND: neutral density. 11/13/2018

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 7.40±0.05 7.40; 6.83±0.38 CHO cells 7.20±0.04 7.1; 7.3±0.2 MCF-7 cells 7.15±0.03 6.80; 6.65±0.4 a) b) c) 11/13/2018

Figure 13: Time Course of cytosolic pH variations in CHO cells after different manipulations 0 5 10 15 20 25 30 Time (min) Time (min) 11/13/2018

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 11/13/2018

Figure 15: In vitro pH-dependant spectral shift and lifetime responsive curves of DND-160 in 3T3 fibroblasts 11/13/2018

Table 2: Comparison of lysosomal pH levels derived from FLIM measurements Low pH indicator From FLIM From literature DND-160 4.6±0.2 4.5-4.5 DM-NERF dextrans 4.3±0.3 4.3-4.7 OG-514 carboxylic acid dextran 4.9±0.1 N/A Fluorescein dextrans N/A 5.14, 4.7-4.8 C-fluorescein dextrans N/A 4.5-4.9 OG-488 dextrans N/A 4.7-4.9 11/13/2018

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 11/13/2018

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 11/13/2018

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 11/13/2018

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. 11/13/2018

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 11/13/2018