1. Are You FRETting? Find Out for Sure With FLIM Frequency Domain FLIM for Your Scope Intelligent Imaging Innovations

2. Fluorescence Fluorophores absorb light Fluorophores absorb light Some energy is lost to heat Some energy is lost to heat Fluorophores emit red shifted light Fluorophores emit red shifted light This process takes time (nanoseconds) This process takes time (nanoseconds) The average time is called the “lifetime” of the probe

3. Fluorescence Lifetime time counts

4. What can FLIM do? Probes the molecular environment (FRET, dimerization, pH, mobility, …) Probes the molecular environment (FRET, dimerization, pH, mobility, …) More specificity (GFP ≠ FITC) More specificity (GFP ≠ FITC) Intensity (concentration) independent Intensity (concentration) independent More quantitative and aberration-free measurements More quantitative and aberration-free measurements FRET does not require multiple filters (only measure donor) FRET does not require multiple filters (only measure donor)

5. FRET FRET only occurs if… the donor fluorescence emission spectrum overlaps with the acceptor absorbance the donor fluorescence emission spectrum overlaps with the acceptor absorbance the donor and acceptor fluorophores are in close proximity the donor and acceptor fluorophores are in close proximity the transition dipole moments of the donor and acceptor fluorophores are not perpendicular. the transition dipole moments of the donor and acceptor fluorophores are not perpendicular.

6. FRET through FLIM Only the lifetime of the donor needs to be measured Only the lifetime of the donor needs to be measured If FRET occurs, the lifetime of the donor decreases If FRET occurs, the lifetime of the donor decreases No extensive correction factors are needed as in sensitized emission FRET No extensive correction factors are needed as in sensitized emission FRET Efficieny can be easily calculated Efficieny can be easily calculated FRET efficiency = (Td- Tda) / Td FRET efficiency = (Td- Tda) / Td

7. excitation target emission filter detector frequency domain time domain Measuring fluorescence lifetimes

8. The Instrument objective Pockel’s Cell laser modulated intensifier camera dichroic target phase shifted images

9. What do we measure? A: Phase lag and demodulation Only in the case of single lifetimes do these have any obvious meaning.

10. Δφ M Homodyne

11. Phase shift

12. Demodulation M M

13. Lifetime Vectors Let the radial distance be M and the angle from the x-axis be φ. Let the radial distance be M and the angle from the x-axis be φ. Call this a lifetime vector. Call this a lifetime vector.

14. Polar Plot M Δφ

15. Polar Plot All single exponential lifetimes lie on the semicircle All single exponential lifetimes lie on the semicircle Multi-exponetinal Lifetime are a linear combination of their components Multi-exponetinal Lifetime are a linear combination of their components The ratio of the linear combination determines the fraction of the components The ratio of the linear combination determines the fraction of the components

16. 5ns Cy3 R101 Mix 1ns R101, CY3, and a mixture CY3 R101 Both

17. Using the polar plot with images

18. Phase Lifetime Mod Lifetime 2 Component Fit Distance from Point 1ns 5ns Cy3 R101 Mix x R101 distort

19. C5A and C5V FRET Standards (Steve Vogel Lab) Intensity single tau Where there is no difference in intensity or color, the lifetimes can be different

20. C5A and C5V FRET Standards (Steve Vogel Lab)

21. Donor Only

22. Donor/Acceptor

23. Donor+ Donor/Acceptor With good data you can determine the % FRET and the fraction of donors that are FRETting

24. How to use SlideBook Start SlideBook Start SlideBook Select focus window Select focus window Focus on your sample Focus on your sample Set Modulation Frequency Set Modulation Frequency Set Pockel Cell Bias Set Pockel Cell Bias Set number of Phases Set number of Phases 4 minimum

25. Calibrate to known Lifetime Take an image of Fluorescein Take an image of Fluorescein or Rhodamine dye and image or Rhodamine dye and image using the aforementioned using the aforementioned protocol protocol After image is collected, select a After image is collected, select a Region and click on calibrate lifetime

26. Capture Lifetime Images Select FLIM filter, capture FLIM image of the donor only Select FLIM filter, capture FLIM image of the donor only Capture FLIM image of the donor/ acceptor Capture FLIM image of the donor/ acceptor Make sure to have a good S/N ratio for the FLIM images Make sure to have a good S/N ratio for the FLIM images 10% precision: Intensity needs 100 photons, Lifetime needs 1000 photons

27. Lifetime Vector Analysis The microscope captures a series of images at different phases The microscope captures a series of images at different phases From these, 3 images are created: From these, 3 images are created: 1. Lifetime X 2. Lifetime Y 3. Fluorescence Intensity Real physical measurements (such as FRET) are calculated from these images Real physical measurements (such as FRET) are calculated from these images

28. Lifetime Vector Analysis Mark a region on the Image Mark a region on the Image Click on get Lifetime Histogram Click on get Lifetime Histogram Double click on the point on the polar plot Double click on the point on the polar plot  ns

29. Lifetime Vector Analysis Mark a region on the Lifetime Histogram Create Mask (Region of Interest) from selection Highlighted regions within the cell of the lifetime are given

30. FRET Efficiency Measurement Enter the Donor Lifetime Click on Apply and purple portion shows up on unit circle Showing range of Fret Efficiencies for the donor to Acceptor Double click on the point on the unit circle to show green Line and Efficiency appears

32. The Lifetime Vector Plot

33. Homodyne

34. Photons Needed 10% precision: Intensity needs 100 photons, lifetime needs 1000

35. FRET Example Lifetime of the Donor with no Acceptor = Td Lifetime of the Donor with no Acceptor = Td Lifetime of the Donor with Acceptor = Tda Lifetime of the Donor with Acceptor = Tda FRET efficiency = (Td- Tda) / Td FRET efficiency = (Td- Tda) / Td