Reading Fluorescence with a Microplate Reader BioTek Instruments, Inc.

1. Reading Fluorescence with a Microplate ReaderBioTek Instruments, Inc.

2. Fluorescence • FI- Fluorescence Intensity • FP- Fluorescent Polarization • TRF- Time Resolve Fluorescents

3. What is fluorescence? • Reading fluorescence in a microplate • Excitation and Emission parameters • How to ...

4. Excitation (light) Emission (light) Excited State Ground State Ground State Nucleus Inner Orbital Inner Orbital Outer Orbital What is Fluorescence? • Electrons moving around the nucleus of atoms or molecules can be excited by light (they absorb light). • A fluorescent molecule (fluorophore or fluorochrome) will give light emission just after excitation to come back to its ground state.

5. WL Excitation Spectrum Emission Spectrum What is Fluorescence? (Continued) • Mostly (99% of applications), excitation WL is smaller for than emission WL • The difference between excitation WL and emission WL is called the “Stokes shift”

6. Four important elements to detect Fluorescence • Source of Excitation (Light Source) • Fluorophore • Wavelength selectors (most often Filters) to isolate the excited photons from the emitted photons • Detector to register the emitted photons and to convert them into a electronical Output-signal (PMT-element) or into a picture

7. Reading Fluorescence in a Microplate • What is fluorescence? • Reading fluorescence in a microplate • Excitation and Emission parameters • How to ...

8. Light PMT Filters Filters Fiber Optics Reading Fluorescence in a Microplate • General Principle

9. Reading Fluorescence in a Microplate Halogen light bulb vs. Xenon Flash Light • Tungsten – Halogen light bulb • Provides strong and even excitation across the spectrum • Xenon Flash Lamp • You will need a very high energy Flash lamp to receive similar energy • levels compared to Halogen, therefore you will need a very big strong • power supply, thus instruments are often very big and heavy. • Flash light will not send uniform, stable light. >> Sensitivity

10. Fluorescence, light source (Xenon Flash Lamp) Tungsten Halogen Lamp Tg Relative Energy Xe Wavelength 100 250 1100 750 500 • Continuous and strong light source (versus flash) gives more repeatable excitation. CV’s are reduced and S/N ratios improved.

11. Reading Fluorescence in a Microplate Filter vs. Monochromator • Filter-based • Filters are assay specific (e.g. 400/10; 635/32; 580/50) • Transmit 50% of light vs. 15% for a monochromator • Monochromator-based • Much more flexible • Spectral scans possible >> Sensitivity

12. Filter-based Fluorescence • Filters transmit more light than monochromators • Typically 50% of target wavelength goes through a filter vs. 15% through a monochromator. • Monochromator-based systems require big expensive light sources to achieve similar sensitivity. • Filters are dye-specific • Depending on the Stokes shift, the bandwidth is exactly tailored to the assay (e.g. 400/10; 635/32; 580/50 nm filters). • Monochromator-based systems have typically a fixed bandwidth (or only a few choices around 10 nm): the sensitivity is assay dependent. >> Sensitivity

13. 340 380 360 WL (nm) Filter-based Fluorescence (Continued) • Filters have much sharper wavelength profiles than monochromators. They give a much better wavelength specificity, allowing to bring more light to the sample with less risk of interference with emission measurement system. • In red: filter profile of a 360/40 nm light. Because of the straight cut-off, the bandwidth can be large. • In blue: monochromator profile of a 360 nm light. Because of the cut-off slope, bandwidth of monochromator systems is typically around 10 nm. Less excitation light goes to the sample, risk of interference with emission is higher with dyes with small Stokes shift. >> Sensitivity

14. Reading Fluorescence in a Microplate Quartz Fiber Optics against PVC Fiber optics • High quality fibers will transmit even and constant light even at low UV • Top and Bottom mapped quartz fiber optics Even illumination of sample, maximum signal transfer and light collection, low reader-to-reader variation >> Sensitivity

15. Cross Section Excitation Emission Bifurcated Mapped Fiber Optic Probe

16. Cross Section of Fiber Probe Excitation Fibers 5.0 mm Emission Fibers Consistent, even illumination and capture of Fluorescence Bifurcated Mapped Fiber Optic Probe (Continued)

17. Other Probe Configurations Older Instruments 5.0 mm 5.0 mm Random Fibers Concentric Rings

18. Reading Fluorescence in a Microplate • What is fluorescence? • Reading fluorescence in a microplate • Excitation and Emission parameters • How to ...

19. Excitation and Emission Parameters • Filters selection • Light intensity • Fiber optics diameter • Distance from sample • Top/Bottom reading

20. Excitation Parameters Filters If white light is used to excite the sample, many things may happen, such as: • Fluorescence of the target molecule • Fluorescence of other molecules • Reflection of incident light • ...

21. Excitation filter Excitation Parameters (Continued) Filters

22. Excitation filter Excitation filter Emission filter Excitation and Emission Parameters Filters

23. Excitation Parameters Light intensity • Power of light source (most important!) • Quality of the optics • Fiber Optics diameter • Distance between excitation source and sample

24. Multi-Detection Microplate Reader • Tungsten Halogen light bulb: high output for a strong excitation of fluorescent molecules, and high sensitivity • Xenon Flash lamp: strong output from UV to near IR to cover all absorbance applications and TR Fluorescence

25. Excitation and Emission Parameters Diameter of the probe

26. Excitation and Emission Parameters Distance between probe and sample, Light collection

27. Excitation and Emission Parameters Top/Bottom reading Bottom reading Top reading

28. Excitation and Emission Parameters Top reading • Is usually recommended when the fluorescent molecule is in solution. • Will give lower back ground than bottom reading • Will also give lower results (the distance to the sample is bigger)

29. 6-Well Plate 96-Well Plate Adjustment of the distance of the optics Optimum position of probe close to the top of the plate.

30. Excitation and Emission Parameters Bottom reading • Is usually recommended for cellular assays (cell are located on the bottom of the wells). • Will give higher back ground than top reading because of the reflection of excitation light on the well (~4% of incident light). • But will also give higher results than top reading.

31. Transparent White Black Emission Parameters Type of plate

32. False positive measurement Positive Negative Emission Parameters Transparent plates Conclusion: High CROSS TALK in Fluorescence But: ... Recommended for use in Colorimetry (Absorbance)

33. Emission filter Emission Parameters White plates Conclusion: No cross talk, High sensitivity, but : HIGH BACK GROUND • But:…Mainly used in luminescence, may be used in fluorescence (~20%)

34. Emission filter Emission Parameters Black plates Conclusion: NO CROSS TALK LOW BACK GROUND • Black plate: most common in fluorescence (~80%) • Black Plates with clear bottoms are used for bottom • readings (cell assays)

35. Plates to Use (e.g. from Corning Costar) Emission Parameters • If a simple container is needed • Solid black • Corning 3915 • Black sides with clear bottom • Corning 3615 • Clear plates (for Absorption) • Corning 3635 (UV Plates)or 3679 (Half area) or 3675 (for 384) • If cell culture treatment is needed • Solid Black • Corning 3916 • Black sides with clear bottom • Corning 3614 • Corning 3603 • If immunoassay binding is required • Solid Black ●Corning 3925 • Black sides with clear bottom ●Corning 3601

36. Plates to Use (e.g. from Nunc) Emission Parameters • Nunc 442587 • Translucent • Polypropylene • Useful for all wavelengths, especially those under 400 nm • V bottom • For assays simply requiring a container. Not for cell culture or immunoassays. • Read from bottom or top. • Very low background when reading from the bottom.

37. Light PMT Filters Filters Excitation Emission Emission Parameters Adjustment of the PMT

38. Emission Parameters Adjustment of the PMT Low Voltage Low Signal (FU’s) High Signal (FU’s) High Voltage

39. Highly Fluorescent Sample Low Fluorescent Sample 99999 Max Out FU 0 0 255 PMT adjustment : Sensitivity Setting Emission Parameters Adjustment of the PMT0

40. Excitation and Emission Parameters Summary • Filters selection: to avoid interference and to read only the WL of interest • Light intensity: to have a strong excitation (Fiber optics diameter, Distance between probe and sample) • Light collection (Distance, Fiber Optics diameter): to collect a lot of energy • Top/Bottom: to have the best excitation and emission for the current application • Type of plate • Adjustment of the PMT: to optimize dynamic range of results

41. Reading Fluorescence in a Microplate • What is fluorescence? • Reading fluorescence in a microplate • Excitation and Emission parameters • How to ...

42. How to ... • Select the right filter set • Adjust the distance of the optics • Select Top/Bottom reading • Select the right fiber optics diameter • Select the proper type of plate • Adjust the PMT • ...

43. How to select the right filter set Excitation Spectrum Emission Spectrum

44. 340 380 How to select the right filter set Band pass filter parameters Example of a 360/40 filter 1. Theory 40 360 WL (nm)

45. 340 380 How to select the right filter set Band pass filter parameters Example of a 360/40 filter 2. Reality 40 50 % of Max. 360 WL (nm)

46. High result Good sensitivity How to select the right filter set Correct selection Excitation Emission

47. Excitation Emission Low result Bad sensitivity How to select the right filter set • Poor selection • Wrong excitation filter • Good emission filter

48. Very high back ground signal How to select the right filter set Poor selection: interference between filters

49. Efficiency of excitation is not 100% But there is no choice How to select the right filter set Correct selection: no interference between filters

50. How to adjust the distance of the optics • FLx800: thumb screw on the top of the reader. • Synergy HT: automatic, just select the plate type to be read in Gen5™ . Synergy Selecting a plate type in Gen5 automatically moves the fiber optics to the correct height for that plate.