Practical aspects of fluorescence microscopy

1. Practical aspects of fluorescence microscopy

2. I want to improve …. Colour separation Sensitivity Resolution What can I do?

3. How to avoid cross-talk between channels

4. How does cross-talk happen? Antigen1 Primary antibody1 Secondary antibody1 Fluorescent dye1 Excitation light 1 Emission light 1 Antigen2 Primary antibody2 Secondary antibody2 Fluorescent dye2 Excitation light 2 Emission light 2

5. How can you test cross-talk? Remove each primary antibody. Corresponding signals should disappear.

6. How to choose antibodies Primary antibodies Use two antibodies made by different species. (can label primary antibodies directly if you are desperate) Secondary antibodies Secondary antibodies should not recognise immunoglobulin of host species of other primary or secondary antibodies. Use antibodies cross-absorbed with immunoglobulin of the other species (careful! goat~sheep, mouse~rat) 2. Use antibodies with fluorescent dyes matched with fliter sets. Donkey secondary anti-sheep Goat secondary anti-rabbit eg. Sheep primary antibody Rabbit primary antibody

7. How to choose filter sets and fluorescent dyes A combination of Excitation filters (lights) avoid exciting other dyes 2. Emission filters avoid passing emission lights from other dyes.

8. How to choose filter sets. Excitation Cy2 (=FITC) Cy3 (=Rhodamin) Emission 400 600nm 500

9. How to choose filter sets. Cy3 is also excited Excitation Cy2 Cy3 Emission 400 600nm 500

10. How to choose filter sets. Cy3 is also excited Excitation Cy2 Cy3 Cy3 emission is blocked Emission 400 600nm 500

11. How to choose filter sets. Excitation Cy2 Cy3 Cy2 emission is not blocked Emission 400 600nm 500

12. How to choose filter sets. Cy2 is not excited Excitation Cy2 Cy3 Cy2 emission is not blocked Emission 400 600nm 500

13. How to choose filter sets. Excitation Cy2 Cy3 Emission 400 600nm 500

14. Choosing filter sets is very important for sensitivity and avoiding cross talk. Filters are the cheapest component but paid least attention can make a huge difference Do NOT trust a microscope rep! Check if you use a new dye or fluorescent proteins.

15. How to tell the property of filters Long pass (LP) filter Band pass (BP) filter Short pass (SP) filter LP500 500nm BP500-530 or BP515/30 500 530nm Multiband pass filter

16. Sensitivity how to get brighter images

17. Sensitivity: how to get brighter images • For immunofluorescence • use bright/stable dyes • Cy or Alexa (not FITC, rhodamine, Texas Red) • use a higher concentration of antibodies or dyes • use a longer exposure time/ gain. • use contrast enhancement (post-capture)

18. Live-imaging: sensitivity objective lens filters camera fluorecent molecules contrast enhancement

19. Objective lens Brightness: propotional to (NA)4 / (magnification)2 X63 NA1.4 vs X63 NA1.2 (>1.8X brighter) X63 NA1.4 vs X100 NA1.4 (>2.5X brighter) "resolution": propotional to 1/NA Use a lens with a high NA (and low magnification). Consider a lens with less correction. (Corrected lens has more internal lenses which absorbe light) Avoid phase contrast lenses (use DIC lenses if required)

20. Filters If single channel, consider a long-pass filter(broad-band pass) NB, blocking auto-fluorescence may increase contrast Emission spectra You are loosing these light! GFP 500 600

21. Camera – sensitivity High quantum efficiency Monochrome Low noise camera cooling the chip, or on-chip amplification (EMCCD) Large pixel size 13 m is 4X brighter than 6.5m (sacrifices resolution) Binning 2X binning gives 4x brighter images (sacrifices resolution) Gain increase noise as well

22. Avoid photobleaching Lower excitation light use a neutral density filter Shorter time for excitation (only excite when capturing images) Use stable/bright molecules "enhanced" FP (eGFP …), tandem GFP

23. Getting bright live images objective lens high NA, low mag, less correction, no phase filtersLong pass, or broader band pass longer exposure time vs photo bleaching/speed only expose when capturing camera binning, gain, high quantum efficiency, monochrome, large pixel size use bright/stable fluorecent molecules "enhanced" FP (eGFP, …), tandem GFP contrast enhancement after capture

24. Resolution How to get finer images

25. Resolution 200nm Theoritical limit ~200nm Limited by NA, not magnification ~0.6 x (wave length)/NA NA1.4 ~200nm Camera pixel size need a half size of the optical resolution eg, To get 200nm resolution, you need 100nm pixel size (=10m on chip for 100x lens) 2 dots 200nm apart under 'scope Low NA High NA pixel 200nm pixel 100nm

26. Resolution in reality Resolution is often limited by the quality of your samples and optical system! Out-of-focus light Camera noise Dirty lens Bad illumination Burnt out filters High sample background Bad fixation

27. Time resolution Facters affecting time resolution Exposure time (vs sensitivity, resolution) can reduce by increasing sensitivity (eg, binning) Readout time from camera can reduce by a subarray readout or binning Computer (software) speed Filter/laser switch time filter cube (~1s), filter wheel (~100ms), laser (<1ms) Focus moving time reduce by a Piezo driven focus

28. Fancy microscopy Choose a method according to your sample and purpose

29. Various fluorescence microscopy Deconvolution : good for point signals high sensitivity, slow to process Confocal : good for diffused signals in thick samples low sensitivity, slow capture Spinning disc confocal : high speed, low bleaching (good for live imaging) TIRF (total internal reflection fluorescence) microscopy : imaging only the surface (with low background) Molecular dynamics study FRAP, FLIM, FLIP, iFRAP, FRET, FCS ….. Super-resolution microscopy (nanoscopy) PALM, STED, SIM ….

30. END Now go back to your lab and improve your images !