Troubleshooting Two-Photon Excitation With Filipin III Labeling


I’ve been working on imaging cholesterol localization in human endothelial cells using Filipin III. Because the excitation for Filipin is around 350, we have to use Two-photon excitation because no one on campus has a confocal capable of such short wavelengths. It has been going well, except lately I’ve been having odd, inconsistent imaging over time.

Despite all samples being prepped at the same time with the same reagents, there is significant variation that I do not believe is real. Additionally, signal localization for the exact same sample changed from junctional to nuclear after going back to it an hour later (1st and 17th images below). All samples are fixed in 4% PFA and not permeabilized to prevent disruption of cholesterol.

Here are summary Max IP images:

My usual protocol is as follows:

  1. Fix in 4% PFA for 10-15 minutes.
  2. Wash in Tris-Glycine for 10 minutes (2X).
  3. Remove Filipin stock (abcam) from -80 C and dilute 1:100 in either PBS (or buffer provided by abcam).
  4. Incubate in the dark 30 - 60 minutes.
  5. Wash in PBS for 5 minutes (2X).
  6. Image same day.

Everything was kept in the dark and DAPI-free throughout the process. To be sure the signal is not unique to the two-photon laser, I checked the signal afterwards with our epifluorescent microscope and observed the same loss of signal/nuclear labeling.

One concern we’ve considered is the laser intensity is too high and is cooking my samples, leading to degradation of Filipin and autofluorescence of the nuclei. While it’s possible there was uneven labeling with the Filipin between coverslips, that doesn’t explain why the same coverslip showed completely different localization after only 1 hour, despite fixation.

Any feedback is appreciated!

Hi Tim,

Fluorophores are very bleachy under two-photon excitation. See reference below.
If your samples are a monolayer of tissue culture cells, I’d recommend you try widefield.

Patterson, G.H. and Piston, D.W. (2000) Photobleaching in two-photon excitation microscopy. Biophys. J. 78, 2159–2162

Best, Jennifer

Thanks for the suggestion. These are monolayers so I know two-photon is somewhat overkill, but we were limited by wavelength constraints. We do get decent images with epifluorescence, they’re just messier and lack some of the sharp detail we want to highlight - but perhaps we’re being too picky, haha.

I’m just surprised that bleaching would reveal the nuclei - I’ve never seen that with standard immunofluorescence.

  • Tim

Are these mounted on a slide? If so, in addition to the bleaching issue, the sample will cook very fast as the localized heat can’t be dissipated very well into the mountant/glass, as compared to animal or thick tissue. Jennifer’s suggestion is good, try a widefield, its been a long time since I’ve imaged fillipin, do you get no excitation at 405nm? Regarding the multiphoton, I don’t know the instrument and what you’ve tried, but lower power, high gain, and resonance scanning with some averaging could help. I wonder if having the cells in water/buffer in a dish (is it an upright?) and using a water dipping objective could help with the heat if there is a decent volume of water/buffer?

We’ve tried both mounted and immersed with PBS. The two-photon objective is 25x water immersion. The trouble with the mounting media we have is it’s designed to reduce overall signal intensity (presumably to add longevity), so we lose a lot of vibrant Filipin signal. Because Filipin is very unstable, I’ve found it’s best to just label and image all in the same day.

The Filipin we have gives no excitation at 405, as tested on our confocal microscope, but like I mentioned I can still get by with epifluorescence at around 380 - it just lacks the detail that we were originally getting with the two-photon.

I’m currently just a beta-tester for this new instrument as well, so it is very possible some settings are set way too high.