Q1: Will FocusClear™ work on live plant tissue?

In general, FocusClear™ is suitable for fixed samples. However, we have few experiences to treat FocusClear™ on living plant tissues, and the short-term treatment did not cause acute killing.

Q2: Does FocusClear™ work with Drosophilae embryos?

Definitely.

Q：I just had my first look at a FocusClear™ treated brain on the multi-photon microscope. There was no DAPI staining. Does FocusClear™ interact with DAPI? I used DAPI and PI as these are good fluorochromes to stain nuclei.

Since the binding between DAPI and DNA is weak and often dissociate during long-term storage, the post-fixation is needed (4 % paraformaldehyde). You can also shorten the FocusClear™ treatment time. In general, a cockroach brain with a thickness of 500 micrometer can be cleared within 1~2 hours. If the specimen is thin like fly brains, the clearing time is about 10 minutes.

Q4：After applying FocusClear™ to my specimen, I found that bleaching was a lot faster than my control. Why did this happen? And how should I avoid it?

Because of more efficient excitation one would expect faster bleaching. The solution of this is to use lower laser power. For example, use 488 laser equipped in Zeiss LSM 510 confocal microscopes at 10-15% attenuation of 75% laser power. This would prevent most bleeching problem. Since the fluorescence signal is much stronger in FocusClear™, even with low excitation laser, the image quality as well as resolution is greatly improved comparing with samples in glycerol.

Q5：Why was there increased background fluorescence on my sample when I used FocusClear™?

Since FocusClear™ treated samples will become completely transparent, it is expected to see more fluorescence from focus as well as out-of-focus planes. This becomes a problem when using a conventional fluorescence microscope because it often becomes too bright. The increased background fluorescence is coming mostly from out-of-focus planes. In fact, in some cases, fluorescence signals become so strong that we cannot even look at the samples under a conventional fluorescence microscope directly without using a ND filter. Thus, for the observation of thick samples under conventional fluorescence microscope, FocusClear™ is only useful for increasing detection sensitivity from samples with weak signals, which is often the case for many samples. On the other hand, strong fluorescence signals become a great advantage when using a confocal microscope that allows the use of a smaller pinhole size to filter out all the out-of-focus signals. Under this situation, in fact, many of our users found that the background fluorescence of the focus plane of samples mounted in FocusClear™ is much lower than the samples mounted in glycerol.

Q6: How really bad does FocusClear degrade by the expiration date? What exactly happens? Will the clearing effect be reduced, or lessened?

As you know, we recommend an 1 year expiration. However, if the solution remains clear, you should still be able to use it. You should discard the solution if it turns brown or appears cloudiness.

Q7：Could pre-fixing or post-fixing pose any additional problems with FocusClear™?

In some cases like samples for immunohistochemistry, a short fixation is required for the following immunolabeling treatment. In that case, you should then do a post-fixation at the end of immunohistochemistry before the FocusClear™ treatment. Fixation before or after dissection or even after immunohistochemical staining should not make much difference for the effect of FocusClear™. Most importantly, your sample should be well fixed before clearing.

Q8：We would like to know the approximate thickness of mouse brain which you used FocusClear. (Please see attached file.) Could you let me know?

By clearing the mouse brain slice in the FocusClear, it is possible to see GFP-labeled structures up to 1 mm beneath the surface. However, image quality is largely compromised beyond 500μm in depth. The demonstrated mouse brain slice was 200μm in thickness under a Zeiss LSM710 confocal microscope with a 40X water immersion lens (1.2 N.A.). All neuronal fibers could be clearly imaged and 3D reconstructed.