Agree with C.J.'s answer above. I have found that coverslips sold by Warner instruments to be the cleanest ones off the package. Also, I am assuming that you are using TIRF imaging. Make sure that you are in the right imaging plane by using beads. Sometimes you may be looking at the defects in glass that will light up under TIRF conditions if they are not optimal. Finally, when I had this problem a few years ago, I used to irradiate my buffers etc. in Hg lamp illumination for 30 mins and that made it work. I know it is very frustrating when you can't get rid of impurities from your coverslips, but keep trying. There is definitely hope!

Best wishes,

-Saumya

There are a few protocols that you can use for cleaning:

- Sonication in 1M KOH is very useful, followed by thorough rinsing with water.

- Cleaning the coverslip in a Plasma cleaner can also be helpful, and it also makes the glass more hydrophilic.

These protocols you can also find in the book "Single molecule techniques" by Selvin & Ha.

The most powerful method (cleaning by flaming with a blue Bunsen burner flame) unfortunately does not work for glass coverslips (only quartz), so for this you would have to change your coverslips.

Good luck!

We usually sonicate 1 hour with 5M NaOH, then 1 hour sonication in DI water (use lots of water to make sure all NaOH will be removed), a couple of times spray 100% ethanol and rinse off with the DI water (instead, some people sonicate 1 hour with acetone) and finally blow off the cover-slip with compressed air or nitrogen. It works every times for us.

Just in addition to the former comments:

Quarz slides can be heated up to 1000°C to get rid of fluorescence contaminations (we use an electric furnace). Normal glass coverslips (150(micro)m) are a bit more tricky, as they melt at temperatures higher than 540°C. Here, you should use one of the former suggestions. We go for KOH as well.

After cleaning your slides by one of the above mentioned protocols, ozone clean for at least 10min. You will get zero impurity for sure because this kills all your organic stuffs left behind on the slide. I always do ozone cleaning and this also keeps your surface hydrophilic which is helpful for further functionalization of your surface.

good luck

Thank you for these kind advice above, till now I have tried several treatments including Piranha solution, KOH, NaOH, UV-light, etc. But none of which was useful, the fluorescence spot still exsited on the cover slips. Are there any other effective treatments? I am looking forward to receive your reply, thank you very much.

Since you have used a variety of methods to clean the coverslips, perhaps you could try a different brand of coverslips, or try purifying your reagents and cleansers too. Also, is it possible there is fluorescence that appears in a conjugate specimen plane of your optical system (not coming from the coverslip)? You can move the coverslip laterally and see if the position of the fluorescence changes.

Agree with C.J.'s answer above. I have found that coverslips sold by Warner instruments to be the cleanest ones off the package. Also, I am assuming that you are using TIRF imaging. Make sure that you are in the right imaging plane by using beads. Sometimes you may be looking at the defects in glass that will light up under TIRF conditions if they are not optimal. Finally, when I had this problem a few years ago, I used to irradiate my buffers etc. in Hg lamp illumination for 30 mins and that made it work. I know it is very frustrating when you can't get rid of impurities from your coverslips, but keep trying. There is definitely hope!

Best wishes,

-Saumya

I use a serial wash/sonication procedure for 20 minutes each, starting with Toluene , than dichloromethane, than ethanol, than water mQ 2 times. I store the coverslip in mQ water and before the experiment, I put it under Air stream to dry than Plasma treatment !

Influence of impurities could be reduced by FRET.

In attached paper is example of FRET between fluorescent protein and conducting polymer.

Article Immunosensor based on fluorescence quenching matrix of the c...

I am facing a similar issue. In my research, I came across this D263M borosilicate coverslips that apparently have a lower autofluorescence.

http://www.schott.com/advanced_optics/english/products/optical-materials/thin-glass/cover-glass-d-263-m/index.html?so=india&lang=english

I have not personally used them, but am going to try it out. 

Does anyone have any experience using this? 

Regards, 

Avaneesh 

Hi,Weidong Huang. Now I meet the same problem with you, and I want to know what you did to solve the problem.

I would appreciate if you can answer my question.