Why is my yellow dye signal very weak in dSTORM imaging?

Probes & Prep
1

Dear all,

I’m suffering from a very weak signal of yelow dye, such as CF568 and JF549, in my dSTORM imaging. I used yellow dye-conjugated Halo tag or antibody to stain my samples. Althrough I can see a very bright and specific staining under TIRF or confocal microscope, the brightness of localizations under dSTORM is very weak and make it impossible to identify a single molecules. Does anyone has an experience on that? Any suggestions will be highly appreciated.

Attached is an example of my dSTORM image.

Thanks!

image

2

Can you tell us a little more about your imaging conditions? Illumination laser power? Microscope, and microscope objective (NA? oil? water)? Imaging buffer? Depth into the sample you are trying to image? Camera?

-Hazen

1 Like
3

Thanks for your reply, Hazen!

I tried to image membrane proteins in fixed cell. The microscope I used is Nikon Ti2 TIRF N-STORM with objective of CFI SR HP Apochromat TIRF 100XAC Oil and iXON Ultra 897 EMCCD camera. Under TIRF view, I can successfully get SMLM image using AF647 labeled antibody. My issue is I can not get bright blinking events when I used CF568 and JF549 dye. I’m trying to do a multiple-color STORM, thus I want to use CF568 or JF549 combined with AF647. For CF568 and JF549 dye, I always got a very dim blinking event just above background. I did not test the power of laser, but I usually used 100% power of 561nm laser controlled by an Agilent laser box. The excitation light is reflected by a quad dichroic and the emission is filtered by a quad emission filter (open at 422 – 478 nm, 502 – 549 nm, 581 – 625 nm, and 674 – 786 nm).

I thought it might be due to the buffer. So far, I have tried two kinds of buffer below, but none of them worked.

MEA/OS buffer: 50 mM Tris (pH 8) +10 mM NaCl + 10% glucose + 100mM MEA + 0.56 mg/mL Glucose Oxidase + 0.034 mg/mL Catalase

OxEA buffer: 1xPBS (pH 8) + 20% (v/v) of sodium DL-lactate solution + 50 mM MEA + 3% (v/v) OxyFlour™

It would be highly appreciated if you have any suggestions to solve this problem.

Many thanks!

Yunlong

4

If you non-specifically absorb an antibody with CF568 or JF549 to a coverslip and try to image it does this work well?

My recollection of the N-STORM setup is that it doesn’t have particularly high power lasers with the exception of the 647nm laser (but this has probably changed). For a 40x40um FOV you should have something like 50mW going into the objective, which given typical system efficiencies means starting with a 100mW or more laser.

5

Keep in mind you are comparing to the best dSTORM dye around, so you won’t get the same quality/number of photons/stability/blinking conditions out of other dyes.

One way to get more signal out of these dyes is to increase your exposure time, i.e. image less fast. You have to be in a regime to see single molecules but also get enough photons.

Also if you do see quite some signal in the beginning of your acquisition lowering the laser power might also prevent bleaching to keep getting blinking events longer.