My goal is to see two kinds of particles mixed well in an aggregated form.
My experiment was as follows:
1. Prepared Microparticles 1 + EDC/NHS + RhoB, and Microparticles 2 + EDC/NHS + FITC.
2. Mixed them, centrifuged them, placed them on a glass slide, and then checked them with a fluorescence microscope using different filter sets.
3. Merged the image channels (green and red).
The outcome was not satisfying. There seems to be crosstalk between the particles. When the images were merged, the green and red colors overlapped, making it difficult to distinguish the particles. I troubleshooted by checking each type separately, and they both showed successful labeling (Particle 1 showed red with no green, and Particle 2 showed green with no red).
ChatGPT-4 recommended the following steps:
1. Wash each particle after labeling.
2. Use blocking agents (BSA, FBS, and Tween-20) to prevent non-specific binding.
3. Check the filter set range when measuring.
I did all these steps (including overnight blocking), but the crosstalk between particles 1 and 2 still occurs.
I would be appreciated if someone can recommend me the solution to solve this problem.
Thank you.
Sequential Excitation and Detection (Multi-tracking):
Excite and detect each dye sequentially rather than simultaneously. This approach ensures that the emission from one dye doesn’t interfere with the detection of another. For example, if you label particles with Alexa 488 (green) and Alexa 568 (red), excite and detect them one after the other to avoid crosstalk.
Spectral Separation:
Use additional spectral information to separate emission signals. If there’s overlap between the emission spectra of two dyes, choose laser lines and detection channels that minimize this overlap. Spectral imaging systems can help with this.
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