I have synthesized core-shell microgels which are FITC-labelled. The core is FITC rich and the shell FITC poor. But when I take a z-stack (or even just an image below the microgel center) a black spot appears right in the middle. It looks like theres a huge dent on the bottom of the microgels, which can't be because if that were the case the dent should be statistically distributed and not face down across the whole sample. Photo bleaching can also be ruled out. I assume it's a CLSM artifact.
The pictures were taken in water and the microgel size is around 230 µm. I had no such issue with other microgel samples. I'm appreciative of any help that I can get!
Hi Thomas,
Can you post an image, please? If possible, information about the setup (hardware, Z step size, lasers, objective with NA information, post-processing software, etc.) would be useful.
Sathya Srinivasan
Hi Sathya,
thanks for your reply! I have attached a video of a z-stack to my original question but I will also post an image of the fluorescent and bright-field channel here.
The hardware was a Leica TCS SP8 microscope. I've done three different z stacks with step sizes of 4.2 µm (System optimized) and 2.5 µm.
We used a 488 nm Argon Laser, a PMT detector, and the objective HC PL FLUOTAR 10x/0.30 DRY. The software LAS AF / LAS X 3.5.7.23225 was used.
Thomas Bissing
- Badges
- Science topic
- Similar topics
- Physical Sciences
- Physical Phenomena
- Luminescence
- Fluorescence