Are gaps in the focus ring really a problem?
At the initial training for our OneMP, I was told that gaps in the focus ring due to air bubbles in the objective oil were detrimental and should be removed prior to a measurement by lifting then resettling the coverslip. I recently made a measurement with gaps in the ring and it appeared to be fine. Can you explain how gaps in the focus ring affect measuring accuracy?
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Hi Chase,
Bubbles have different refractive index than oil, which causes light to bend differently than intended. This results in less light reaching to the particles of interest and reducing the amount of scattering from the particles. Since we capture the interference between particle scattering and background reflection, less scattering from particle caused by oil would reduce the interference, thus lower contrast and mass.
If it's a small gap in the autofocus ring, it's likely not a huge mass shift. If the gap gets wider (more bubble), you will likely see a bigger shift. A simple test you can do is to measure the sample before and after introducing bubble to the oil. Move some oil to a microcentrifuge tube and shake it to add bubbles.
If you noticed your measurements tend to create gaps in the autofocus ring, you can heat the oil at 60 C for 2-8 hours and then cool to room temperature.
Amy
Sr. FAS