NetNotes
means possible. Waiting for self-equilibration takes a loooong time. My company, Bioptechs, has developed micro-environmental control systems for both steady state and quick thermal ramping applications such as gene expression experiments. Terefore, I believe we can precisely meet your needs. In addition to typical fixed physiological temperatures, our systems are being used in the temperature ranges of, 15°C to 25°C in 20 seconds, 4°C to 40°C in 3 minutes, and 15°C to 60°C in 2 minutes while maintaining stability in the Z-axis within our FCS2 flow cell. If an actual laminar flow cell is not needed, we have a number of other products and technologies for fast and efficient thermal control as well as Z-axis stability to choose from, including a coverslip-bottomed dish with intrinsic temperature control at the coverslip. Temperature control for the objective is a specialty of ours. Our patented Objective Heater incorporates the objective temperature at the specimen plane in the control loop and compensates for the heat- sinking effect of the nosepiece. It has a detachable cable from the heater so that you can rotate the nosepiece. If you need to obtain temperatures below ambient, we have products to do that as well. Special note, we developed a thermal control system used on all our products that takes the specimen from initial to target temperature typically in seconds then steady thereaſter and it does not induce the variations typical with PID. Setting up a micro-environmental control system for imaging is not trivial. Dan Focht
dan@bioptechs.com
Multiple-Position Long-Term Time-Lapse Imaging with Oil Immersion Lens Confocal Listserver One of our users is doing long-term time-lapse imaging in multi-well plates
with reflection based autofocus. Te sample is quite flat, so an oil immersion lens seemed the best choice in terms of light collection efficiency and image quality. Our first run worked well. However, during the second experiment the focusing failed quite early during the time-lapse and the sample remained out of focus for most of the experiment. I suspect that an air bubble in the immersion oil could have interfered with the focusing and because of high viscosity of the oil, a bubble can persist there for quite some time. I’m wondering if the hypothesis of the bubble is correct. How likely is such an incident to happen - whether we were particularly lucky in the first experiment or particularly unlucky in the second? What is your experience with the use of oil immersion in such experiments? Radek Machan
radek.machan@
ntu.edu.sg
If the sample is in water, I would use a water objective and have
oil with RI=1.33 as the immersion medium. You will get better image quality since your sample is very likely to be thicker than 10μm. On top of that the oil with low RI is less viscous so less likely to have
bubbles. I would apply the oil on the objective with a rod, not with a bottle that needs to be flipped and squeezed. You will need to check that the autofocus works with the water objective. Depending on how many weeks you are imaging, another reason for the loss of focus might be that the oil was spread too thin on the plate. Sylvie Le Guyader
sylvie.le.guyader@
ki.se
We have quite some experience with multi-well plates and
immersion oil objective lenses. An air bubble could have caused the auto focus to have failed, but excess or too little oil can also cause the auto- focus system to fail as the oil runs off the lens or is spread too thin shortly aſter starting acquisition. Most microscope vendors include the auto- focus status in the raw file metadata for each timepoint and position. You could check back and see when the auto-focus system failed by looking at the status code to trace if the focus or another malfunction occurred. Here are some tips that might prevent this issue in the future: 1) We use a folded piece of lens paper to distribute a thin oil layer on the glass and then remove the lens paper on a part of the glass you will not image (air bubbles are introduced when liſting the paper off the glass!); 2) Move around to all xy positions a few times to evenly smear the immersion oil and then start setting up auto-focus; 3) use 96-well or 348-well plates if possible and space samples close together, also this allows more conditions to be imaged simultaneously; 4) slow the xy-stage movement down if hardware supports this and if it doesn’t impact the experimental setup too much. Timo E.F. Kuijt
t.kuijt@hubrecht.eu
I think your hypothesis is very likely. Additionally, what could have
happened is that during the stage movement the oil was dragged and did not cover the top of the lens during acquisition. Or the focal plane across the plate plane was not the same. In general, multipoint time-lapse with immersion oil is very challenging because of the issues you bring up. Plates aren’t completely flat and slight changes in Z will either make the experiment fail or damage the lens. Tings you could do is to reduce the speed of the stage, “paint” immersion oil on the glass (this will make a huge mess and can potentially damage the stage if oil gets inside), or determine a map of Z positions throughout the plate so that the nosepiece can adjust for the different focal plane and hopefully damage the lens less. Paula De La Milagrosa
paula_monterollopis@hms.harvard.edu
I do that experiment quite a lot and yes, bubbles have been the
bane of my existence. Before I start an experiment, I always do a quick bubble test - just move the stage around and look carefully at the meniscus. You can spot bubbles because they move like bearings, at about half the speed the stage is moving. If you spot a bubble, take the
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