NetNotes


Edited by Bob Price University of South Carolina School of Medicine Bob.Price@uscmed.sc.edu


Selected postings are from recent discussion threads included in


the Microscopy (http://www.microscopy.com), Confocal Microscopy (https://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy), and 3DEM (https://mail.ncmir.ucsd.edu/mailman/listinfo/3dem) listservers. Postings may have been edited to conserve space or for clarity. Complete listings and subscription information can be found at the above websites.


Microscope Incubation Chambers: Cage or Stage? Confocal Listserver In common with many of you, I manage a facility where users


want to use many different model organisms. We mostly have cage-type incubation systems on our microscopes, which is less than ideal when one user wants the microscope at 26°C, and the next user wants it at 37°C. I have found that cage-type incubators do have the advantage of suppressing temperature fluctuation caused by thermostat control AC, so it’s good to have them around the machine. However, I would like to know whether there are stage-type incubation systems I could use inside the chambers that would allow us to quickly switch between imaging specimens at 20, 26 and 37°C (possibly below ambient, so with cooling) and also provide a reliable way of maintaining focus with high NA immersion lenses. I have used objective lens heaters before and some (but not all) have been very good, but are there any that won’t clash with the stage or microscope when the nose piece is rotated? I’d like to leave them in position rather than take them off all the time. Any suggestions would be most welcome. Tanks, Andrew Vaughan andrew.vaughan@ucl.ac.uk


What kind of microscope did you use? I think it is an inverted one,


but what were the conditions? Pascal Weber pascal.weber@univ-amu.fr Te microscopes are inverted. Te rooms are just standard


microscope laboratories. Te conditions vary according to the model organism. Some users have mammalian cells which need 37°C, some have yeast which are happy around 20°C and some people are using Drosophila tissue at 26°C. Andrew Vaughan andrew.vaughan@ucl.ac.uk


We use hotbox-type boxes too, but we have an LSM 710 with


a Pecon stagetop incubation system for Ibidi microwell plates that rapidly equalizes to the required temperature. It has excellent XY repositioning and the Zen autofocus works well for slower time-lapse experiments, though Definite focus/Perfect focus/ZDC type hardware autofocusing might be a faster means of getting back to a stable Z position. Hotboxes are great, but the expansion and contraction time for a stable microscope setup under different conditions is tedious. Te stagetop design works reasonably well for our needs. All the best. Darran Clements dkc25@cam.ac.uk


We carry a brand-new product by a startup company called


Linnowave which is out of the Max-Planck Institute in Erlangen, Germany. Teir novel solution is to have the actual cover slip be


62 doi:10.1017/S1551929520001480


the heating and measuring element using transparent, conductive coatings. It measures and generates heat where you need it: near the focal plane at the coverslip surface! It comes as a 18×18mm coverslip size and is available also with a PDMS liquid reservoir. All you need is a humidity chamber which can be equilibrated much faster than conventional heating elements based on equilibration of an entire sample system, including objective. Since the temperature control with the Linnowave VAHEAT is targeted at the very volume where your focus is it can adapt within seconds. For more information please visit https://linnowave.com/ and https://shop.boselec.com/ products/vaheat-microscope-temperature-control. Jens Breffke jens@boselec.com


Having looked at the literature, I am not clear where the tempera-


ture is measured. Is an integrated measurement across the whole coverslip being made? If so, any immersion objective will act as a heat sink and reduce the local temperature for the actual cells being imaged, but this will contribute little to an overall temperature measurement - and not be compensated. If compensated and the heating increased, then cells at a distance from the objective will be heated above 37°C. I have a strong preference for heating everything to 37°C so local gradients around the cells/objective cease to be an issue. Have a look at J Microscopy 210 (2003) 131-137. We found some wonderful distortions when a slow Z-series had a similar timescale to the room temperature oscillations. Jeremy Adler jeremy.adler@igp.uu.se


Let me help to clarify: Our VAHEAT system is specifically designed


to compensate the effects you were mentioning: 1) Local heat sink in the FOV and 2) thermal driſt. We have a highly sensitive temperature probe sitting in the FOV that enables fast feedback to adjust for temperature variations in the environment. Te specific geometrical arrangement of the heating element, temperature probe and the immersion objective insure a homogeneous temperature distribution in the FOV. Additionally, the heat load injected into the objective via the immersion oil is kept to a minimum and the quality of the optical imaging system can be maintained, even at elevated temperatures (up to 80°C). Tis allows for fast, reproducible temperature changes and long-term temperature stability without warming the whole microscope to the desired temperature (which takes several minutes to hours and usually doesn’t improve on the temperature accuracy). All in all VAHEAT can, thus, be combined with various highly sensitive measurements such as AFM/confocal, STORM or PALM, PAINT, STED, TIRF. Pierre Türschmann pierre@linnowave.com


Here is how I address the micro-environmental issues you


described. I agree the box approach does reduce the thermal expansion of the whole scope that occurs due to room temp variations. However, in that you describe the need to use immersion lenses for and during temperature transitions, I recommend keeping the thermal mass of the specimen containment structure to a minimum and applying or removing heat to that immediate area in the most efficient


www.microscopy-today.com • 2020 November


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