NetNotes
5 mm deep, attached to the bottom of a 24-well plate with media filling the well. Tis has kept multiple organoids per matrigel cylinder viable for the full timecourse. Running non-imaged controls alongside has not shown any noticeable phototoxicity. We’ve been taking Z stacks up to approximately 1.5 mm from the plate bottom without any problems or need to increase gain or laser power through the stack, so we’re not finding the matrigel to be a problem. Te caveat here is that we’ve been able to get away with using a 10x/0.45 objective for our needs, but if you have the working distance in the objective you need, it should be worth a try. Richard Lisle
richard.lisle@
ludwig.ox.ac.uk
Glycerol/Type G Immersion Liquid Confocal Listserver Does anyone know if glycerol immersion liquid is in fact merely
glycerol, and if off-the-shelf (molecular biology grade) glycerol can be used instead of designated glycerol immersion liquid? If so, can the refractive index be lowered simply by adding water and thoroughly vortexing/ sonicating to get immersion liquid similar to Type G? Jan Tonnesen
janton@gmail.com
I have in the past used various solutions as immersion liquids for
my glycerol immersion lens. You can straight up use glycerol, but with a refractive index of n=1.47 you ideally want to bring it down (with water) to n=1.45 to match the specifications of the lens. You can correct for some mismatch using the correction collar, but this takes quite some time and practice, so best keep it to a minimum, if possible. Tis works perfectly fine, with two main caveats. First, over time the water in the mixture evaporates, causing the refractive index to tick up slowly. Tis induces increasing aberrations, or requires frequent AO, correction collar readjustment, or reapplication of fresh immersion liquid - all of
which you would like to avoid when performing live imaging. If I am correct, branded immersion liquids (e.g., Leica Type G) include additives aimed at slowing the evaporation of water. Tis would slow the process of RI change, requiring less frequent realignment. Also, this means the refractive index of the “stock” immersion liquid is more constant and predictable, leading to less time spent on pre-aligning the correction collar/AO at the beginning of an experiment. Especially if the vial of pre- mixed immersion liquid sits on a shelf for long periods of time in between imaging. Tat being said, I have imaged with both, and you can easily make both work. Especially if the RI is tweaked a little to pre-compensate aberrations (such as are incurred by imaging deeper within living tissue), this is best done by either pre-mixing the immersion liquids, or by simply buying RI-matched liquids with the appropriate RI. I have used Cargille immersion liquids in a range of RIs to do this and they worked fine. Tey are slightly pungent, however, and can be more unpleasant to work with. Nicolai Urban
nicolai.urban@mpfi.org
We have used glycerol objectives for many years. Te advice
from Leica has always been to use glycerol/water 80:20. Konstantin
microscopia-ibis@us.es
Phase Contrast Microscopy Microscopy and Confocal Listservers I thought I understood how phase contrast microscopy works,
but then I was reading MicroscopyU (
https://www.microscopyu.com/ techniques/phase-contrast/introduction-to-phase-contrast-microscopy) and other sites and now I am confused. My understanding is that it works because of refractive index differences in different parts of the light path (cells) leading to retardation of refracted light and eventual phase differences relative to un-refracted (surround) light that passes through
2020 November •
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