NetNotes


Try this paper: DL Becker et al., https://doi.org/10.1113/


jphysiol.2007.138776. Figure 3 shows the decrement in DiI signal with and without Phenol red. Kevin F. Webb kevin.webb@nottingham.ac.uk


Tanks, Kevin. Yes, good stuff in Figure 3, but this is 840nm excita-


tion. I wonder if the same occurs with single photon excitation. Anyway, this is a good one! Tanks! Sylvie Le Guyader sylvie.le.guyader@ki.se


If my memory serves me well, there was a chapter in Jim Pawley’s


book: Handbook of Biological Confocal Microscopy, Tird edition: Confocal Microscopy of Living Cells, Michael E. Dailey, Erik Manders, David R. Soll, and Mark Terasaki, pages 381-403. Franco Del Principe franco.delprincipe@lis.ch


Phenol red seems to have a strong absorbance at 550nm only at


alkaline pH (https://www.researchgate.net/publication/221925346_ Plastic_Optical_Fiber_pH_Sensor_Using_a_Sol-Gel_Sensing_ Matrix/figures?lo=1). But, if leſt at room temperature, bicarbonate- based media will turn alkaline, so it is possible that some quenching by energy transfer occurs. Mike Model mmodel@kent.edu


Phenol red (and other cyclic compounds) has been shown to increase


background fluorescence (its peak is 440nm), which can create issues for users depending on the channels being used. One such reference is https://link.springer.com/protocol/10.1385/1-59259-826-9:395. Hence, if using colors in that spectrum, it is best to use Phenol red-free medium. Additionally, Phenol red’s cyclic nature is similar to that of estrogen, so it can bind to and activate estrogen receptors of estrogen-sensitive cells (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC323325/). If doing work in those areas it is also a good idea to remove Phenol red. John Fisk techsupport@thermofisher.com


Te reference given by John from Termo Fisher shows a 20%


increase in background fluorescence in the green and red channel when using Phenol red compared to medium without. Tere is also information about estrogen which I did not know. Absolutely nothing about quenching fluorescence. However, I saw that the measurements were done on plastic dishes. I did measure background fluorescence in the green and red channels with and without Phenol red some years ago and never found any difference. I wonder if one would get the same results on glass bottom dishes. Pawley’s confocal handbook (p. 361) mentions that the problem with Phenol red is fluorescence quenching but there is no reference. Sylvie Le Guyader sylvie.le.guyader@ki.se


My understanding is that the autofluorescence could also be due


to riboflavins. I have noticed higher levels of murky autofluorescence when higher levels of fetal calf serum are used, particularly in the 488 channel, for example, GFP-expressing cells. Tis is a rather old flow cytometry paper, but I find it quite useful: https://journals.sagepub. com/doi/pdf/10.1177/27.1.438504. Please have a look at Figure 4. Jacqui Ross jacqui.ross@auckland.ac.nz


Te main problem with Phenol red is usually given as


autofluorescence. In its presence, cells and tissues show a diffuse fluorescence. Given that it appears red, we might also assume that Phenol red absorbs red and blue light. Which raises the possibility that Phenol red could differentially reduce the excitation produced by light in this range at increasing depths. Tat Phenol red absorbs light might mean that what is described as autofluorescence may actually be fluorescence from Phenol red associated with the tissue. Tis would explain why it is a larger problem with some cells type - T cells are


66


mentioned. Apologies for not being able to reference this. Jeremy Adler jeremy.adler@igp.uu.se


Springer protocol: Stadtfeld 2005 in Methods in Molecular


Medicine, Vol. 105: Developmental Hematopoiesis: Methods and Protocols: 2.3. Phenol Red-Free Medium and Glass Bottom Vessels Improve Image Quality. Te second parameter that contributes to image quality is background fluorescence. To test the influence of Phenol red in the culture medium on background fluorescence, we acquired a series of images in media with and without this pH indicator. As shown in Figure 3, Phenol red dramatically increases the background levels, especially when visualizing GFP and RFP with the Endow GFP and TRITC filters, respectively. As a result, the relative signal intensity is decreased, hampering the detection of weak signals (when visualizing YFP with the Yellow GFP and JP2 filters, this effect is only minor). Terefore, we recommend using Phenol red-free medium (we routinely fill a chamber in an eight-chamber slide or a row in a 96-well plate with Phenol red-containing medium to visually monitor the pH of the cultures). “Phenol red dramatically increases the background levels”…Figure


3 shows under 20% increase


in intensity. See Lambert et al., 2020: (https://journals.plos.org/ plosbiology/article?id=10.1371/journal.pbio.3000936). No need for Phenol red in culture media on a microscope, and bicarbonate ions are not just a buffer: they are a substrate for CFTR and several other ion transporters. Evrogen has been selling DMEMgfp for nearly a decade (URL is for DMEMgfp-2): https://evrogen.com/products/medium_ DMEM_gfp/medium_DMEM_gfp.shtml and TermoFisher and abcam (acquired Marker Gene Tech): https://www.abcam.com/opti- kleartrade-live-cell-imaging-buffer-1x-ab275938.html sell similar media. George McNamara geomcnamara@earthlink.net


Stable Stochastic Optical Reconstruction Microscopy (STORM) Buffer Reagents Confocal Listserver We have a Nikon STORM system at our facility. It is used


extensively for short periods when a user needs it and then several months (or years) may pass before the next user pops up. When the time comes to demo STORM at a course, we oſten realize that our ‘new’ reagents have gone bad. Typically, mercaptoethylamine (MEA) buffer has absorbed water, despite the fact that we aliquot it in screw cap tubes in nitrogen. I assume that others are in the same situation. Does anyone have a solution? How long does MEA stay good and if it is more than 1 year, how should it be stored? I can see that there are new fluorophores that blink without oxygen scavenger buffers for live cell STORM, but as far as I know these fluorophores are not yet commercialized. Sylvie Le Guyader sylvie.le.guyader@ki.se


nature of the MEA. We typically make up our MEA at 100mM in PBS (correcting the pH), and store aliquotted and frozen at -80o


We have had the same issues you describe due to the hygroscopic C.


For fluorophores that require scavengers, we then make them up in a final concentration of 50mM. Imaging then takes place with sealed samples, excluding the atmosphere to increase their duration. We can get 6 months from the frozen MEA and 3-4 weeks for the samples. Colin Rickman c.rickman@hw.ac.uk


Has anyone tried the buffer detailed in this BioRxiv paper:


https://www.biorxiv.org/content/10.1101/465492v1, which potentially gets around some of these issues (although probably not TIRF compatible). We are planning on giving it a go in the next few weeks. Simon Walker simon.walker@babraham.ac.uk


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