NetNotes


single-layer disk indeed requires more laser power than dual-layer disks that include a microlens array, but lasers are oſten relatively powerful these days. Josh Vaughan jcv2@uw.edu


Tis DIY spinning disk is quite impressive. I was wondering, the


commercial disks are made of identical sectors (I mean “angular” sectors, to distinguish it from the radial sectors in the preprint) - this helps with short exposures (there are typically 8 identical sectors). Is the multiple spiral approach somehow equivalent to this? My other comment goes to fluorescein. I’ve spent a lot of time with fluorescein, and…let me put it this way…we’re not friends. Tere is a dramatic decrease of absorbance at 488nm between 1mM and 10mM concentrations, as well as a drop in quantum yield to almost zero. It’s quite possible that at 1M the fluorescence only originates from the coverslip surface (nobody really knows), but if you extrapolate the dilute fluorescein extinction coefficient, that’s not right. And its sodium salt of fluorescein for that matter, as fluorescein is insoluble in water. Also, why didn’t the disk vendor drill the hole to the typical engineering tolerances (10μm should be possible)? Zdenek Svindrych zdedenn@gmail.com


Yes, the product number we referenced is indeed for the disodium


salt of fluorescein. Do you have a literature reference on the absorbance behavior of fluorescein at high concentration? We could revise or remove our ∼62nm 1/e estimate, but the exact value is not important for our purposes. Tat is, the ∼800nm FWHM of the fluorescein signal we measured with the water lens matches the 100nm-bead-measured axial PSF so the emission layer of 1M fluorescein seems thin enough for measuring background rejection out to +/- 10μm. Is it 8 sectors per revolution in commercial disks? As to your question, we just didn’t study partial revolutions since for most of our experiments we use >0.1 second exposures in multiples of a revolution. Fortunately, the photomasks are relatively affordable and can be easily customized for speed or other priorities. Tere may be more precise ways to mount the disk, but our solution seems adequate. We aren’t sure about the tolerance for hole placement by the photomask manufacturer (it might have been as low as 10μm for all we know) but we still think it is good to use a design that tolerates being off-center. Josh Vaughan jcv2@uw.edu


Regarding fluorescein absorbance, all the relevant research is


very old, and I don’t have good references. What’s worse, I was *totally wrong* about the absorbance decreasing appreciably with increasing concentration at any wavelength. I was misled by our microvolume- spectrophotometer that reads zero when the absorbance is above ∼50. I was getting double peaks like in Figure 5 of [https://onlinelibrary. wiley.com/doi/10.1111/j.1365-2818.2008.02026.x], just much deeper. Other references related to this figure might be of interest to you. If anything, the absorption increases faster than linearly with concentration, or at least shiſts to longer wavelengths. You can see it readily when comparing 100mM Na-Fl in 1mm cuvette versus 10mM Na-Fl in a 10mm cuvette, see, for example, this image (https:// drive.google.com/file/d/1dwi18lhBxjC2fwq8hysDfwHgX8-Zi1S_/ view?usp=sharing); the darker one is the 1mm cuvette with 10x the concentration. I have no means to measure the absorbance other than the long-wavelength shoulder, which shiſts to longer wavelengths with higher concentrations. I know, there are machines that can go to OD 6, I just don’t have one. Te quantum yield drop with increasing concentration is a well-known feature used in many “dequenching” experiments, see, for example, Figure 3A in (https://www.nature. com/articles/srep29460). Sorry for the confusion brought about by my previous post, I know it can’t be undone and will remain in the internet’s history for eternity. Zdenek Svindrych zdedenn@gmail.com


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Crossword Puzzle Answers See puzzle on page 58.


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