NetNotes
I had been in discussion with Abberior quite a while ago concerning
mounting media for thick 3D samples, so we talked in length about the hardening vs non-hardening media. (As far as I recall) you are completely correct in assuming that the “3D-suitability” of specific mounting media (such as the Abberior TDE) are its non-hardening properties. I love working with ProLong Gold/Diamond, but it does shrink when hardening, so effectively squashes the tissue samples in the axial direction specifically. Obviously, this makes much more of a difference when imaging truly thick samples (such as brain slices) vs. monolayers or cells, but if you really need quantitative measurements of axial distance, then use non-hardening media. (So SlowFade, TDE, or similar). But then again, if you are imaging thick samples using mounting media with the incorrect RI, then you are going to run into problems anyway. Concerning ProLong Glass, Abberior also warned me that they had observed significant spectral red shiſts of fluorescent dyes, yet I cannot confirm this in the samples I have been imaging. We prepared 3-colored green, red and far-red samples (STAR 488, AF 594, STAR RED) in either Mowiol, ProLong Gold, or ProLong Glass, and imaged them side-by-side. We observed different brightness levels of the dyes (but this might have to do with antibody concentration more than with the actual mounting media), but the STED efficiencies of all dyes were very similar, especially comparing ProLong Glass vs. Gold. If the spectral shiſts were as pronounced as stated, the STED efficiencies (i.e., how much STED power do you need to achieve a specific resolution) should have also changed. Tis was not the case. I haven’t recorded the spectra of the dyes, considering that the samples were extremely densely labeled in 3 colors, but I expect it would prove my point. Nicolai Urban
nicolai.urban@mpfi.org
Regarding SlowFade Diamond— I did not see any significant
aberrations when using it in with Leica’s 100×/1.4 NA oil immersion STED objective for 2D and 3D STED, but without any AO of course. However, my samples were either for imaging cell compartments in cell monolayers or adhered individual virus particles so not very deep imaging. Regarding its stability one time, due to a conference, I had to image a series of fixed cell samples (3D STED) 2 days and 2 weeks aſter labelling and I got great results in both sessions with no discernible signal degradation. Samples were kept in the fridge over this time. So, while as always this may be sample-dependent, I think that SlowFade Diamond can last longer than just few days. Jakub Chojnacki
jakubcho@gmail.com
Photostimulation: Andor MicroPoint or Mosaic Confocal Listserver We have an Andor Dragonfly spinning disk confocal, but no
FRAP scanner. We are interested in performing experiments to recruit fluorescently tagged proteins to DNA damage sites using laser micro- irradiation and live-cell imaging. For this, we plan to micro-irradiate points and line patterns, as described for example in:
JJ Kim et al., (2019) Methods in Molecular Biology, vol 1999. M Tampere and O Mortusewicz (2016) Bio-protocol 6(23): e2039. JF Haince (2008) J Biol Chem. 2008; 283(2):1197–1208.
We would prefer the 405 nm wavelength to induce DNA damage.
Andor offered 2 different tools for photostimulation: MicroPoint and Mosaic. We have several questions:
− Does anybody have experience with one or the other regarding the recruitment of proteins to DNA damage sites?
− How fast is the MicroPoint system for the creation of line patterns? 2020 September •
www.microscopy-today.com
− As far as we know, neither is integrated with the Andor Fusion soſtware. So how well do they work using iQ soſtware or swapping between Fusion and iQ?
− What are their pros and cons? Christian Kukat
christian.kukat@age.mpg.de
Mosaic is really the system intended for patterns, although I don’t
have much experience with it. It is a projector, so you may trade off a little bit of sharpness in your pattern and some limitation in peak intensity for truly simultaneous illumination. Te MicroPoint is a dye laser that maxes out at a 15 Hz repetition rate which is not practical for quickly painting shapes. A few vendors sell 355 nm pulse lasers with kHz rep rates that can paint shapes well, but I don’t know how soſtware integration works with a Dragonfly. Gataca’s iLas and Rapp’s Firefly systems come to mind. I moved from a MicroPoint to a Rapp system and have been extremely impressed with its capabilities. 355 nm illumination will also produce DNA damage more readily than 405 nm. We mostly do point ablations, but it also handles painting very well. Pavak Shah
pavak@ucla.edu
We have both Mosaic and MicroPoint on the same spinning
disk (not a Dragonfly), but I haven’t used them for DNA damage. Te MicroPoint (basically a dye laser pumped by a nitrogen pulsed laser) produces quite strong (50–100 μJ) nanosecond pulses that will ablate anything. But in our configuration, it can only do single-shot ablation, the laser is focused to a small spot on the sample with no galvo. Te repetition rate of the laser itself is limited to 15 Hz, so it may not be suitable for your application. Mosaic is a 100–200 mW 405 nm laser with a DMD projection, so arbitrary patterns are possible, but if you want to irradiate a small point or thin line, the power is of course much lower. Mosaic can be controlled from MetaMorph (I haven’t tried) or IQ. IQ should be able to control Dragonfly quite well, but I don’t remember if there is any critical functionality missing (maybe the ASI stage? Perfect Focus?). I use the Fusion soſtware almost exclusively. But IQ is also capable of grabbing the screen output of another program (instead of getting data directly from the camera), so it should be possible to run both IQ and Fusion simultaneously with Mosaic control through IQ and everything else through Fusion. Andor also offers (or used to offer) the FRAPPA—a galvo-based unit for illuminating arbitrary shapes. Tis can deliver much stronger illumination into small regions (point, line), but you need second (single mode) fiber output from your laser module, and it’s again controlled through IQ. I used it with the good old Yokogawa X1, and since the FRAPPA sits in between the spinning disk unit and the microscope stand, it’s quite possible that it limits the field of view. Zdenek Svindrych
zdedenn@gmail.com
SEM Filaments Blowing Microscopy Listserver I am using Hitachi S3500-N SEM, it runs on tungsten filament. Te
filament is getting blown as I start the HV, it shows 0 current. I lost three brand new filaments in a row. Can anyone help me how to troubleshoot this? Ravi Takkar
ravi.thakkar369@
gmail.com
We had exactly the same problem with a JEOL JSM5600 and we
found that the high tension control board had a transistor blown, we changed it and then it was OK. Yorgos Nikas
eikonika@otenet.gr
Tere might be 3 causes: 1. Poor vacuum. Check the vacuum
gauges before starting the HV. If the gauges are faulty opening the HV will lead to burn out of the filaments. Check the tip using a light microscope and based on how the filament’s ends appear it may indicate
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