NetNotes
and wanting to respectfully follow the rules for commercial responses, I will comment in as balanced a way as I can. Disk designs without microlenses with, let’s say, 50 μm pinhole and 25 μm spacing gives you about a 4% pinhole transmission (∼96% excitation light has to be rejected). Microlens-free designs can overcome this using two or more of the following approaches: 1) have a high density of pinholes to either overcome their lower excitation transmission efficiency, and/ or to be able to capture at exceptionally high frame rates for extreme cell dynamins like calcium sparks and puffs; 2) have larger pinholes; 3)simply use higher power lasers; or 4) instead of using single mode fibers, as is the case for CSU, use multimode fibers. Te limitation of (1) is lower blocking of out-of-focus light so higher background in multicell thick cultures or thicker samples (for example, tissue and model organisms). Tis may also mean higher light intensity for running at such high speeds, therefore optimal for shorter term imaging (due to phototoxicity). Te limitation of (2) will be on resolution, but the importance of this also depends on your needs. Microlenses can improve excitation throughput to around 60%, plus the fact that this design also has a dichroic between the microlens disk and the pinhole disk helps further isolate rejected light, so reducing background and improving signal-to-noise. Ten, in our case, we can combine with multimode fiber input giving an additional boon for efficiency, uniformity, and signal-to-noise. Sample crosstalk from out-of-focus planes impacts background in both systems in a similar manner because of their pinhole size and spacing. Overcoming this factor, which gets significant quickly with thicker specimens like embryos, can only be achieved with greater pinhole spacing and an element we chose to focus on as a key design parameter in Dragonfly. On the emission path, assuming the same power density of light at the sample, then sensitivity and signal-to-noise performance becomes more about management of internally reflected light, dichroic and emission filter performance, and finally, the sensitivity of the camera you use. Tis is something we paid particular attention to when we designed Dragonfly. Ten there is the pinhole size itself. For those who prefer to image with the typical “live-cell” 60/63x (water or glycerol immersion) objectives, we use a different pinhole size that optimally matches to numerical aperture. Basically, how the different technologies match up is somewhat dependent on your specific needs. We all offer something different (illumination optics, with/without microlens, pinhole size and spacing, filter specifications, reflected light management) which you should match to the samples you will work with, and the spatial and temporal resolution you require. Your decision is best shaped by detailed conversations with specialists from the vendors, peers like the forum, and testing (if that’s feasible in the current restrictions). Obviously, us companies may well have examples of our technology in publications studying similar or the same cell physiology. Geraint Wilde
g.wilde@andor.com
pinhole-based spinning disc confocals, I thought to throw information about
Although the original post asked for comparison of two classical a
less-known spinning disc confocal method: Aperture
Correlation Microscopy. You can find a very nice explanation of the general principle, and, in particular, to the spinning disc variant here:
http://zeiss-campus.magnet.fsu.edu/articles/opticalsectioning/
aperturecorrelation/
introduction.html. In essence, the spinning disc ACM system uses a structured illumination disc with a grid-like structure where each region (pixel) of the image is randomly excited at rapid sequence (disc rotates at 3000 rpm). Te disc itself, consisting of 50% transmission/50%reflection zones, basically passes 50% of the emitted light from the focal plane. Additionally, the 50% of non- sectioned light is also recorded to generate a final high-intensity
2021 May •
www.microscopy-today.com
confocal image. Due to the high transmission of the disc, the system does not need lasers but can use standard mercury/metal halide/ LED sources for non-saturating excitation of
the fluorochromes.
Mika Ruonala
mika@icit.bio To add to Mika’s comment, we have been using Aurox’s thick tissue sections, cellular delivery targets,
Unity and Clarity confocal modules for the last few months. We have imaged cells,
and microspheres with samples ranging from 200 nm to 1 mm.
http://www.aurox.co.uk. Te system was easy to install and use. Kirti Prakash
kirtiprakash2.71@gmail.com
Ganesh (#2): I’ve encountered no issues with hardware triggering
on a Nikon scope with the X-Light V2 LFOV, and the light engine was the Lumencor CELESTA with 1W laser lines. Tanks, Alessandra, for covering #1. Dave: I concur. I’ve never wanted/needed more power in the
context of live cell imaging, and I’ve been pleased with the V2 LFOV. If you had a power meter that could ensure the same power density at the sample plane, I’d love to see what the difference between your systems would be. But it does sound like there are too many things that would have to be estimated to make the comparison conclusive. Zdenek: Yes, I’ve heard many people suggesting at least 100mW
lasers for the W1. Some have also suggested (for when you’re fine with diffraction-limited resolution) using the SoRa microlensed emission disk with 1x magnification to collect roughly 3x the light versus the standard 50 μm W1 disk. But I’d need more than a couple of $600 relief checks for the SoRa disk. For the X-Light V2 LFOV, I believe the emission filters have high (6+) OD to block (reflected) laser light from making it to the detector. I never really considered if this reflected laser light would excite out-of-focus regions of the sample, and either increase background or induce unnecessary photodamage, but maybe there’s a little bit of that going on. I suppose the V3’s soſtware-controlled square iris also partially sidesteps the issue of cranking up the laser power.
Craig: as Alessandra said, they use multimode fiber coupled light
engines (Lumencor CELESTA or 89 North LDI) which are cheaper than the Yokogawa SDs, which require single-mode optical fiber input. Tanks all for your responses. William Giang
wgiang@emory.edu
Number of Fields of View (FOV) Required in Publications Microscopy Listserver I received feedback from a reviewer asking me to add in the Methods
& Materials section how many fields of view were taken for each specimen. Te journal is PLoS ONE. I am surprised by this request because we all know that we publish representative pictures of our samples, so what is the point of precisely stating how many fields of view were taken? How will this type of information be interpreted? Are 10 fields of view too few, are 20 enough?? What about 15? I am interested in your comments. Tank you in advance! Stephane Nizet
nizets2@yahoo.com
Te reviewer has a valid question. Tey want to know what the
sampling fraction in the total area that you analyzed is, otherwise known as the area sampling fraction in Stereology. Let us say, for example, if you can cover the ROI on the section with 100 FOVs, and you have only analyzed 1 FOV, then it is just 1% of the total, and what is the reason for them to believe that you have not based your representative FOV in a biased way. Tis becomes more complicated if you also have sections. What is the section sampling fraction? Same logic, except that it is with the number of sections within your
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