jamesdmanton.bsky.social
Applied physicist & microscopist/microscopologist
335 posts
1,253 followers
523 following
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Because the field stop is part of the microscope, not the objective?
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...everything is still bleaching weirdly quickly. They measure mScarlet-H's half-life to be 146 s under 5.1 W / cm^2, while Bindels et al. measure it to be 574 s under 6.9 W / cm^2. If they haven't normalised, then their results lead to a normalised half-life of 89 s!
bsky.app/profile/jame...
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I haven't tried it yet, but the results presented in the paper are a bit odd. Either they've normalised the half-lives (which would be good) without saying so, or they've measured everything as bleaching _much_ faster than everyone else's measurements. Even if they have normalised...
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Funnily enough, the professionally processed camera arrived this morning. We've not tested it properly yet, but from a brief inspection under the stereo it indeed looks like they've done a nicer job than we managed!
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We're big fans of Ximea's cameras. That tiny one in your hand is from the same xiMU series that we used for the DvOPM, and their xiX series made controlling and synchronising the eight cameras for our multispectral system a doddle. Everything is very reasonably priced, too. Would highly recommend.
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The smallest pixel size we found was 600 nm on a chip by Samsung, but you can't buy it unless you want a shipping container full of them. I'm a bit surprised they even went that small, but it'd be great if we could somehow convince a manufacturer to make really tiny pixels. Probably many €€€, though
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No, I just chucked in 510 nm rather than the sodium D-line into refractiveindex.info. The datasheet doesn't really give much information about what's under the coverglass, and the photon loss measurements suggests that it's not just Fresnel reflections from silicon going on.
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If you're scared about removing the coverglass, and don't want to pay a company to do it, Jacob did find that Allied Vision sell a version of the IMX226 chip (Alvium 1800 U-1240m) without a coverglass. The downsides are larger pixels (1.85 µm) and higher cost.
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Oh no, sorry that we scooped you! That said, in the image below, it looks like you're using a mirror in front of the camera chip. That's different, and seems like a novel & inventive idea worthy of publication to me! Let me know if you want to chat more about the differences in our respective works.
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Thanks!
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Thanks! It took a bit of editing to get it to fit within the strict page limit. We'll probably have to remove it for 'proper' publication, but we thought it was important to have a complete record of our methods somewhere.
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We did also find a few companies that would remove the coverglass for us (this is fairly popular for increasing IR sensitivity, for example), but there were some issues with sending them a PO that took a long time to resolve. In the end, we were too impatient and tried ourselves.
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Physically, it was fairly straightforward and required just some forceps, a razor blade and a stereomicroscope. Mentally, it was fairly taxing (we alternated attempts to remove more and more to share the blame in case it all went horribly wrong at some point) and definitely needed a tea afterwards!
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I see your 1.91 and raise you 4.25...
bsky.app/profile/jame...
(we're ignoring anti-reflection coatings, right...?)
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We hope our concept will significantly democratise access to high-throughput imaging of tissue samples at sub-cellular resolution and, with continued developments in camera technologies, might become a viable technique for high-resolution single-cell OPM imaging without the need for a 3rd objective.
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As a proof-of-concept, Jacob Lamb built a direct-view mesoscopic OPM using a €700 Ximea MU196MR-ON camera with 1.4 μm pixels. We could then image expanded cerebral organoids, created by @miguelcmestre.bsky.social, with a 2 μm × 2 μm × 22 μm resolution over a 5.3 mm × 2.88 mm field of view.
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In our work, we just put the camera directly into the remote refocus volume to detect the oblique plane directly, without the need for a tertiary microscope system!
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Hoffmann et al. have also shown how an image transfer fibre plate can be used to achieve a similar goal without the photon losses of diffractive elements, but this requires a costly custom-made optical element.
doi.org/10.1038/s414...
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More recent work, e.g. by Shao et al. and Daetwyler et al., have extended this concept, but the relatively low photon efficiency and difficult alignment procedure remain.
doi.org/10.1364/OPTI...
doi.org/10.1364/OPTI...
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In 2019, Hoffmann and Judkewitz showed that a diffraction grating could be used to redirect the light into the tertiary objective, but this led to a distorted point spread function and low photon efficiency.
doi.org/10.1364/OPTI...
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This leads to all the light collected by the primary objective and directed into the remote refocussing volume by the secondary objective missing the entrance aperture of the tertiary objective! Over the past few years, a number of groups have proposed neat solutions to this problem.
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Fundamentally, this is because of the need for a third microscope system to image a tilted plane in the remote refocus volume created by the two microscope systems nearest the sample. Due to constraints in lens design, low magnification objective lenses necessarily have a low numerical aperture.
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Oblique plane microscopy (OPM) is a well-established sub-cellular imaging technique that combines the volumetric imaging power of light sheet microscopy with the convenience and ease of use of traditional inverted microscopes. However, applying this to mesoscopic samples has proven challenging.
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In general, I think Microsoft Office peaked with the 97 release, but this is one of the few modern advantages I give the later developers kudos for.
And no, I'm not as old as this statement makes me sound.
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If you're on Windows just rename the file to have a .zip extension. I'm not sure if that works directly on a Mac, but I can check in the morning.
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If you have a .pptx file then it's really a ZIP file in disguise. You can change the file extension, or equivalent, and then extract all the original photos and videos and convert as you desire.
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ffmpeg (www.ffmpeg.org) and Handbrake (handbrake.fr) are, in my opinion, the best tools to use for such conversions. Handbrake is easier to get started with, but ffmpeg is much more powerful. It's particularly good for converting 'standard' MP4s into 'lesser' MP4s, such as those used by WhatsApp.
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Not a direct fix, as such, but I always convert my AVIs to some form of H.264 MP4. That way you don't have to worry about AVI being a container format, which means it'll even play on all 21st century potatoes.
Incidentally, the latest Windows Update killed my 8k display. I wonder if it's related...
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For comparison, mCherry is at around 542,000 photons, while mScarlet-H (i.e. the same mutation as in mYongHong, but in mScarlet not mScarlet3) is at around 828,000.
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Usual caveats about using single exponentials to model photobleaching, and of comparing in vitro to cellular measurements apply! There's also no guarantee I've done my sums correctly, so you might want to double check (e.g. for mYongHong):
beryl.mrc-lmb.cam.ac.uk/calculators/...
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Note that we are considering both regular tenure track appointments and potentially also more junior appointees (~research fellows) in this recruitment round. Please share/apply/contact with any queries. Thanks! Greg.
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What is life without meaning?