Doug Shepherd
recovering alpinist and scientist.
- We're still exploring how to improve LSFM performance with "big" detection air objectives, where field curvature often limits achievable FOV. Here, Steven introduces curvedASLM, a method to dynamically correct field curvature (along one axis) in axially scanned setups www.biorxiv.org/content/10.6...
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- Thanks! Your benchtop mesoSPIM paper was very helpful!! Confirmed a lot of what we were seeing and gave us the kick to finish this off.
- There are a lot of nice papers dealing with field curvature in LSFM, including work that from @nvladimus.bsky.social, @adamkglaser.bsky.social, @aakinwo.bsky.social, and others whose bluesky handles I don't know.
- Additionally, @adamezracohen.bsky.social group's paper that used this beast of an objective for widefield was very helpful when thinking about how to optimize the light path to retain as much performance as possible.
- If you've seen anyone from the lab talk about RNA-FISH over the past years, they've shown data somehow related to this preprint. After a wandering road writing it up, happy to put out some thoughts on scalable 3D imaging-based spatial transcriptomics data processing. www.biorxiv.org/content/10.1...
- Special thanks to @moffittlab.bsky.social for a lot of help and advice along the way! More to come soon on speeding up the experimental side of large scale 3D spatial omics.
- We are really enjoying having Nick Galati visit us on sabbatical for some fun protist work, including our first forays into expansion microscopy! Max projection xy&xz views of expanded paramecium and tetrahymena cells. Scale bar is ~5.5 um accounting for expansion. Epifluorescence + deconvolution.
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- We tried your FEP chamber setup and the results are great with an Olympus 60X/NA 1.0 water dipping and U-ExM ciliate samples. Thanks for putting it out there!
- @ptbrown1729.bsky.social and the rest of the team's work on high-speed optical diffraction tomography plus structured illumination microscopy is finally out! www.science.org/doi/10.1126/...
- Accompanied by a nice perspective piece from our colleague Lei Tian, whose own work on high-information content computational imaging was a strong inspiration for our own. www.science.org/doi/10.1126/...
- first data from the new LLSM in PertzLab @olivierpertz.bsky.social! i always thought nuclei were spherical but they are pancakes???? so much more to see in 3d, but data volume produced is insane, we will have to rethink our analysis pipelines. tips welcome! here shown in @napari.org
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- Tensorstore is much faster for read/write than vanilla zarr. Tools are here: github.com/QI2lab/opm-p... Currently we only support our custom pymmcore-plus OPM format and ASI’s OPM format. Happy to work with you to support CZI LLSM files if you open an issue and provide some example data.
- We have a bunch of fast skewed data handling tools (flatfield, decon, deskew, stitch) in Python, but they are not meant for real-time lazy viewing - we do that in our own custom acq. software. We use Tensorstore to write with compression at reasonably fast speeds.
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- We use a number of FLIR and Basler cameras with the high end Sony CMOS chips. For short exposures, they can work well. Another thing to look out for is that the hot pixels fluctuate on top of the true dead pixels. We correct a static map and then do a second correction that looks for outliers.
- Our experience is the dark current (no cooling) and dead pixels (quality control) are higher than your standard sCMOS. We've mapped the pixel by pixel gain for a few different industrial CMOS chips and often find some non-linearities.
- Spending some time at #lsfm25 cleaning up code. It now has distributed GPU gradient-consensus RL decon (@jamesdmanton.bsky.social @andrewgyork.bsky.social) our fast orthogonal deskew, and stitching/fusion to ome-ngff v0.5. Example AO-OPM data on uncleared mouse brain slice github.com/QI2lab/opm-p...
- Steven has our Snouty-OPM running again after our lab move. He added projection mode, sensorless AO, and a new control GUI based on @talley.codes + crew's pymmcore-plus/pymmcore-gui. Here are some IF-labeled microglia (orange) in the middle of an uncleared 60um mouse brain slice. No deconvolution!
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- Thanks! We use a Nikon 100x/1.35 silicone oil for O1. The objectives in the setup are still the same as our previous Snouty-OPM papers.
- As usual, all the code is developed in the open at our github. Control: github.com/QI2lab/opm-v2 and processing: github.com/QI2lab/opm-p.... The control code is fully hardware triggered and pretty performant already. We are actively working on speeding parts of the processing (fusion to ome-zarr).
- Out of nowhere, our institute just froze all indirect accounts - no matter when or where the indirects were generated. With completely unclear direction on how this works going forward. No words…
- In a brief respite from current events, I got to see a California Condor for the first time while running in the Grand Canyon this weekend! What an amazing animal. Unfortunately, public lands and those who protect them are also targets of this admin. Keeping fighting…
- I have spent many hours over the past two days speaking with reporters from major outlets, linking them to public and not-so-public documents, and connecting them with other folks. I hope there will be lots coming out soon.
- Thank you!
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- Super cool! Would be fun to play with at some point.
- My partner told me today many community health centers (she works at one) may need to declare bankruptcy if the Federal funding freeze is allowed to continue. If you aren't familiar with CHCs, here is a good overview: www.kff.org/medicaid/iss...
- CHCs served roughly 31 million people in 2023, 90% of which were at or below 200% the federal poverty level. That includes roughly 9.1 million pediatric patients. Despite the court orders and the executive branch's claim that Medicaid is not effected, their Medicaid funding portals remain frozen.
- This is yet another reason (in addition to MANY others) to call your representatives and senators. Likely more is going to be required, but it's a starting point.
- A nice treat in this craziness. Ayush’s work on SIM reconstructions using physical noise modeling is out: www.nature.com/articles/s44... The low signal (avg of 16 photons/pixel) reconstructions, achieved without deep learning, are quite impressive in my opinion. We’re working on a faster GPU version
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- Yeah, lead spheres and torsion bar suspended by 20 micron diameter tungsten wire. It's a really nice setup, but tricky for them to get right because of how long it takes to settle down before you can take data.
- Fixed the Cavendish experiment for our second semester advanced lab, fun with gravity!
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- Finally got the promised lab space >5 years after I uprooted the lab from Colorado…so a bit of column A and a bit of column B?
- Having just rebuilt due to a lab move, I also vote for two galvo scanning for easier alignment.
- We just got asked (aka told) to pause or cancel any HHS grant funded travel. Time to scramble to find flexible funds to send people places...
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- A number of us as graduate students were involved in one in condensed matter physics. Nearly all of us (across many institutions) bailed from that field, but managed to stay in science somehow. I don’t think it moved science forward and just created a lot of hard feelings.
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- I got an e-bike a few years ago to help deal with the Arizona summers and added work/grocery panniers. >6k miles later, I only use a car for longer trips. One of the best investments I've ever made.
- Anyone successfully dealt with chronic, at times crippling, facilities issues? It has really impacted the team's productivity, even though it's not their fault. I'm open to any and all advice beyond moving spaces. We already did that twice in 5 years with new (broken) promises each time.
- A bit of good fun at the gym helps to lighten up an insane week…
- Nick Galati (wp.wwu.edu/galatilab/) and I are recruiting a postdoc. The project is to study organelle distribution across protists with wildly different swimming strategies using bench work, expansion microscopy, and advanced light sheet microscopes. Full ad to come, please reach out if curious!
- This position will be part of the ASU Center for Biological Physics, ASU Center for Mechanisms of Evolution, and an NSF Biological Integration Institute - with plans to spend some time at Western Washington University working with the Galati lab on ciliates from the Salish Sea!
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- Oof, looks familiar!
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- We are happy with the interleaved stabilization approach we currently use, if we build a new OPM we’ll definitely try it out.
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- Yes, the drift is almost all temperature for us. We recently moved labs and things are much better now. We are doing some long experiments where we need to keep the instrument stable for week or more.
- Nice! We detect a shuttered 532 nm beam reflected off the back of the dichroic with the main camera, but have to stop fluorescence lasers and run the correction in between acquisition volumes. The continuous feedback in this approach is very elegant.
- Nice morning run up Mt. Lowe in the San Gabriel mountains while visiting family. I love how many people, including families, get out and use these relatively steep trails in the Pasadena area. #running #trailrunning
- It’s not all science all the time! Beautiful late Fall run in the Grand Canyon today. #trailrunning #running
- We haven't imaged big samples in a few years. Recently Steven brought our custom ASLM, with some fun/interesting detection optics, back to life. Tatz Murakami graciously sent us a beautifully cleared and vasculature labeled mouse brain for imaging. Fused max projections of raw data.
- Paired with getting the instrument running again (he's writing that up now), Steven recently published some thoughts on modeling and optimizing remote focus pathways with large RI mismatches (opg.optica.org/oe/fulltext....)
- Today's adventure in academia. How many layers of IT do I need to contact to reset the fancy new Zoom TV system they put in our conference room. I'm betting greater than ten, but there are a still a few hours left in the day!
- Fluorescence Friday! 3D timelapse of MOLM-14 cells expressing GFP-LC3-RFP embedded in collagen. Orthoview rendering of GFP (cyan) and RFP (orange) imaged every 30 seconds using our Snouty-OPM.
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- Can you please add me? Thanks!
- Slowly getting things settled in our (finally!) renovated labs. Part of the beam path in our multimodal setup for multicolor 2D-SIM and kilohertz 3D QPI.
- It's woefully incomplete, but I've made the start of a starter pack for folks interested in microscopy. Suggestions for who else to include (including yourself!) are very welcome. go.bsky.app/SaYfAX3at://did:plc:5t5g4zki76auoldxeo7wkfxt/app.bsky.graph.starterpack/3l4jewk43z32o
- I would like to added please!
