Tanner Fadero

Playing around with how to represent volume in #Snouty light sheet data. Here is a dividing hiPSC with endogenously tagged histone in all three orthogonal projections. Bluer is further away. If anything, it distracts from politics.

Yes, there's a nice trick to compensating for transferring possession to the table from the crane. But there's also certain steps you can take before giving possession to the crane that decrease tension, leading to less severe shifts during future transfers. I'd love to walk you through them!

the anticipation is killing me

(I see nothing)

@jsdaniel02.bsky.social can make it happen!

@tanner-fadero.bsky.social told me there was a discussion about the unofficial name for #snouty V3. We have had Mr. Snouty and King Snouty, and there were people suggesting Emperor Snouty. I think it is time for a Lady or EMPRESS snouty. I have created Art to support this.

www.ascb.org/society-news...

What if we could use 'any' immersion objective and get good 3D imaging? Normally we can't as we must refractive index match the immersion to the sample (previous post).

-> However, I realized a few years ago that we can get good 3D imaging with any immersion using remote refocus optics!

But yeah, a modest tilt angle between projections looks similar!

Oh neat! You're looking at a projection from the "native" angle. Interestingly, the closer your sheet tilt angle is to 0°, the more similar the native projection is to the normal Z projection.

If that projection is from the deskew plug-in, then it's a true max intensity Z projection (where Z is O1's optical axis).

The deskew plug-in deskews AND rotates your data.

Some first raw (not deskewed) data of live human induced pluripotent stem cells from #Snouty now ready for some serious live imaging. Light sheet moving at a 38-degree angle through the cell layer; coverslip is on the bottom.

Bluesky now platform of choice for science community It’s not just you. Survey says: “Twitter sucks now and all the cool kids are moving to Bluesky”…

Bluesky is now the platform of choice for the scientific community.

From @jenlucpiquant.bsky.social on @arstechnica.com

arstechnica.com/science/2025...

Chroma said no, but thorlabs seems interested!

Optics folks: does anyone know a manufacturer of a dichroic beamsplitter *cube*?

Essentially, a dichroic version of this (for fluorescence microscopy):

Science hive mind!

Does anyone do hardware tech dev on MRI or CT? Or do you know someone who does? I'd love an introduction, if so. Let me know!

It's playing, you've just learned how to tune it out after so many builds

Also, this build doesn't actually take six months. I was mostly waiting on parts!

Here's a six-month time-lapse of me building the single objective light sheet fluorescence microscope at the UCSF Center for Advanced Light Microscopy!

Also pictured is @nicostuurman.bsky.social and @kahsage.bsky.social.

#snouty

You should get @jsdaniel02.bsky.social to help you add in a 808 laser!

I noticed that too! StayGold seems to have a very short triplet lifetime, suggesting that it's already doing a similar triplet-quenching mechanism to what is happening with infrared. StayGold should definitely be a first target for directed evolution. Imagine StayGold with infrared depletion!

Interesting! What's more interesting to me is not "what proteins does this work in?" and instead "can this be engineered with directed evolution?"

We got this effect in GFP by *accident*, and the primary sequence of the proteins seems to have huge influence over how strong this effect is.

In summary, for < $10,000 USD and less than a day's work, you can more than double the efficiency of your light sheet fluorescence microscope!

Unlike confocal, light sheet also doesn't need to deplete triplets outside of the focal plane, because light sheet doesn't *generate* out-of-plane triplets.

That brings us to light sheet fluorescence microscopy. Unlike widefield, it's easy to achieve the infrared intensity needed to reduce photobleaching in light sheet because the infrared laser is focused into a line. Here, we're getting a 2.5-fold reduction using a 200-mW 808-nm laser from Vortran.

So the total volumetric bleaching reduction in confocal microscopy will be far less than the two- to ten-fold that Ludvikova et al. demonstrated.

Infrared co-illumination in a confocal microscope will only remove triplets quickly enough from the in-focus plane. But confocal microscopy generates the same number of triplets at every plane!

The less time that green proteins spend in the triplet state, the less likely they are to bleach (and poison your organism)!

Infrared co-illumination works by selectively exciting green proteins in the triplet state, which converts them back to the singlet state more quickly. Triplets interact with dissolved oxygen and can donate their energy to create reactive oxygen species.