Jon Daniels

Pan-ASLM: a high-resolution and large field-of-view light sheet microscope for Expansion Microscopy Expansion microscopy, a super-resolution fluorescence microscopy technique in which samples are expanded up to ∼8,000 times (after 20-fold expansion) their original volume, places high demands on the ...

BTW these objectives generally work for water/ExM though not specifically optimized for it. For instance the 54-12-8 -- NA 0.64 in water -- was used as the illumination objective for ASLM imaging of pan-ExM samples in www.biorxiv.org/content/10.1...

of course

In the past this (and the glycerol one) was available for purchase only with Olympus/Evident confocal systems, not to homebuilders. Not sure if they have changed their policy.

for cleared tissue see these four objectives with different NA/WD/FOV tradeoffs: asiimaging.com/docs/multi-i...

Note this would be a dipping objective and basically a regular microscope objective lens, compared to the specialized ExaSPIM lenses which are awesome but unconventional optically.

I have some thoughts in this direction... I'm thinking NA 0.75 or NA 0.8 with >10mm WD.

I'm hoping to talk with researchers at Seeing Is Believing and SfN (or by email/DM) and then start the detailed design.

Please reach out to me jon@asiimaging.com.

Here is what it will look like:

OK not sure why it's not posting as either png or jpg...

I like Empress but I don't want to be the arbiter of nicknames.

Official PN is 57-14-7 and it will be colloquially "AMS-AGY v3" on our website.

It's 10mm shorter than v2. OD is flared near the threads: 30mm OD for most and 37mm OD near the threads, whereas v2 was 35mm OD all the way.

Details here: bsky.app/profile/jsda...

Tagging @amsikking.bsky.social @andrewgyork.bsky.social @tanner-fadero.bsky.social @retof.bsky.social @loicaroyer.bsky.social and I'm sure I'm missing more but hopefully this will make the rounds...

High-res cellular imaging uses O1 > 1.0 with O2 being 40x/0.95 02. Short O2 WD means the grind has to sacrifice FOV in the depth axis, but FOV in the other direction (parallel the coverslip) is still 600um.

For larger samples e.g. zebrafish the grind position is wider to capture the entire FOV.

4. 55° grind so compatible with any-immersion concept.

5. Two different options for grind position: one for situations where O2 is NA ~0.95 and another for NA ~0.8. This one requires a bit more explanation.

A few nerdy details:

1. Generally better imaging quality especially for those pushing the FOV. I can send plots to those interested.

2. The image plane is a few microns off the glass tip to mitigate problem with dust.

3. Improved capture of high-angle rays despite slightly reduced NA of 0.99.

comparison table for v2 and v3

Production has started for a new #Snoutscope objective, AMS-AGY v3. It has significantly larger FOV and some other tweaks from v1 and v2. Currently accepting pre-orders. The price will need to increase modestly after deliveries start late October.

OK, makes sense. There is a loss in perfect 3D focusing away from the native focal plane described in the Botcherby paper and seemingly there in practice. Can that also be derived from this analytic approach?

Really cool! Could this be extended to focal planes other than the "native" one?

The equation from the paper is equivalent to 4.676 pixels per "Rayleigh diffraction limit square" (i.e. change that one number and my calculation lines up)

Even if you don't use all the "pixels" in such an objective, there is speed/brightness benefit to having such a large NA over such a large FOV.

Can you explain a bit more how you calculate it? I'm probably missing a factor.

FOV is π/4*(7200^2) in units of μm^2

Rayleigh diffraction limit is 0.61*λ/NA in units of μm

Can support 4 pixels per (diffraction limit)^2 (is this right?)

At 500nm: π/4*(7200^2)/(0.61*0.5/0.5)^2*4 = 438 MPix

I'm excited to be able to be in Zurich for the mesoSPIM symposium. This is a really fantastic project.

We're about to start sending quotes, should be delivered late summer / early fall. Happy to discuss via email.

v3 is coming which will functionally replace v2, and should be modestly better in a few respects. So no reason to make more v2, at least for now.

I'll have to think about that one

Correct. We have precision-thickness 1.5 coverglass AR-coated and that is what we cut to size. If the objective you are gluing to has a correction collar -- many do -- then it doesn't matter anyway.

We routinely see PSFs indistinguishable before/after gluing (flipping the coverslip that the beads are supported on).

We use silicone-based glue with working time of 10s of minutes but hardens overnight. It can be carefully peeled off the objective after hardening, but coverslip isn't recoverable.

(yes this is verbatim a Twitter post from 2022, but I've basically stopped using Twitter and wanted to be able to refer to it here)

Iterative leveling is guided by bead stacks: take stack of beads, inspect reliced images, poke again. It’s tedious but algorithmic once you determine which coma direction corresponds to which tilt direction. In our hands, once the coma is gone then other aberrations are gone too.

If you want to do this yourself the process we’ve come to is 1) cut AR-coated coverslip to size; 2) apply silicone glue around the edge of the objective tip; 3) place coverslip 4) level it by iteratively pushing the edge with a toothpick; 5) double-check with beads after curing.