Structured light, deep imaging and automated super resolution

Morning everyone. Everyone is looking pretty fresh and ready for the day, and appear to have entirely dodged jetlag. I myself am dangerously full of caffeine and ready to go.

The lecture hall is filling up, with little biophysical interactions (ha) going on all around…we await the introduction by Jung-Chi Liao.

Jung-Chi asks out thanks for bringing all of the rain from across Asia – funny as it has been raining here for 4 days straight now [note – I ask someone later and this is not normal in Taipei!]. He reminds us of the upcoming Shilin night market and cultural tours plus the banquet at the end. A big thanks to the other organisers, particularly Wei-Chun.

Suliana Manley – Aesop’s fable by SIM

IMAG0173

Achilles Kapanidis and Suliana Manley 

And up first is Suliana Manley : she uses Super Resolution live microscopy to reveal that cell wall manipulating proteins are also responsible for changing cell size after division.

Suliana opens with the popular Aesop’s story the ‘tortoise and the hare’. She uses the story to describe the ability of individual bacteria to decide how big they should be, a bit like the hare, choosing his/her moment to race through the cell cycle.

The model is Caulobacter crescentus – and she shows us that population of such bacterial cells plateaus to become all the same size – the larger ones get smaller and the smaller get larger. With this in mind, she uses Structured Illumination Microscopy to address the question of how this happens.

Its known, says Suliana, that the divisome, the group of proteins that manage the construction and destruction of the peptidoglycan cell wall are the ones that also form a contractile ring when cells are ready to divide in two. By using SIM, which is really gentle technique, the group can watch several cell cycles and collect ample data while tagging these two proteins.

What’s interesting is that by treating the bacteria with a pedtidoglycan inhibitor (fosfomycin), the cells get longer (by decreasing the rate of pedtidoglycan synthesis). It seems therefore that the availability of peptidoglycan intermediates might be a way to control the size of such cells.

Bi-Chang Chen – lattice light sheet extends to localisation

A swift change then, after some questions, to Bi-Chang chen – well known for his work on the lattice light sheet in Eric Betzig’s lab.

As usual, the lattice images never cease to amaze. Using multiple Bessel beams and a spatial light modulator, the team have been able to image whole cells, and even whole brains – like that of the fly – 185 um maps. That’s because beam energy is spread, photodamage is reduced and signal to noise is maintained. The sample is moved up and down to achieve magnificent images.

Of most interest to me is the single molecular side of things. In particular, swept z stacks of sox2 transcription factors in live cells are impressive. I quite fancied that I could see ‘hop’ diffusion, certainly there were regions where the Sox2 molecules were clustering together in a very interesting consistent looking way. Bi-Chang also describes some new self blinking dyes suitable for super resolution localisation microscopy. This type of dye is probably going to be invaluable for live cell localisation: especially for nanocluster tracking.

Shean-Jen Chen: Adaptive optics in a new guise

Next up Shean-Jen Chen who is going to talk to us about the availability of his multi-photon, which has recently been revamped and outfitter with some sweet new adaptive optics that he calls ‘temporal focusing’.

It’s a multi-photon ‘scope but not as we know it. He’s made it fully biotissue compatible, in all of its messy, undulating glory.

First, he has used a grating to create multiphoton at the sample plane where the now separate wavelength beams converge. Second, he uses a Digital Micromirror Device (DMD) basically a plate with 1000s of tiny controllable mirrors, with which he can structure the light going onto the sample, to get to that high spatial frequency information: only one lens as this is non -linear. The effective resolution laterally is 168 nm and axially is 1.3 um.

Finally, the juicy bit – he uses a deformable mirror to compensate for temporal distortion. He basically measures how the light should look and corrects for deformation in the image. Biotissue has diverse refractive indices, so correcting for them with adaptive optics like this is desirable.

The result of all of that is high speed (30 cell volumes per second) and much nicer resolution, which will be invaluable in years to come, as the biologists dream up new avenues, some of which might be 6 cells deep!

IMAG0167.jpg

Takeharu Nagai and Yemima Riani

 

Masahiro Ueda: super resolution x 1000

OK so the final talk of this session comes from Masahiro Ueda. This one really stands out for me, something that has been in my mind for some time!

Masahiro has some very impressive statistics. That’s because he’s found a way to automate super resolution microscopy, as well as drug treatments of such cells. With this technique, we are looking at 1000s of cells per day – incredible: my record on the dSTORM at King’s is around 80 cells in one (long day). So this really is a big deal.

The set up involves a two 96 well plates, one full of chemicals or drugs, the other full of cells, which both go into a chamber with the microscope at 37 degrees, with 5 % CO2. The drugs are added to the cells at time points with a robot (and extensive washing – the video of this robot washing itself was strangely amusing to us, the jetlagged), while the cells are located using machine learning software, autofocusing (on the side of the aperture) and a quick fluorescence check to avoid unnecessary time wasting.

The example given here is the clear relationship between EGFR trimerization and an activation marker, the adaptor protein Grb2. The Grb2 proteins cluster and hover around the areas of no longer diffusing EGFR trimers. Very promising stuff.

Oh, and yes, they did also engineer an objective oil supply device for all of those stage shifts!

Phooph! That’s the end of that section *runs into the rain to reinvigorate*

Next up, mechanobiology!­

Advertisements

One thought on “Structured light, deep imaging and automated super resolution

  1. […] The first section was about imaging techniques, including structured illumination from Suliana Manley in bacteria, and full automation of super resolution/single molecule microscopy from Masahiro Ueda. Read all about it! […]

Leave a Reply

Fill in your details below or click an icon to log in:

WordPress.com Logo

You are commenting using your WordPress.com account. Log Out / Change )

Twitter picture

You are commenting using your Twitter account. Log Out / Change )

Facebook photo

You are commenting using your Facebook account. Log Out / Change )

Google+ photo

You are commenting using your Google+ account. Log Out / Change )

Connecting to %s