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ref: -2021 tags: FIBSEM electron microscopy presynaptic plasticity activity Funke date: 10-12-2021 17:03 gmt revision:0 [head]

Ultrastructural readout of in vivo synaptic activity for functional connectomics

  • Anna Simon, Arnd Roth, Arlo Sheridan, Mehmet Fişek, Vincenzo Marra, Claudia Racca, View ORCID ProfileJan Funke, View ORCID ProfileKevin Staras, Michael Häusser
  • Did FIB-SEM on FM1-43 dye labeled synapses, then segmented the cells using machine learning, as Jan has pioneered.
    • FM1-43FX is membrane impermeable, and labels only synaptic vesicles that have been recycled after dye loading. (Invented in 1992!)
    • FM1-43FX is also able to photoconvert diaminobenzidene (DAB) into a amorphous highly conjugated polymer with high affinity for osmium tetroxide
  • This allows for a snapshot of ultrastructural presynaptic plasticity / activity.
  • N=84 boutons, but n=7 pairs / triples of boutons from the same axon.
    • These boutons have the same presynaptic spiking activity, and hence are expected to have the same release probability, and hence the same photoconversion (PC) labeling.
      • But they don't! The ratio of PC+ vesicle numbers between boutons on the same neuron is low, mean < 0.4, which suggests some boutons have high neurotransmitter release and recycling, others have low...
  • Quote in the abstract: We also demonstrate that neighboring boutons of the same axon, which share the same spiking activity, can differ greatly in their presynaptic release probability.
    • Well, sorta, the data here is a bit weak. It might all be lognormal fluctuations, as has been well demonstrated.
    • When I read it I was excited to think of the influence of presynaptic inhibition / modulation, which has not been measured here, but is likely to be important.

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ref: -2013 tags: microscopy space bandwidth product imaging resolution UCSF date: 06-17-2019 14:45 gmt revision:0 [head]

How much information does your microscope transmit?

  • Typical objectives 1x - 5x, about 200 Mpix!

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ref: -2019 tags: super-resolution microscopy fluorescent protein molecules date: 05-28-2019 16:02 gmt revision:3 [2] [1] [0] [head]

PMID-30997987 Chemistry of Photosensitive Fluorophores for Single-Molecule Localization Microscopy

  • Excellent review of all the photo-convertable, photo-switchable, and more complex (photo-oxidation or reddening) of both proteins and small molecule fluorophore.
    • E.g. PA-GFP is one of the best -- good photoactivation quantum yield, good N ~ 300
    • Other small molecules, like Alexa Fluor 647 have a photon yield > 6700, which can be increased with triplet quenchers and antioxidants.
  • Describes the chemical mechanism of the various photo switching -- review is targeted at (bio)chemists interested in getting into imaging.
  • Emphasize that critical figures of merit are photoactivation quantum yield Φ pa\Phi_{pa} and N, overall photon yield before photobleaching.
  • See also Colorado lecture

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ref: -0 tags: Airy light sheet microscopy attenuation compensation LSM imaging date: 02-19-2019 04:51 gmt revision:1 [0] [head]

Light-sheet microscopy with attenuation-compensated propagation-invariant beams

  • Ah ... beautiful illustration of the airy light sheet concept.
  • In practice, used a LCOS SLM to generate the beam (as .. phase matters!) plus an AOM to scan the beam.
    • Microscope can operate either in SPIM (single plane imaging microscope) or DSLM (digital scanning light sheet microscope),
  • Improves signal-to-background ratio (SBR) and contrast-to-noise ratio (CNR) (not sure why they don't use SNR..?)

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ref: -2017 tags: calcium imaging seeded iterative demixing light field microscopy mouse cortex hippocampus date: 02-13-2019 22:44 gmt revision:1 [0] [head]

PMID-28650477 Video rate volumetric Ca2+ imaging across cortex using seeded iterative demixing (SID) microscopy

  • Tobias Nöbauer, Oliver Skocek, Alejandro J Pernía-Andrade, Lukas Weilguny, Francisca Martínez Traub, Maxim I Molodtsov & Alipasha Vaziri
  • Cell-scale imaging at video rates of hundreds of GCaMP6 labeled neurons with light-field imaging followed by computationally-efficient deconvolution and iterative demixing based on non-negative factorization in space and time.
  • Utilized a hybrid light-field and 2p microscope, but didn't use the latter to inform the SID algorithm.
  • Algorithm:
    • Remove motion artifacts
    • Time iteration:
      • Compute the standard deviation versus time (subtract mean over time, measure standard deviance)
      • Deconvolve standard deviation image using Richardson-Lucy algo, with non-negativity, sparsity constraints, and a simulated PSF.
      • Yields hotspots of activity, putative neurons.
      • These neuron lcoations are convolved with the PSF, thereby estimating its ballistic image on the LFM.
      • This is converted to a binary mask of pixels which contribute information to the activity of a given neuron, a 'footprint'
        • Form a matrix of these footprints, p * n, S 0S_0 (p pixels, n neurons)
      • Also get the corresponding image data YY , p * t, (t time)
      • Solve: minimize over T ||YST|| 2|| Y - ST||_2 subject to T0T \geq 0
        • That is, find a non-negative matrix of temporal components TT which predicts data YY from masks SS .
    • Space iteration:
      • Start with the masks again, SS , find all sets O kO^k of spatially overlapping components s is_i (e.g. where footprints overlap)
      • Extract the corresponding data columns t it_i of T (from temporal step above) from O kO^k to yield T kT^k . Each column corresponds to temporal data corresponding to the spatial overlap sets. (additively?)
      • Also get the data matrix Y kY^k that is image data in the overlapping regions in the same way.
      • Minimize over S kS^k ||Y kS kT k|| 2|| Y^k - S^k T^k||_2
      • Subject to S k>=0S^k &gt;= 0
        • That is, solve over the footprints S kS^k to best predict the data from the corresponding temporal components T kT^k .
        • They also impose spatial constraints on this non-negative least squares problem (not explained).
    • This process repeats.
    • allegedly 1000x better than existing deconvolution / blind source segmentation algorithms, such as those used in CaImAn

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ref: -0 tags: serial electron microscopy Lichtman reconstruction nervous tissue date: 01-17-2017 23:32 gmt revision:0 [head]

PMID-26232230 Saturated Reconstruction of a Volume of Neocortex.

  • Data presented at Cell "Big Questions in Neuroscience", perhaps the most impressive of the talks.