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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: -0 tags: synaptic plasticity 2-photon imaging inhibition excitation spines dendrites synapses 2p date: 05-31-2019 23:02 gmt revision:2 [1] [0] [head]

PMID-22542188 Clustered dynamics of inhibitory synapses and dendritic spines in the adult neocortex.

  • Cre-recombinase-dependent labeling of postsynapitc scaffolding via Gephryn-Teal fluorophore fusion.
  • Also added Cre-eYFP to lavel the neurons
  • Electroporated in utero e16 mice.
    • Low concentration of Cre, high concentrations of Gephryn-Teal and Cre-eYFP constructs to attain sparse labeling.
  • Located the same dendrite imaged in-vivo in fixed tissue - !! - using serial-section electron microscopy.
  • 2230 dendritic spines and 1211 inhibitory synapses from 83 dendritic segments in 14 cells of 6 animals.
  • Some spines had inhibitory synapses on them -- 0.7 / 10um, vs 4.4 / 10um dendrite for excitatory spines. ~ 1.7 inhibitory
  • Suggest that the data support the idea that inhibitory inputs maybe gating excitation.
  • Furthermore, co-inervated spines are stable, both during mormal experience and during monocular deprivation.
  • Monocular deprivation induces a pronounced loss of inhibitory synapses in binocular cortex.

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ref: -0 tags: Na Ji 2p two photon fluorescent imaging pulse splitting damage bleaching date: 05-31-2019 19:55 gmt revision:5 [4] [3] [2] [1] [0] [head]

PMID-18204458 High-speed, low-photodamage nonlinear imaging using passive pulse splitters

  • Core idea: take a single pulse and spread it out to N=2 kN= 2^k pulses using reflections and delay lines.
  • Assume two optical processes, signal SI αS \propto I^{\alpha} and photobleaching/damage DI βD \propto I^{\beta} , β>α>1\beta \gt \alpha \gt 1
  • Then an NN pulse splitter requires N 11/αN^{1-1/\alpha} greater average power but reduces the damage by N 1β/α.N^{1-\beta/\alpha}.
  • At constant signal, the same NN pulse splitter requires N\sqrt{N} more power, consistent with two photon excitation (proportional to the square of the intensity: N pulses of N/N\sqrt{N}/N intensity, 1/N per pulse fluorescence, Σ1\Sigma \rightarrow 1 overall fluorescence.)
  • This allows for shorter dwell times, higher power at the sample, lower damage, slower photobleaching, and better SNR for fluorescently labeled slices.
  • Examine the list of references too, e.g. "Multiphoton multifocal microscopy exploiting a diffractive optical element" (2003)

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ref: -0 tags: phosphorescence fluorescence magnetic imaging slicing adam cohen date: 05-29-2019 19:41 gmt revision:8 [7] [6] [5] [4] [3] [2] [head]

A friend postulated using the triplet state phosphorescence as a magnetically-modulatable dye. E.g. magnetically slice a scattering biological sample, rather than slicing optically (light sheet, 2p) or mechanically. After a little digging:

I'd imagine that it should be possible to design a molecule -- a protein cage, perhaps a (fully unsaturated) terpine -- which isolates the excited state from oxygen quenching.

Adam Cohen at Harvard has been working a bit on this very idea, albeit with fluorescence not phosphorescence --

  • Optical imaging through scattering media via magnetically modulated fluorescence (2010)
    • The two species, pyrene and dimethylaniline are in solution.
    • Dimethylaniline absorbs photons and transfers an electron to pyrene to produce a singlet radical pair.
    • The magnetic field represses conversion of this singlet into a triplet; when two singlet electrons combine, they produce exciplex fluorescence.
  • Addition of an aliphatic-ether 12-O-2 linker improves things significantly --
  • Mapping Nanomagnetic Fields Using a Radical Pair Reaction (2011)
  • Which can be used with a 2p microscope:
  • Two-photon imaging of a magneto-fluorescent indicator for 3D optical magnetometry (2015)
    • Notably, use decay kinetics of the excited state to yield measurements that are insensitive to photobleaching, indicator concentration, or local variations in optical excitation or collection efficiency. (As opposed to ΔF/F\Delta F / F )
    • Used phenanthrene (3 aromatic rings, not 4 in pyrene) as the excited electron acceptor, dimethylaniline again as the photo-electron generator.
    • Clear description:
      • A molecule with a singlet ground state absorbs a photon.
      • The photon drives electron transfer from a donor moiety to an acceptor moiety (either inter or intra molecular).
      • The electrons [ground state and excited state, donor] become sufficiently separated so that their spins do not interact, yet initially they preserve the spin coherence arising from their starting singlet state.
      • Each electron experiences a distinct set of hyperfine couplings to it's surrounding protons (?) leading to a gradual loss of coherence and intersystem crossing (ISC) into a triplet state.
      • An external magnetic field can lock the precession of both electrons to the field axis, partially preserving coherence and supressing ISC.
      • In some chemical systems, the triplet state is non-fluorescence, whereas the singlet pair can recombine and emit a photon.
      • Magnetochemical effects are remarkable because they arise at a magnetic field strengths comparable to hyperfine energy (typically 1-10mT).
        • Compare this to the Zeeman effect, where overt splitting is at 0.1T.
    • phenylanthrene-dimethylaniline was dissolved in dimethylformamide (DMF). The solution was carefully degassed in nitrogen to prevent molecular oxygen quenching.

Yet! Magnetic field effects do exist in solution:

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ref: -2019 tags: three photon imaging visual cortex THG chirp NOPA mice GCaMP6 MIT date: 03-01-2019 18:46 gmt revision:2 [1] [0] [head]

PMID-30635577 Functional imaging of visual cortical layers and subplate in awake mice with optimized three photon microscopy

  • Murat Yildirim, Hiroki Sugihara, Peter T.C. So & Mriganka Sur'
  • Used a fs Ti:Saphirre 16W pump into a non-colinear optical parametric amplifier (both from Spectra-Physics) to generate the 1300nm light.
  • Used pulse compensation to get the pulse width at the output of the objective to 40 fS.
    • Three-photon cross section is inverse quadratic in pulse width:
    • NP 3δ(τR) 2(NA 22hcλ) 3 N \sim \frac{P^3 \delta}{(\tau R)^2} (\frac{NA^2}{2hc\lambda})^3
    • P is power, δ\delta is 3p cross-section, τ\tau is pulse width, R repetition rate, NA is the numerical aperture (sixth power of NA!!!), h c and λ\lambda Planks constant, speed of light, and wavelength respectively.
  • Optimized excitation per depth by monitoring damage levels. varied from 0.5nJ to 5 nJ.
  • Imaged up to 1.5mm deep! All the way to the white matter / subplate.
  • Allegedly used a custom scan and tube lens to minimize aberrations in the excitation path (hence improve 3p excitation)
  • Layer 5 neurons are more broadly tuned for orientation than other layers. But the data is not dramatic.
  • Used straightforward metrics for tuning, using a positive and negative bump gaussian fit, then vector averaging to get global orientation selectivity.
  • Interesting that the variance between layers seems higher than between mice.

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ref: -2015 tags: CWEETS amplified Fourier imaging raman amplification date: 02-19-2019 06:46 gmt revision:1 [0] [head]

Amplified dispersive Fourier-Transform Imaging for Ultrafast Displacement sensing and Barcode Reading

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ref: -2011 tags: HiLo speckle imaging confocal boston university optical sectioning date: 02-19-2019 06:18 gmt revision:2 [1] [0] [head]

PMID-21280920 Optically sectioned in vivo imaging with speckle illumination HiLo microscopy

  • Ah, brilliant! Illuminate a sample with a speckle pattern from a laser, and use this to optically section the data -- the contrast of the speckle pattern shows how in focus the sample is.
    • Hanece, the contrast indicates the in-focus vs out-of-focus ratio in a region.
  • The speckle statistics are invariant even in a scattering media, as scattering only further randomizes an already random laser phase front. (Within some limits.)
  • HiLo microscopy involves illuminating with a speckle pattern, then illuminating with standard uniform illumination, resulting in a diffraction-limited optically sectioned image. PMID-18709098
  • Algorithm is :
    • Take the speckle image and subtract the uniform image δI\delta I
    • Bandpass δI\delta I
    • Measure the standard deviation of the δI\delta I to get a weighting function C δs 2C^2_{\delta s}
    • Debias this estimate based on sensor..
    • Generate low-passed image from the weighted uniform image, LP[C δsI u] LP[C_{\delta s} I_u] , and high-pass from the difference HP=1LPHP = 1 - LP
    • Resultand image is a weighted sum of highpassed and lowpassed images.
  • Looks about as good as confocal.
  • Cited by...

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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 >= 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: superresolution imaging scanning lens nanoscale date: 02-04-2019 20:34 gmt revision:1 [0] [head]

PMID-27934860 Scanning superlens microscopy for non-invasive large field-of-view visible light nanoscale imaging

  • Recently, the diffraction barrier has been surpassed by simply introducing dielectrics with a micro-scale spherical configuration when using conventional optical microscopes by transforming evanescent waves into propagating waves. 18,19,20,21,22,23,24,25,26,27,28,29,30
  • The resolution of this superlens-based microscopy has been decreased to ∼50 nm (ref. 26) from an initial resolution of ∼200 nm (ref. 21).
  • This method can be further enhanced to ∼25 nm when coupled with a scanning laser confocal microscope 31.
  • It has achieved fast development in biological applications, as the sub-diffraction-limited resolution of high-index liquid-immersed microspheres has now been demonstrated23,32, enabling its application in the aqueous environment required to maintain biological activity.
  • Microlens is a 57 um diameter BaTiO3 microsphere, resolution of lambda / 6.3 under partial and inclined illumination
  • Microshpere is in contact with the surface during imaging, by gluing it to the cantilever tip of an AFM.
  • Get an image with the microsphere-lens, which improves imaging performance by ~ 200x. (with a loss in quality, naturally).

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ref: -0 tags: photoacoustic tomography mouse imaging q-switched laser date: 05-11-2017 05:23 gmt revision:1 [0] [head]

Single-impulse panoramic photoacoustic computed tomography of small-animal whole-body dynamics at high spatiotemporal resolution

  • Used Q-switched Nd:YAG and Ti:Sapphire lasers to illuminate mice axially (from the top, through a diffuser and conical lens), exciting the photoacuostic effect, from which they were able to image at 125um resolution a full slice of the mouse.
    • I'm surprised at their mode of illumination -- how do they eliminate the out-of-plane photoacoustic effect?
  • Images look low contrast, but structures, e.g. cortical vasculature, are visible.
  • Can image at the rep rate of the laser (50 Hz), and thereby record cardiac and pulmonary rhythms.
  • Suggest that the photoacoustic effect can be used to image brain activity, but spatial and temporal resolution are limited.

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ref: -0 tags: photoacoustic tomography mouse imaging q-switched laser date: 05-11-2017 05:21 gmt revision:0 [head]

Single-impulse panoramic photoacoustic computed tomography of small-animal whole-body dynamics at high spatiotemporal resolution

  • Used Q-switched Nd:YAG and Ti:Sapphire lasers to illuminate mice axially, exciting the photoacuostic effect, from which they were able to image at 125um resolution a full slice of the mouse.
  • Images look low contrast, but structures, e.g. cortical vasculature, are visible.
  • Can image at the rep rate of the laser (50 Hz), and thereby record cardiac and pulmonary rhythms.
  • Suggest that the photoacoustic effect can be used to image brain activity, but spatial and temporal resolution are limited.

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ref: -0 tags: super resolution imaging PALM STORM fluorescence date: 09-21-2016 05:57 gmt revision:0 [head]

PMID-23900251 Parallel super-resolution imaging

  • Christopher J Rowlands, Elijah Y S Yew, and Peter T C So
  • Though this is a brief Nature intro article, I found it to be more usefully clear than the wikipedia articles on super-resolution techniques.
  • STORM and PALM seek to stochastically switch fluorophores between emission and dark states, and are parallel but stochastic; STED and RESOLFT use high-intensity donut beams to stimulate emission (STED) or photobleach (RESOLFT) fluorophores outside of an arbitrarily-small location.
    • All need gaussian-fitting to estimate emitter location from the point-spread function.
  • This article comments on a clever way of making 1e5 donuts for parallel (as opposed to rastered) STED / RESOLFT.
  • I doubt stetting up a STED microscope is at all easy; to get these resolutions, everything must be still to a few nm!

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ref: -0 tags: Anna Roe optogenetics artificial dura monkeys intrinisic imaging date: 09-30-2013 19:08 gmt revision:3 [2] [1] [0] [head]

PMID-23761700 Optogenetics through windows on the brain in nonhuman primates

  • technique paper.
  • placed over the visual cortex.
  • Injected virus through the artificial dura -- micropipette, not CVD.
  • Strong expression:
  • See also: PMID-19409264 (Boyden, 2009)

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ref: -0 tags: brain micromotion magnetic resonance imaging date: 01-28-2013 01:38 gmt revision:0 [head]

PMID-7972766 Brain and cerebrospinal fluid motion: real-time quantification with M-mode MR imaging.

  • Measured brain motion via a clever MR protocol. (beyond my present understanding...)
  • ventricles move at up to 1mm/sec
  • In the Valsava maneuver the brainstem can move 2-3mm.
  • Coughing causes upswing of the CSF.

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ref: -0 tags: optical imaging neural recording diamond magnetic date: 01-02-2013 03:44 gmt revision:0 [head]

PMID-22574249 High spatial and temporal resolution wide-field imaging of neuron activity using quantum NV-diamond.

  • yikes: In this work we consider a fundamentally new form of wide-field imaging for neuronal networks based on the nanoscale magnetic field sensing properties of optically active spins in a diamond substrate.
  • Cultured neurons.
  • NV = nitrogen-vacancy defect centers.
    • "The NV centre is a remarkable optical defect in diamond which allows discrimination of its magnetic sublevels through its fluorescence under illumination. "
    • We show that the NV detection system is able to non-invasively capture the transmembrane potential activity in a series of near real-time images, with spatial resolution at the level of the individual neural compartments.
  • Did not actually perform neural measurements -- used a 10um microwire with mA of current running through it.
    • I would imagine that actual neurons have far less current!

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ref: -0 tags: neural imaging recording shot noise redshirt date: 01-02-2013 02:20 gmt revision:0 [head]


  • Shot Noise: The limit of accuracy with which light can be measured is set by the shot noise arising from the statistical nature of photon emission and detection.
    • If an ideal light source emits an average of N photons/ms, the RMS deviation in the number emitted is N\sqrt N .
    • At high intensities this ratio NN\frac{N}{\sqrt N} is large and thus small changes in intensity can be detected. For example, at 10^10 photons/ms a fractional intensity change of 0.1% can be measured with a signal-to-noise ratio of 100.
    • On the other hand, at low intensities this ratio of intensity divided by noise is small and only large signals can be detected. For example, at 10^4 photons/msec the same fractional change of 0.1% can be measured with a signal-to-noise ratio of 1 only after averaging 100 trials.

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ref: Grutzendler-2011.09 tags: two-photon imaging in-vivo neurons recording dendrites spines date: 01-03-2012 01:02 gmt revision:3 [2] [1] [0] [head]

PMID-21880826[0] http://cshprotocols.cshlp.org/content/2011/9/pdb.prot065474.full?rss=1

  • Excellent source of information and references. Go CSH!
  • Possible to image up to 400um deep. PMID-12490949[1]
  • People have used TPLSM imaging for years in mice. PMID-19946265[2]


[0] Grutzendler J, Yang G, Pan F, Parkhurst CN, Gan WB, Transcranial two-photon imaging of the living mouse brain.Cold Spring Harb Protoc 2011:9, no Pages (2011 Sep 1)
[1] Grutzendler J, Kasthuri N, Gan WB, Long-term dendritic spine stability in the adult cortex.Nature 420:6917, 812-6 (2002 Dec 19-26)
[2] Yang G, Pan F, Gan WB, Stably maintained dendritic spines are associated with lifelong memories.Nature 462:7275, 920-4 (2009 Dec 17)

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ref: notes-0 tags: two-photon laser imaging fluorescence lifetime imaging FRET GFP RFP date: 01-21-2008 17:23 gmt revision:0 [head]