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ref: -0 tags: adaptive optics two photon microscopy date: 10-26-2021 18:17 gmt revision:1 [0] [head]

Recently I've been underwhelmed by the performance of adaptive optics (AO) for imaging head-fixed cranial-window mice. There hasn't been much of an improvement, despite significant optimization effort. This begs the question: where are AO microscopes used?

When the purpose of a paper is to explain and qualify an novel AO approach, the improvement is always good, >> 2x. Yet, in the one paper (first below) when the purpose was neuroscience, not optics, the results are less inspiring. Are the results from the optics papers cherry-picked?

Thalamus provides layer 4 of primary visual cortex with orientation- and direction-tuned inputs Wenzhi Sun, Zhongchao Tan, Brett D Mensh & Na Ji 2016 https://www.nature.com/articles/nn.4196

  • This is the primary (only?) paper where AO was used, but the focus was biology: measuring the tuning properties of thalamic boutons in mouse visual cortex. Which they did, well!
  • Surprisingly, the largest improvement was not from using AO, but rather from thinning the cranial window from 340um to 170um.
  • "With a 340-μm-thick cranial window, 70% of all boutons appeared to be non-responsive to visual stimuli and only 7% satisfied OS criteria. With a thinner cranial window of 170-μm thickness, we found that 31% of boutons satisfied OS criteria (of total n = 1,302, 5 mice), which was still substantially fewer than 48% OS boutons as determined when the same boutons (n = 1,477, 5 mice) were imaged after aberration correction by adaptive optics"

Direct wavefront sensing for high-resolution in vivo imaging in scattering tissue Kai Wang, Wenzhi Sun, Christopher T. Richie, Brandon K. Harvey, Eric Betzig & Na Ji, 2015 https://www.nature.com/articles/ncomms8276

  • Direct wavefront sensing using indocayanine green + Andor iXon 897 EMCCD Shack-Hartmann wavefront sensor (read: expensive).
  • Alpao DM97-15, basically the same as ours.
  • Fairly local wavefront corrections, see figure 2.
  • Also note that these wavefront corrections seem low-order, hence should be correctable via a DM

Multiplexed aberration measurement for deep tissue imaging in vivo Chen Wang, Rui Liu, Daniel E Milkie, Wenzhi Sun, Zhongchao Tan, Aaron Kerlin, Tsai-Wen Chen, Douglas S Kim & Na Ji 2014 https://www.nature.com/articles/nmeth.3068

  • Use a DMD (including a dispersion pre-compensator) to amplitude modulate phase ramps on a wavefront-modulating SLM. Each phase-ramp segment of the SLM was modulated at a different frequency, allowing for the optimal phase to be pulled out later through a Fourier transform.
  • Again, very good performance at depth in the mouse brain.

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ref: -2011 tags: two photon cross section fluorescent protein photobleaching Drobizhev gcamp date: 11-04-2020 18:07 gmt revision:9 [8] [7] [6] [5] [4] [3] [head]

PMID-21527931 Two-photon absorption properties of fluorescent proteins

  • Significant 2-photon cross section of red fluorescent proteins (same chromophore as DsRed) in the 700 - 770nm range, accessible to Ti:sapphire lasers ...
    • This corresponds to a S 0S nS_0 \rightarrow S_n transition
    • But but, photobleaching is an order of magnitude slower when excited by the direct S 0S 1S_0 \rightarrow S_1 transition (but the fluorophores can be significantly less bright in this regime).
      • Quote: the photobleaching of DsRed slows down by an order of magnitude when the excitation wavelength is shifted to the red, from 750 to 950 nm (32).
    • See also PMID-18027924
  • Further work by same authors: Absolute Two-Photon Absorption Spectra and Two-Photon Brightness of Orange and Red Fluorescent Proteins
    • " TagRFP possesses the highest two-photon cross section, σ2 = 315 GM, and brightness, σ2φ = 130 GM, where φ is the fluorescence quantum yield. At longer wavelengths, 1000–1100 nm, tdTomato has the largest values, σ2 = 216 GM and σ2φ = 120 GM, per protein chain. Compared to the benchmark EGFP, these proteins present 3–4 times improvement in two-photon brightness."
    • "Single-photon properties of the FPs are poor predictors of which fluorescent proteins will be optimal in two-photon applications. It follows that additional mutagenesis efforts to improve two-photon cross section will benefit the field."
  • 2P cross-section in both the 700-800nm and 1000-1100 nm range corresponds to the chromophore polarizability, and is not related to 1p cross section.
  • This can be useflu for multicolor imaging: excitation of the higher S0 → Sn transition of TagRFP simultaneously with the first, S0 → S1, transition of mKalama1 makes dual-color two-photon imaging possible with a single excitation laser wavelength (13)
  • Why are red GECIs based on mApple (rGECO1) or mRuby (RCaMP)? dsRed2 or TagRFP are much better .. but maybe they don't have CP variants.
  • from https://elifesciences.org/articles/12727

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ref: -0 tags: synaptic plasticity 2-photon imaging inhibition excitation spines dendrites synapses 2p date: 08-14-2020 01:35 gmt revision:3 [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 label 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: -2013 tags: 2p two photon STED super resolution microscope synapse synaptic plasticity date: 08-14-2020 01:34 gmt revision:3 [2] [1] [0] [head]

PMID-23442956 Two-Photon Excitation STED Microscopy in Two Colors in Acute Brain Slices

  • Plenty of details on how they set up the microscope.
  • Mice: Thy1-eYFP (some excitatory cells in the hippocampus and cortex) and CX3CR1-eGFP (GFP in microglia). Crossbred the two strains for two-color imaging.
  • Animals were 21-40 days old at slicing.

PMID-29932052 Chronic 2P-STED imaging reveals high turnover of spines in the hippocampus in vivo

  • As above, Thy1-GFP / Thy1-YFP labeling; hence this was a structural study (for which the high resolution of STED was necessary).
  • Might just as well gone with synaptic labels, e.g. tdTomato-Synapsin.

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ref: -0 tags: Na Ji 2p two photon fluorescent imaging pulse splitting damage bleaching date: 03-10-2020 21:44 gmt revision:6 [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)

  • In practice, a pulse picker is useful when power is limited and bleaching is not a problem (as is with GCaMP6x)

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ref: -0 tags: two photon scanning microscope mirror relay date: 01-31-2020 02:46 gmt revision:1 [0] [head]

PMID-24877017 Optimal lens design and use in laser-scanning microscopy

  • Detail careful design of a scanning two-photon microscope, with custom scan lens, tube lens, and standard 25x objective.
  • Near diffraction limited performance for both the scan and tube lenses across a broad excitation range -- 690 to 1400nm.
  • Interestingly, use a parabolic mirror relay to conjugate the two galvos to each other; seems like a good idea, why has this not been done elsewhere?

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ref: -2019 tags: non degenerate two photon excitation fluorophores fluorescence OPO optical parametric oscillator date: 10-31-2019 20:53 gmt revision:0 [head]

Efficient non-degenerate two-photon excitation for fluorescence microscopy

  • Used an OPO + delay line to show that non-degenerate (e.g. photons of two different energies) can induce greater fluorescence, normalized to input energy, than normal same-energy excitation.

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ref: -0 tags: ETPA entangled two photon absorption Goodson date: 09-24-2019 02:25 gmt revision:6 [5] [4] [3] [2] [1] [0] [head]

Can we image biological tissue with entangled photons?

How much fluorescence can we expect, based on reasonable concentrations & published ETPA cross sections?

Start with beer's law: A=σLN A = \sigma L N AA = absorbance; LL = sample length, 10 μm, 1e-3 cm; NN = concentration, 10 μmol; σ\sigma = cross-section, for ETPA assume 2.4e18cm 2/molec2.4e-18 cm^2 / molec (this is based on a FMN based fluorophore; actual cross-section may be higher). Including Avogadro's number and 1l=1000cm 31 l = 1000 cm^3 , A=1.45e5A = 1.45e-5

Now, add in quantum efficiency ϕ=0.8\phi = 0.8 (Rhodamine); collection efficiency η=0.2\eta = 0.2 ; and an incoming photon pair flux of I=1e12photons/sec/modeI = 1e12 photons / sec / mode (which roughly about the limit for quantum behavior; n = 0.1 photons / mode; will add this calculation).

F=ϕησLNI=2.3e6photons/secF = \phi \eta \sigma L N I = 2.3e6 photons/sec This is very low, but within practical imaging limits. As a comparison, incoherent 2p imaging creates ~ 100 photons per pulse, of which 10 make it to the detector; for 512 x 512 pixels at 15fps, the dwell time on each pixel is 20 pulses of a 80 MHz Ti:Sapphire laser, or ~ 200 photons.

Note the pair flux is per optical mode; for a typical application, we'll use a Nikon 16x objective with a 600 μm Ø FOV and 0.8 NA. At 800 nm imaging wavelength, the diffraction limit is 0.5 μm. This equates to about 7e57e5 addressable modes in the FOV. Then an illumination of 1e121e12 photons / sec / mode equates to 7e177e17 photons over the whole field; if each photon pair has an energy of 2.75eV,λ=450nm2.75 eV, \lambda = 450 nm , this is equivalent to 300 mW. 100mW is a reasonable limit, hence scale incoming flux to 2.3e172.3e17 pairs /sec.

Hence, the imaging mode is power limited, and not quantum limited (if you could get such a bright entangled source). And right now that's the limit -- for a BBO crystal, circa 1998 experimenters were getting 1e4 photons / sec / mW. So, 2.3e172.3e17 pairs / sec would require 23 GW. Yikes.

More efficient entangled sources have been developed, using periodically-poled potassium titanyl phosphate (PPPTP), which (again assuming linearity) puts the power requirement at 23 MW. This is within the reason of q-switched lasers, but still incredibly inefficient. The down-conversion process is not linear in intensity, which is why Goodson pumps with SHG from a Ti:sapphire to yield ~1e7 photons; but this of induces temporal correlations which increase the frequency of incoherent TPA.

Still, combining PPPTP with a Ti:sapphire laser could result in 1e13 photons / sec, which is sufficient for scanned microscopy. Since the laser is pulsed, it will still be subject to incoherent TPA; but that's OK, the point is to reduce the power going into the animal via larger ETPA cross-section. The answer to above is a tentative yes. Upon the development of brighter entangled sources (e.g. arrays of quantum structures), this can move to fully widefield imaging.

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ref: -0 tags: ETPA entangled two photon absorption Goodson date: 09-19-2019 15:49 gmt revision:13 [12] [11] [10] [9] [8] [7] [head]

Various papers put out by the Goodson group:

And from a separate group at Northwestern:

  • Entangled Photon Resonance Energy Transfer in Arbitrary Media
    • Suggests three orders of magnitude improvement in cross-section relative to incoherent TPA.
    • In SPDC, photon pairs are generated randomly and usually accompanied by undesirable multipair emissions.
      • For solid-state artificial atomic systems with radiative cascades (singled quantum emitters like quantum dots), the quantum efficiency is near unity.
    • Paper is highly mathematical, and deals with resonance energy transfer (which is still interesting)

Regarding high fluence sources, quantum dots / quantum structures seem promising.

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ref: -2017 tags: two photon holographic imaging Arch optogenetics GCaMP6 date: 09-12-2019 19:24 gmt revision:1 [0] [head]

PMID-28053310 Simultaneous high-speed imaging and optogenetic inhibition in the intact mouse brain.

  • Bovetti S1, Moretti C1, Zucca S1, Dal Maschio M1, Bonifazi P2,3, Fellin T1.
  • Image GCamp6 in either scanned mode (high resolution, slow) or holographically (SLM, redshirt 80x80 NeuroCCD, activate opsin Arch, simultaneously record juxtasomal action potentials.

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ref: -2017 tags: neuromorphic optical computing nanophotonics date: 06-17-2019 14:46 gmt revision:5 [4] [3] [2] [1] [0] [head]

Progress in neuromorphic photonics

  • Similar idea as what I had -- use lasers as the optical nonlinearity.
    • They add to this the idea of WDM and 'MRR' (micro-ring resonator) weight bank -- they don't talk about the ability to change the weihts, just specify them with some precision.
  • Definitely makes the case that III-V semiconductor integrated photonic systems have the capability, in MMACs/mm^2/pj, to exceed silicon.

See also :

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ref: -0 tags: nanophotonics interferometry neural network mach zehnder interferometer optics date: 06-13-2019 21:55 gmt revision:3 [2] [1] [0] [head]

Deep Learning with Coherent Nanophotonic Circuits

  • Used a series of Mach-Zehnder interferometers with thermoelectric phase-shift elements to realize the unitary component of individual layer weight-matrix computation.
    • Weight matrix was decomposed via SVD into UV*, which formed the unitary matrix (4x4, Special unitary 4 group, SU(4)), as well as Σ\Sigma diagonal matrix via amplitude modulators. See figure above / original paper.
    • Note that interfereometric matrix multiplication can (theoretically) be zero energy with an optical system (modulo loss).
      • In practice, you need to run the phase-moduator heaters.
  • Nonlinearity was implemented electronically after the photodetector (e.g. they had only one photonic circuit; to get multiple layers, fed activations repeatedly through it. This was a demonstration!)
  • Fed network FFT'd / banded recordings of consonants through the network to get near-simulated vowel recognition.
    • Claim that noise was from imperfect phase setting in the MZI + lower resolution photodiode read-out.
  • They note that the network can more easily (??) be trained via the finite difference algorithm (e.g. test out an incremental change per weight / parameter) since running the network forward is so (relatively) low-energy and fast.
    • Well, that's not totally true -- you need to update multiple weights at once in a large / deep network to descend any high-dimensional valleys.

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ref: -0 tags: 3D SHOT Alan Hillel Waller 2p photon holography date: 05-31-2019 22:19 gmt revision:4 [3] [2] [1] [0] [head]

PMID-29089483 Three-dimensional scanless holographic optogenetics with temporal focusing (3D-SHOT).

  • Pégard NC1,2, Mardinly AR1, Oldenburg IA1, Sridharan S1, Waller L2, Adesnik H3,4
  • Combines computer-generated holography and temporal focusing for single-shot (no scanning) two-photon photo-activation of opsins.
  • The beam intensity profile determines the dimensions of the custom temporal focusing pattern (CTFP), while phase, a previously unused degree of freedom, is engineered to make 3D holograph and temporal focusing compatible.
  • "To ensure good diffraction efficiency of all spectral components by the SLM, we used a lens Lc to apply a small spherical phase pattern. The focal length was adjusted so that each spectral component of the pulse spans across the short axis of the SLM in the Fourier domain".
    • That is, they spatially and temporally defocus the pulse to better fill the SLM. The short axis of the SLM in this case is Y, per supplementary figure 2.
  • The image of the diffraction grating determines the plane of temporal focusing (with lenses L1 and L2); there is a secondary geometric focus due to Lc behind the temporal plane, which serves as an aberration.
  • The diffraction grating causes the temporal pattern to scan to produce a semi-spherical stimulated area ('disc').
  • Rather than creating a custom 3D holographic shape for each neuron, the SLM is after the diffraction grating -- it imposes phase and space modulation to the CTFP, effectively convolving it with a holograph of a cloud of points & hence replicating at each point.

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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: -0 tags: optical neural recording photon induced electron transfer date: 01-02-2013 04:25 gmt revision:2 [1] [0] [head]

PMID-22308458 Optically monitoring voltage in neurons by photo-induced electron transfer through molecular wires.

  • Photoinduced electron transfer.
    • About what you would think -- a photon bumps an electron into a higher orbital, and this electron can be donated to another group or drop back down & fluoresce a photon.
  • Good sensitivity: ΔF/F\Delta F/F of 20-27% per 100mV, fast kinetics.
  • Not presently genetically targetable.
  • Makes sense in terms of energy: "A 100-mV depolarization changes the PeT driving force by 0.05 eV (one electron × half of 100-mV potential, or 0.05 V). Because PeT is a thermally controlled process, the value of 0.05 eV is large relative to the value of kT at 300 K (0.026 eV), yielding a large dynamic range between the rates of PeT at resting and depolarized potentials.
  • Why electrochromic dyes have plateaued:
    • "In contrast, electrochromic dyes have smaller delta G values, 0.003 (46) to 0.02 (47) eV, and larger comparison energies. Because the interaction is a photochemically controlled process, the energy of the exciting photon is the comparison energy, which is 1.5–2 eV for dyes in the blue-to-green region of the spectrum. Therefore, PeT and FRET dyes have large changes in energy versus their comparison energy (0.05 eV vs. 0.026 eV), giving high sensitivities; electrochromic dyes have small changes compared with the excitation photon (0.003–0.02 eV vs. 2 eV), producing low voltage sensitivity."

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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]