Ultrawidefield microscope for high-speed fluorescence imaging and targeted optogenetic stimulation

Werley, Christopher A.; Chien, Miao‐Ping; Cohen, Adam E.

doi:10.1364/boe.8.005794

Cited by 89 publications

(97 citation statements)

References 48 publications

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“…Large-aperture array: The combination of a large pixel pitch and high pixel count leads to large device sizes, in our case an active array area of 30.7 x 30.7 mm. Although this necessitates larger optics in the system, some photostimulation setups are already moving to low-magnification high-NA imaging that require microscope objectives with back apertures of 30 mm or more [13][14][15] . Because a SLM is typically imaged onto a microscope objective's back aperture, a closer match between SLM and microscope objective aperture means less (or even zero) magnification change, and fewer of the aberrations associated with magnification change.…”

Section: Stabilitymentioning

confidence: 99%

Designing a new spatial light modulator for holographic photostimulation

Shane

McKnight

Hill

et al. 2019

Optical Trapping and Optical Micromanipulation XVI

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Driven by the demands for speed and field of view in the holographic photostimulation community, we designed, built, and tested a liquid crystal on silicon (LCoS) spatial light modulator (SLM) with a 1536x1536 square pixel array and high-voltage LC drive. We discuss some of the engineering work that made the MacroSLM possible, including the custom FPGA board for handling huge data rates, the large pixel size for minimizing rolloff and crosstalk, and the temperature control to handle heating effects from the high-voltage controls and high-power laser illumination. We also designed an FPGA implementation of the overdrive method for increasing liquid crystal switching speed, allowing us to overcome the significant data bottlenecks that limit frame rates for large arrays. We demonstrate 500 Hz hologram-tohologram speed at 1064 nm operating wavelength, and discuss the new science that these speeds and array sizes have enabled.

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Section: Stabilitymentioning

confidence: 99%

Designing a new spatial light modulator for holographic photostimulation

Shane

McKnight

Hill

et al. 2019

Optical Trapping and Optical Micromanipulation XVI

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show abstract

“…By patterning the illumination light, one can activate or suppress specific neural ensembles in order to map neural circuits, investigate neural dynamics, or attempt to draw links between specific patterns of neural activity and behavior [3][4][5][6][7][8] . Groups have developed both onephoton 3,4,[9][10][11] and two-photon photostimulation approaches 5,[12][13][14][15] . Two-photon photostimulation has the advantage of high spatial resolution for precise neural circuit control and relative immunity to light scattering from tissue, enabling control over deep brain circuits.…”

Section: Introductionmentioning

confidence: 99%

Three-dimensional Multi-site Random Access Photostimulation (3D-MAP)

Xue

Waller

Adesnik

et al. 2020

Preprint

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AbstractHigh precision optical control of neural ensemble activity is essential for understanding brain function and may ultimately revolutionize the treatment of brain disease. Currently only multiphoton holographic optogenetics achieves the requisite spatial resolution for precise neural network control. However, it suffers from critical drawbacks that limit the number of addressable neurons and prevent the development of implantable or wireless devices. Therefore, achieving high-resolution optogenetic control with one-photon activation is essential to catalyze a dramatic leap in spatiotemporally precise multi-site optogenetic technology for research and clinical use. To overcome this challenge, we developed a new light sculpting technique that can synthesize custom illumination patterns in 3D by rapidly projecting structured illumination patterns along with novel computational methods for precise optogenetic control, termed ‘3D-MAP’, for Three-dimensional Multi-site random Access Photostimulation. This technology enables the optogenetic synthesis of complex spatiotemporal sequences of neural activity in the intact brain. As a one-photon photostimulation technology, 3D-MAP can be widely adopted for custom optogenetic applications by the neuroscience research community, and opens the door to scalable, wireless high precision optical brain interfaces.

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“…Cells were imaged in Tyrodes Buffer (10mM HEPES, 125mM NaCl, 2mM KCl, 3mM CaCl 2 , 1mM MgSO 4 , 30mM Glucose, pH 7.40) in the presence of synaptic blockers (10μM NBQX (Sigma), 25μM D-AP5 (Tocris) and 20μM Gabazine (Sigma) to block AMPA, NMDA and GABA currents, respectively) to allow for measurements of intrinsic spontaneous and evoked neuronal activity. Optopatch imaging was performed on a custom built, ultra-wide field fluorescence microscope described previously (38,73). Briefly, samples were illuminated with ∼100 W/cm 2 635 nm laser excitation to monitor changes in membrane potential through changes in QuasAr3 fluorescence.…”

Section: Methodsmentioning

confidence: 99%

Impaired M-current in KCNQ2 Encephalopathy Evokes Dyshomeostatic Modulation of Excitability

Simkin¹,

Searl²,

Piyevsky³

et al. 2019

Preprint

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35Mutations in KCNQ2, which encodes a pore-forming K + channel subunit responsible for 36 neuronal M-current, cause neonatal epileptic encephalopathy, a complex disorder presenting 37 with severe early-onset seizures and impaired neurodevelopment. The condition is exceptionally 38 difficult to treat, partially because the effects of KCNQ2 mutations on the development and 39 function of human neurons are unknown. Here, we used induced pluripotent stem cells and gene 40 editing to establish a disease model, and measured the functional properties of patient-derived 41 neurons using electrophysiological and optical approaches. We find that while patient-derived 42 excitatory neurons exhibit reduced M-current early, they develop intrinsic and network 43 hyperexcitability progressively. This hyperexcitability is associated with faster action potential 44 repolarization, larger afterhyperpolarization, and a functional enhancement of large conductance 45 Ca 2+ -activated K + (BK) channels. These properties facilitate a burst-suppression firing pattern 46 that is reminiscent of the interictal electroencephalography pattern in patients. Importantly, we 47 were able to phenocopy these excitability features in control neurons only by chronic but not 48 acute pharmacological inhibition of M-current. Our findings suggest that dyshomeostatic 49 mechanisms compound KCNQ2 loss-of-function and lead to alterations in the 50 neurodevelopmental trajectory of patient-derived neurons. Our work has therapeutic implications 51 in explaining why KCNQ2 agonists are not beneficial unless started at an early disease stage. 52 53 KEYWORDS 54 KCNQ2, Kv7.2, epileptic encephalopathy, human induced pluripotent stem cells, excitatory 55 neurons, M-current, epilepsy, dyshomeostatic and homeostatic plasticity, burst firing, disease 56 modeling 57 58 59 60 61 62 63 64 65 66 92However, some variants associated with severe clinical phenotypes produce gain-of-function 93 effects (23,24). Enhanced K + conductance, specifically in the AIS, could hyperpolarize the AIS 94 membrane, decreasing steady state inactivation for sodium channels. This would increase the 95 rate of action potential activation and action potential repolarization (25). 96 The mechanisms by which developmental expression of KCNQ2 channels impact 97 neuronal excitability are not clear. What remains elusive is how the defects in M-current affect 98 the electrophysiological properties of human neurons leading to impaired neurodevelopment. 99 The use of patient-specific induced pluripotent stem cell (iPSC) technology has enabled a new 100 4 approach for elucidating pathogenic mechanisms of genetic disorders such as the epileptic 101 channelopathies as it allows for the generation of otherwise inaccessible human neurons (26-102 28). Here, we use KCNQ2-NEE patient-specific and isogenic control iPSC-derived excitatory 103 neurons to elucidate the dynamic functional effects of a prototypical KCNQ2 mutation during 104 differentiation and maturation in culture.

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Ultrawidefield microscope for high-speed fluorescence imaging and targeted optogenetic stimulation

Cited by 89 publications

References 48 publications

Designing a new spatial light modulator for holographic photostimulation

Designing a new spatial light modulator for holographic photostimulation

Three-dimensional Multi-site Random Access Photostimulation (3D-MAP)

Impaired M-current in KCNQ2 Encephalopathy Evokes Dyshomeostatic Modulation of Excitability

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