Photon counting, censor corrections, and lifetime imaging for improved detection in two-photon microscopy

Driscoll, Jonathan D.; Shih, Andy Y.; Iyengar, Satish; Field, Jeffrey J.; White, G.; Squier, Jeffrey A.; Cauwenberghs, Gert; Kleinfeld, David

doi:10.1152/jn.00649.2010

Cited by 39 publications

(30 citation statements)

References 50 publications

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“…Animals were imaged (32) using a custom twophoton microscope (33) that employed photon counting detection (34) and was controlled by MPScan software (35). We further used a 10× 0.…”

Section: Methodsmentioning

confidence: 99%

Fluctuating and sensory-induced vasodynamics in rodent cortex extend arteriole capacity

Drew

Shih

Kleinfeld

2011

Proc. Natl. Acad. Sci. U.S.A.

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266

388

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Neural activity in the brain is followed by localized changes in blood flow and volume. We address the relative change in volume for arteriole vs. venous blood within primary vibrissa cortex of awake, head-fixed mice. Two-photon laser-scanning microscopy was used to measure spontaneous and sensory evoked changes in flow and volume at the level of single vessels. We find that arterioles exhibit slow (<1 Hz) spontaneous increases in their diameter, as well as pronounced dilation in response to both punctate and prolonged stimulation of the contralateral vibrissae. In contrast, venules dilate only in response to prolonged stimulation. We conclude that stimulation that occurs on the time scale of natural stimuli leads to a net increase in the reservoir of arteriole blood. Thus, a "bagpipe" model that highlights arteriole dilation should augment the current "balloon" model of venous distension in the interpretation of fMRI images.ocalized changes in the flow and volume of oxygenated blood in the brain are commonly used as a correlate of heightened neural activity. For two important imaging modalities, blood oxygen level-dependent functional magnetic resonance imaging (BOLD fMRI) (1) and intrinsic optical signal imaging (IOS) (2), the signals are generated by a complex interplay of the rate of oxidative metabolism, the flux of blood in the underlying angioarchitecture, and changes in vascular volume (3). The locus for the increase in vascular volume that follows sensory stimulation (i.e., arterioles or venules) is an enduring controversy that bears directly on interpreting and quantifying fMRI signals (4-6). To resolve this question, we used in vivo two-photon laserscanning microscopy to image spontaneous and sensory evoked vascular dynamics in the vibrissa area of parietal cortex of awake, head-fixed mice. All data were collected through a reinforced thin-skull window (7) (Fig. 1A); this method obviates potential complications from inflammation or changes in cranial pressure that may occur with a craniotomy (8). ResultsImages of the pial surface and measurements of the diameter of the lumen of surface arterioles and venules were performed while the mouse sat passively (Fig. 1B). Dilations greater than 5% of the baseline diameter occurred with frequencies of 0.07 ± 0.05 Hz (mean ± SD, n = 118 arteries in six mice). The diameters of short segments of arterioles (red in Fig. 1B) exhibited relatively large spontaneous increases in the spectral range between 0.1 Hz and 1 Hz ( Fig. 1 C and D). The peak amplitude of these fluctuations was 23% ± 10% of the initial vessel diameter across all arterioles, with instances of a 50% increases in diameter. Further, these lowfrequency oscillations were strongly coherent and synchronous over a distance of several hundred micrometers across cortex (198 pairs of vessels, in six mice, that were separated by 20-315 μm; slope of coherence = 0.001 μm −1 [nonsignificant (NS)] and slope of phase shift = 0.0009 rad/μm

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“…Animals were imaged (32) using a custom twophoton microscope (33) that employed photon counting detection (34) and was controlled by MPScan software (35). We further used a 10× 0.…”

Section: Methodsmentioning

confidence: 99%

Fluctuating and sensory-induced vasodynamics in rodent cortex extend arteriole capacity

Drew

Shih

Kleinfeld

2011

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

266

388

View full text Add to dashboard Cite

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“…Photo currents were amplified using a 700 MHz pulse amplifier (ZPUL-30P; MiniCircuits), the output of which was digitized through a comparator (PRL-350TTL; Pulse Research Lab) (Driscoll et al, 2011). The threshold for the comparator was calibrated slightly above the dark current elicited by the PMT.…”

Section: In Vivo Fiber-optic Photometrymentioning

confidence: 99%

Central Amygdala Somatostatin Neurons Gate Passive and Active Defensive Behaviors

et al. 2016

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The central amygdala (CeA) has a key role in learning and expression of defensive responses. Recent studies indicate that somatostatinexpressing (SOM ϩ ) neurons in the lateral division of the CeA (CeL) are essential for the acquisition and recall of conditioned freezing behavior, which has been used as an index of defensive response in laboratory animals during Pavlovian fear conditioning. However, how exactly these neurons participate in fear conditioning and whether they contribute to the generation of defensive responses other than freezing remain unknown. Here, using fiber-optic photometry combined with optogenetic and molecular techniques in behaving mice, we show that SOM ϩ CeL neurons are activated by threat-predicting sensory cues after fear conditioning and that activation of these neurons suppresses ongoing actions and converts an active defensive behavior to a passive response. Furthermore, inhibition of these neurons using optogenetic or molecular methods promotes active defensive behaviors. Our results provide the first in vivo evidence that SOM ϩ neurons represent a CeL population that acquires learning-dependent sensory responsiveness during fear conditioning and furthermore reveal an important role of these neurons in gating passive versus active defensive behaviors in animals confronted with threat.

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“…PMTs provide an excellent balance of quantum efficiency, response time, wavelength sensitivity, and linearity. Unfortunately, PMTs do not discriminate between multiplying the signal and the noise and thus multiply the uncertainty due to the shot effect ([340,341], as cited by Driscoll et al [342]). Additionally, dark current or thermal noise—the signal produced by the PMT in the absence of any signal light—is also multiplied by the dynode chain and degrades the SNR of the measurements.…”

Section: Photon-counting Detectionmentioning

confidence: 99%

“…Quantum efficiency is a ratio of the number of photons incident on the PMT that produce an electron-hole pair to the total number of incident photons. Additionally, it is important to consider the rise time of the PMT’s electronics as, during this window, any detection of additional photons is censored [342,343]. …”

Section: Photon-counting Detectionmentioning

confidence: 99%

A pragmatic guide to multiphoton microscope design

et al. 2015

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Multiphoton microscopy has emerged as a ubiquitous tool for studying microscopic structure and function across a broad range of disciplines. As such, the intent of this paper is to present a comprehensive resource for the construction and performance evaluation of a multiphoton microscope that will be understandable to the broad range of scientific fields that presently exploit, or wish to begin exploiting, this powerful technology. With this in mind, we have developed a guide to aid in the design of a multiphoton microscope. We discuss source selection, optical management of dispersion, image-relay systems with scan optics, objective-lens selection, single-element light-collection theory, photon-counting detection, image rendering, and finally, an illustrated guide for building an example microscope.

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Photon counting, censor corrections, and lifetime imaging for improved detection in two-photon microscopy

Cited by 39 publications

References 50 publications

Fluctuating and sensory-induced vasodynamics in rodent cortex extend arteriole capacity

Fluctuating and sensory-induced vasodynamics in rodent cortex extend arteriole capacity

Central Amygdala Somatostatin Neurons Gate Passive and Active Defensive Behaviors

A pragmatic guide to multiphoton microscope design

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