Efficient large core fiber-based detection for multi-channel two-photon fluorescence microscopy and spectral unmixing

Ducros, Mathieu; Hoff, Marcel van ‘t; Evrard, Alexis; Seebacher, Christian; Schmidt, Elke; Charpak, Serge; Oheim, Martin

doi:10.1016/j.jneumeth.2011.03.015

Cited by 19 publications

(20 citation statements)

References 35 publications

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“…Color shifts also arise because illumination strength is generally uneven across the imaging field and differs among lasers. This effect can be attenuated by intensity or shading correction for each channel 46, 49 . The resulting composite RGB images provide maximum color separation for viewing by eye (Fig.…”

Section: Resultsmentioning

confidence: 99%

Improved tools for the Brainbow toolbox

et al. 2013

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In a recently introduced transgenic multicolor labeling strategy called “Brainbow,” Cre-lox recombination is used to create a stochastic choice of expression among fluorescent proteins (XFPs), resulting in the indelible marking of mouse neurons with multiple, distinct colors. This method has been adapted to non-neuronal cells in mice and to neurons in fish and flies, but has yet to realize its full potential in the mouse brain. Here, we present several new lines of mice that overcome limitations of the initial lines and an adaptation of the method for use in adeno-associated viral (AAV) vectors. We also provide technical advice about how best to image Brainbow transgenes.

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Section: Resultsmentioning

confidence: 99%

Improved tools for the Brainbow toolbox

et al. 2013

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“…Others have shown that significant gains in collection efficiency can be realized through efficient back aperture expansion systems that capture light originating from outside the point of geometric focus, thus increasing the effective N.A. of collection (Ducros et al , 2011, Zinter & Levene, 2011). The large dichroic mirror in the nosepiece and light collection system of the c-TED arrangement has already realized gains akin to those revealed by Ducross et al 2011 and Zinter and Levine et al 2011 under our “control” conditions, without the parabola contribution (PMT aperture masking data not shown).…”

Section: Discussionmentioning

confidence: 99%

“…of collection (Ducros et al , 2011, Zinter & Levene, 2011). The large dichroic mirror in the nosepiece and light collection system of the c-TED arrangement has already realized gains akin to those revealed by Ducross et al 2011 and Zinter and Levine et al 2011 under our “control” conditions, without the parabola contribution (PMT aperture masking data not shown). Thus, our quoted gain factors for parabola addition are versus the ‘objective alone’ – but the latter includes the enhanced N.A.…”

Section: Discussionmentioning

confidence: 99%

Compact non‐contact total emission detection for in vivo multiphoton excitation microscopy

Combs

Смирнов

Karamzadeh

et al. 2013

Journal of Microscopy

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Summary We describe a compact, non-contact design for a Total Emission Detection (c-TED) system for intra-vital multi-photon imaging. To conform to a standard upright two-photon microscope design, this system uses a parabolic mirror surrounding a standard microscope objective in concert with an optical path that does not interfere with normal microscope operation. The non-contact design of this device allows for maximal light collection without disrupting the physiology of the specimen being examined. Tests were conducted on exposed tissues in live animals to examine the emission collection enhancement of the c-TED device compared to heavily optimized objective-based emission collection. The best light collection enhancement was seen from murine fat (5×-2× gains as a function of depth), while murine skeletal muscle and rat kidney showed gains of over two and just under two-fold near the surface, respectively. Gains decreased with imaging depth (particularly in the kidney). Zebrafish imaging on a reflective substrate showed close to a two-fold gain throughout the entire volume of an intact embryo (approximately 150 μm deep). Direct measurement of bleaching rates confirmed that the lower laser powers (enabled by greater light collection efficiency) yielded reduced photobleaching in vivo. The potential benefits of increased light collection in terms of speed of imaging and reduced photo-damage, as well as the applicability of this device to other multi-photon imaging methods is discussed.

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“…1), and projected onto a PMT (R6357-SELECT; Hamamatsu) as previously described in ref. 44. A near infrared blocking filter (E700 sp 2ph; Chroma Technologies) suppressed scattered laser light.…”

Section: Methodsmentioning

confidence: 99%

Encoded multisite two-photon microscopy

Ducros

Houssen

Bradley

et al. 2013

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

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The advent of scanning two-photon microscopy (2PM) has created a fertile new avenue for noninvasive investigation of brain activity in depth. One principal weakness of this method, however, lies with the limit of scanning speed, which makes optical interrogation of action potential-like activity in a neuronal network problematic. Encoded multisite two-photon microscopy (eMS2PM), a scanless method that allows simultaneous imaging of multiple targets in depth with high temporal resolution, addresses this drawback. eMS2PM uses a liquid crystal spatial light modulator to split a highpower femto-laser beam into multiple subbeams. To distinguish them, a digital micromirror device encodes each subbeam with a specific binary amplitude modulation sequence. Fluorescence signals from all independently targeted sites are then collected simultaneously onto a single photodetector and site-specifically decoded. We demonstrate that eMS2PM can be used to image spike-like voltage transients in cultured cells and fluorescence transients (calcium signals in neurons and red blood cells in capillaries from the cortex) in depth in vivo. These results establish eMS2PM as a unique method for simultaneous acquisition of neuronal network activity.multipoint | multiplexing | voltage-sensitive dyes | scanless two-photon microscopy A persistent and challenging demand in neuroscience is the ability to monitor activity from defined populations of cellular targets in depth in the brain. Imaging with two-photon microscopy (2PM) of cells labeled with calcium-sensitive reporters (1, 2) has become the most popular approach to indirectly report spikes and yields micrometer-scale spatial resolution in vivo (3) to depths up to 1,000 μm (4). Conventional 2PM uses relatively slow scanning mechanisms, however, which do not permit the simultaneous acquisition of multiple millisecond-range signals emitted by cellular ensembles. In the last several years various attempts have been made to overcome this drawback. For example, using a mirror-based targeted path scanning technique that drastically reduced the background scanning time, Lillis et al. monitored the dynamics of spatially extended neuronal networks (5). Another approach consisted of using acousto-optic deflectors to steer the laser beam in less than 10 μs between cells or subcellular sites of interest (6-9). Both of these sequential scanning methods, however, suffer from the fundamental trade-off between signal-tonoise ratio (SNR) and fast temporal resolution (which should be maximized for detection of fast events such as spikes). Increasing the SNR typically requires integrating more photons per pixel, which can be achieved by increasing either the excitation laser intensity or the pixel dwell time. Regarding the former, there is a ceiling beyond which the average laser power cannot be increased (2.5-10 mW), typically referred to as the photo-damage limit (10, 11). Regarding the latter, decreasing the scanning rate will increase photon count but with the cost of lowering temporal resolution, ultim...

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Efficient large core fiber-based detection for multi-channel two-photon fluorescence microscopy and spectral unmixing

Cited by 19 publications

References 35 publications

Improved tools for the Brainbow toolbox

Improved tools for the Brainbow toolbox

Compact non‐contact total emission detection for in vivo multiphoton excitation microscopy

Encoded multisite two-photon microscopy

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