Optimization of multiphoton excitation microscopy by total emission detection using a parabolic light reflector

Combs, Christian A.; Смирнов, Александр; Riley, Jason; Gandjbakhche, Amir; Knutson, Jay R.; Balaban, Robert S.

doi:10.1111/j.1365-2818.2007.01851.x

Cited by 36 publications

(42 citation statements)

References 32 publications

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“…Vučinić et al suggest constructing a reflective shroud around the refractive lens components and predict that collection efficiency may be improved two to four-fold [20]. Combs et al take this idea one step further by implementing a parabolic reflector and second detector to achieve three times higher signal-to-noise than a standard 0.75 NA lens [21]. While these methods are promising, they rely on nonstandard components and restrict possible sample geometries.…”

Section: Resultsmentioning

confidence: 99%

Comparison of objective lenses for multiphoton microscopy in turbid samples

Singh¹,

McMullen²,

Doris³

et al. 2015

Biomed. Opt. Express

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Optimization of illumination and detection optics is pivotal for multiphoton imaging in highly scattering tissue and the objective lens is the central component in both of these pathways. To better understand how basic lens parameters (NA, magnification, field number) affect fluorescence collection and image quality, a two-detector setup was used with a specialized sample cell to separate measurement of total excitation from epifluorescence collection. Our data corroborate earlier findings that lowmag lenses can be superior at collecting scattered photons, and we compare a set of commonly used multiphoton objective lenses in terms of their ability to collect scattered fluorescence, providing guidance for the design of multiphoton imaging systems. For example, our measurements of epifluorescence beam divergence in the presence of scattering reveal minimal beam broadening, indicating that often-advocated over-sized collection optics are not as advantageous as previously thought. These experiments also provide a framework for choosing objective lenses for multiphoton imaging by relating the results of our measurements to various design parameters of the objectives lenses used.

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Section: Resultsmentioning

confidence: 99%

Comparison of objective lenses for multiphoton microscopy in turbid samples

Singh¹,

McMullen²,

Doris³

et al. 2015

Biomed. Opt. Express

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show abstract

“…The other systems in the literature do not use these successful strategies. [21][22][23] Our current microscope design relies on transmission geometry, rather than the conventional epi-fluorescence detection through the objective. While the conventional epiconfiguration is versatile for whole animal or large-tissue imaging experiments, its deep imaging capabilities are generally constrained to the 100 to 500-μm range.…”

Section: Deep-tissue Fluorescence Imagingmentioning

confidence: 99%

“…One strategy, termed total emission detection (TED), uses a parabolic mirror to reflect the "escaped" photons back to the detector and increase the signal/noise gain (roughly 10×) and imaging performance. 21,22 Alternatively, optical fibers can be used for fluorescence collection in imaging systems. 23 The relatively narrow acceptance angle of optical fibers limits their ability to collect fluorescence photons.…”

Section: Introductionmentioning

confidence: 99%

Deep tissue fluorescence imaging andin vivobiological applications

et al. 2012

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Abstract. We describe a novel technical approach with enhanced fluorescence detection capabilities in twophoton microscopy that achieves deep tissue imaging, while maintaining micron resolution. Compared to conventional two-photon microscopy, greater imaging depth is achieved by more efficient harvesting of fluorescence photons propagating in multiple-scattering media. The system maintains the conventional two-photon microscopy scheme for excitation. However, for fluorescence collection the detection system harvests fluorescence photons directly from a wide area of the turbid sample. The detection scheme relies on a wide area detector, minimal optical components and an emission path bathed in a refractive-index-matching fluid that minimizes emission photon losses. This detection scheme proved to be very efficient, allowing us to obtain high resolution images at depths up to 3 mm. This technique was applied to in vivo imaging of the murine small intestine (SI) and colon. The challenge is to image normal and diseased tissue in the whole live animal, while maintaining high resolution imaging at millimeter depth. In Lgr5-GFP mice, we have been successful in imaging Lgr5-eGFP positive stem cells, present in SI and colon crypt bases.

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“…One approach [14][15][16] describes the use of a parabolic mirror to reflect these missed photons back to detector and that may increase the signal gain by factor of 2 to 5. Alternatively, the additional collection of fluorescence photons by a ring of optical fibers, surrounding an objective, may enhance fluorescence collection efficiency by factor of 5 [17,18].…”

Section: Introductionmentioning

confidence: 99%

Imaging in turbid media: a transmission detector gives 2-3 order of magnitude enhanced sensitivity compared to epi-detection schemes

Dvornikov

Gratton

2016

Biomed. Opt. Express

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Imaging depth in turbid media by two-photon fluorescence microscopy depends on the ability of the optical system to detect weak fluorescence signals. We have shown that use of a wide area detector in transmission geometry allows increasing imaging depth in turbid media due to efficient photon collection. Compared to the conventional epi-detection scheme used in most commercial microscopes, the transmission detector was found to be 2-3 orders of magnitude more sensitive when used for in depth imaging in scattering samples simulating brain optical properties. 5390-5398 (2010). 20. V. Crosignani, A. S. Dvornikov, and E. Gratton, "Enhancement of imaging depth in turbid media using a wide area detector," J. Biophotonics 4(9), 592-599 (2011).

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Optimization of multiphoton excitation microscopy by total emission detection using a parabolic light reflector

Cited by 36 publications

References 32 publications

Comparison of objective lenses for multiphoton microscopy in turbid samples

Comparison of objective lenses for multiphoton microscopy in turbid samples

Deep tissue fluorescence imaging andin vivobiological applications

Imaging in turbid media: a transmission detector gives 2-3 order of magnitude enhanced sensitivity compared to epi-detection schemes

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