Simultaneous time- and spectrum-resolved multifocal multiphoton microscopy

Liu, L.; Qu, Junle; Lin, Ziyang; Wang, L.; Fu, Zhe; Guo, Baoping; Niu, Hanben

doi:10.1007/s00340-006-2314-y

Cited by 26 publications

(15 citation statements)

References 16 publications

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“…The majority of current single beam confocal applications use mirror galvanometers to achieve raster scanning of the focal spot [4,5,24,29]. A pair of orthogonal mirror galvanometers are used to tilt the trajectory of a light beam along two axes.…”

Section: Mirror Galvanometer Scanningmentioning

confidence: 99%

“…Since only a single pixel photon detector and associated readout electronics are needed, this approach allows for the use of a very sensitive photo detection system with low noise at relatively low cost [2]. Single detection modules is particularly attractive in specific modalities such as time-resolved fluorescence [3] or spectroscopy [4,5]. For instance, the cost and complexity of time-resolved imaging can be greatly reduced by using time-correlated single photon counting (TCSPC) methods [4,5].…”

Section: Introductionmentioning

confidence: 99%

“…Single detection modules is particularly attractive in specific modalities such as time-resolved fluorescence [3] or spectroscopy [4,5]. For instance, the cost and complexity of time-resolved imaging can be greatly reduced by using time-correlated single photon counting (TCSPC) methods [4,5]. Point-scanning is also critical in other imaging modalities, such as multiphoton microscopy and stimulated emission depletion (STED) microscopy [6,7].…”

Section: Introductionmentioning

confidence: 99%

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High-speed multifocal array scanning using refractive window tilting

Tsikouras

Berman²,

Andrews

et al. 2015

Biomed. Opt. Express

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Confocal microscopy has several advantages over wide-field microscopy, such as out-of-focus light suppression, 3D sectioning, and compatibility with specialized detectors. While wide-field microscopy is a faster approach, multiplexed confocal schemes can be used to make confocal microscopy more suitable for high-throughput applications, such as high-content screening (HCS) commonly used in drug discovery. An increasingly powerful modality in HCS is fluorescence lifetime imaging microscopy (FLIM), which can be used to measure protein-protein interactions through Förster resonant energy transfer (FRET). FLIM-FRET for HCS combines the requirements of high throughput, high resolution and specialized time-resolving detectors, making it difficult to implement using wide-field and spinning disk confocal approaches. We developed a novel foci array scan method that can achieve uniform multiplex confocal acquisition using stationary lenslet arrays for high resolution and high throughput FLIM. Unlike traditional mirror galvanometers, which work in Fourier space between scan lenses, this scan method uses optical flats to steer a 2-dimension foci array through refraction. After integrating this scanning scheme in a multiplexing confocal FLIM system, we demonstrate it offers clear benefits over traditional mirror galvanometer scanners in scan linearity, uniformity, cost and complexity. 277-296 (2015). 27. T. Nielsen, M. Fricke, D. Hellweg, and P. Andresen, "High efficiency beam splitter for multifocal multiphoton microscopy," J.

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Section: Mirror Galvanometer Scanningmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

High-speed multifocal array scanning using refractive window tilting

Tsikouras

Berman²,

Andrews

et al. 2015

Biomed. Opt. Express

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“…1,2 Multifocal multiphoton microscopy (MMM) which was developed in the late 20th century, can improve the utilization ratio of excitation light energy and achieve video-rate imaging speed at high spatial resolution through the use of microlens array, [3][4][5][6] cascaded beam splitters parallel processing ability of optical system to excite the sample and detect°uorescence emission from multiple foci at the same time. MMM also has almost all the advantages of multiphoton microscopy, such as inherent optical sectioning and less photobleaching and photodamage to the out-offocus sample.…”

Section: Introductionmentioning

confidence: 99%

Monte Carlo Simulation of Multifocal Stochastic Scanning System

Liu

Qian

et al. 2014

J. Innov. Opt. Health Sci.

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Multifocal multiphoton microscopy (MMM) has greatly improved the utilization of excitation light and imaging speed due to parallel multiphoton excitation of the samples and simultaneous detection of the signals, which allows it to perform three-dimensional fast°uorescence imaging. Stochastic scanning can provide continuous, uniform and high-speed excitation of the sample, which makes it a suitable scanning scheme for MMM. In this paper, the graphical programming language -LabVIEW is used to achieve stochastic scanning of the two-dimensional galvo scanners by using white noise signals to control the x and y mirrors independently. Moreover, the stochastic scanning process is simulated by using Monte Carlo method. Our results show that MMM can avoid oversampling or subsampling in the scanning area and meet the requirements of uniform sampling by stochastically scanning the individual units of the N Â N foci array. Therefore, continuous and uniform scanning in the whole¯eld of view is implemented.

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“…Using a set of 3×3 array sampling images and reconstructed with a dedicated software, we reconstructed the spectrally resolved streak image of fluorescent microspheres (Invitrogen, c-14837) [22] . Fig.5 (a-d) show four reconstructed two-photon excitation fluorescence and lifetime images at different central wavelengths, 470, 530, 590 and 650 nm, with 60 nm spectral bandwidth respectively.…”

mentioning

confidence: 99%

Time-resolved two-photon excitation fluorescence spectroscopy and microscopy using a high repetition rate streak camera

Liu

Lin

et al. 2007

Optoelectron. Lett.

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We present a time-resolved two-photon excitation fluorescence spectroscopy and a simultaneous time-and spectrumresolved multifocal multiphoton microscopy system that is based on a high repetition rate picosecond streak camera for providing time-and spectrum-resolved measurement and imaging in biomedicine. The performance of the system is tested and characterized by the fluorescence spectrum and lifetime analysis of several standard fluorescent dyes and their mixtures. Spectrum-resolved fluorescence lifetime images of fluorescence beads are obtained. Potential applications of the system include clinical diagnostics and cell biology etc. DOIFluorescence spectroscopy and microscopy have been applied widely in biomedicine and some other fields to study the interactions between electromagnetic radiation and matters and the properties of excited states of atoms and molecules. There are many fluorescence parameters, including fluorescence intensity, spectral profile, peak wavelength, quantum yield, polarization and fluorescence lifetime etc. whose combined measurement can provide complementary information about the object under study. For example, fluorescence spectrum measurement can distinguish different molecular species and contrast different fluorophores in samples. Because fluorescence lifetime is sensitive to the local environment of the fluorophores, many physiological parameters including pH, Ca 2+ , Na + and pO 2 can be quantified via fluorescence lifetime measurement. Fluorescence lifetime contrast is particularly useful in identifying fluorophores with significantly overlapping spectra. Multiphoton excitation fluorescence spectroscopy and microscopy have particularly gained popularity in biomedicine because of many advantages associated with the nonlinear multiphoton excitation [1] , e.g., reduced photobleaching and photodamage, enhanced penetration depth and high signal-to-noise ratio imaging of biological specimens in three dimensions at submicron resolution. Simultaneous measurement of two-photon excitation fluorescence spectra and lifetimes has been applied widely in biology and clinics, e.g., protein-protein interaction study [2] , early cancer diagnosis [3] and tissue discrimination [4] . In order to make full use of the power from a femtosecond Ti: Sapphire laser and reduce the acquisition time in multiphoton fluorescence microscopy, a novel method that employs parallel excitation and detection has been implemented in multifocal multiphoton microscopy (MMM), which uses a microlens array [5,6] or a diffraction optical element [7] to produce an array of high numerical aperture foci on the sample for simultaneous multiphoton excitation and delivers video-rate imaging at high spatial resolution. Recently, fluorescence lifetime imaging (FLIM) has been demonstrated in MMM configuration by several groups [8,9] .Time-resolved fluorescence spectroscopy and fluorescence lifetime imaging microscopy are usually implemented in three configurations, i.e. time-correlated single-photon counting (TCSPC) [10] , time-ga...

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Simultaneous time- and spectrum-resolved multifocal multiphoton microscopy

Cited by 26 publications

References 16 publications

High-speed multifocal array scanning using refractive window tilting

High-speed multifocal array scanning using refractive window tilting

Monte Carlo Simulation of Multifocal Stochastic Scanning System

Time-resolved two-photon excitation fluorescence spectroscopy and microscopy using a high repetition rate streak camera

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