Two-photon imaging of multiple fluorescent proteins by phase-shaping and linear unmixing with a single broadband laser

Brenner, Meredith H.; Cai, Dawen; Swanson, Joel A.; Ogilvie, Jennifer P.

doi:10.1364/oe.21.017256

Cited by 17 publications

(15 citation statements)

References 57 publications

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“…The femtoJock system implemented the multiphoton intrapulse interference phase scan (MIIPS) method [25, 26] that enabled the applied spectral phase for dispersion compensation and pulse compression of the fiber output to be found. Use of the femtoJock system [27] and similar pulse shapers for two photon microscopy has been reported previously [28]. …”

Section: Methodsmentioning

confidence: 99%

Dependence of Two-Photon eGFP Bleaching on Femtosecond Pulse Spectral Amplitude and Phase

et al. 2015

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Photobleaching is a key limitation in two-photon imaging of fluorescent proteins with femtosecond pulsed excitation. We present measurements of the dependence of eGFP photobleaching on the spectral amplitude and phase of the pulses used. A strong dependence on the excitation wavelength was confirmed and measured over a 800–950 nm range. A fiber continuum light source and pulse shaping techniques were used to investigate photobleaching with broadband, 15 fs transform limited, pulses with differing spectral amplitude and phase. Narrow band pulses, >150 fs transform limited, typical of femtosecond laser sources used in two-photon imaging applications, were also investigated for their photobleaching dependence on pulse dispersion and bandwidth. The bleach rate for broadband pulses was found to be primarily determined by the second harmonic spectrum of the excitation light. On the other hand, for narrow band excitation pulses with similar center wavelengths improvement in bleach rate was found to be mostly dependent on reducing the pulse length. A simple model to predict the relative bleach rates for broadband pulses is presented and compared to the experimental data.

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Section: Methodsmentioning

confidence: 99%

Dependence of Two-Photon eGFP Bleaching on Femtosecond Pulse Spectral Amplitude and Phase

et al. 2015

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“…, spectral deconvolution), the contribution of each of the fluorophores used in the experiment in each of the NDD must be measured. Then, using dedicated algorithms based on maximum-likelihood unmixing (Davis and Shen 2007), each image from each NDD can be spectrally unmixed and then analyzed (Thaler and Vogel 2006; Brenner et al 2013; Zimmermann et al 2014). As an example of the crucial importance of spectral unmixing, Ducros et al have used it to detect small spectral variations of odor-evoked FRET transients up to 250 μm in the olfactory bulb of living mice (Ducros et al 2009).…”

Section: Introductionmentioning

confidence: 99%

Two-photon probes for in vivo multicolor microscopy of the structure and signals of brain cells

Ricard

Arroyo

et al. 2018

Brain Struct Funct

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Imaging the brain of living laboratory animals at a microscopic scale can be achieved by two-photon microscopy thanks to the high penetrability and low phototoxicity of the excitation wavelengths used. However, knowledge of the two-photon spectral properties of the myriad fluorescent probes is generally scarce and, for many, non-existent. In addition, the use of different measurement units in published reports further hinders the design of a comprehensive imaging experiment. In this review, we compile and homogenize the two-photon spectral properties of 280 fluorescent probes. We provide practical data, including the wavelengths for optimal two-photon excitation, the peak values of two-photon action cross section or molecular brightness, and the emission ranges. Beyond the spectroscopic description of these fluorophores, we discuss their binding to biological targets. This specificity allows in vivo imaging of cells, their processes, and even organelles and other subcellular structures in the brain. In addition to probes that monitor endogenous cell metabolism, studies of healthy and diseased brain benefit from the specific binding of certain probes to pathology-specific features, ranging from amyloid-β plaques to the autofluorescence of certain antibiotics. A special focus is placed on functional in vivo imaging using two-photon probes that sense specific ions or membrane potential, and that may be combined with optogenetic actuators. Being closely linked to their use, we examine the different routes of intravital delivery of these fluorescent probes according to the target. Finally, we discuss different approaches, strategies, and prerequisites for two-photon multicolor experiments in the brains of living laboratory animals.

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“…Pulse shapers offer not only a convenient method for pulse compression and characterization, but enable generation of shaped pulses as well as collinear, interferometrically stable pulse sequences. The ability to control ultrashort laser pulses combined with high spatial resolution is used in a variety of experiments, including selective nonlinear microscopy [6,7], coherent control and coherent two-dimensional spectroscopy of plasmonic nanostructures [8][9][10][11][12], observation of energy transfer in single light-harvesting complexes [13], and many others. However, although a pulse shaper opens up new possibilites, it is also a further source of spatial beam profile distortions.…”

Section: Introductionmentioning

confidence: 99%

Shaping and spatiotemporal characterization of sub-10-fs pulses focused by a high-NA objective

Pawłowska¹,

Goetz²,

Dreher³

et al. 2014

Opt. Express

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We describe a setup consisting of a 4f pulse shaper and a microscope with a high-NA objective lens and discuss the aspects most relevant for an undistorted spatiotemporal profile of the focused beam. We demonstrate shaper-assisted pulse compression in focus to a sub-10-fs duration using phase-resolved interferometric spectral modulation (PRISM). We introduce a nanostructure-based method for sub-diffraction spatiotemporal characterization of strongly focused pulses. The distortions caused by optical aberrations and space-time coupling from the shaper can be reduced by careful setup design and alignment to about 10 nm in space and 1 fs in time.

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Two-photon imaging of multiple fluorescent proteins by phase-shaping and linear unmixing with a single broadband laser

Cited by 17 publications

References 57 publications

Dependence of Two-Photon eGFP Bleaching on Femtosecond Pulse Spectral Amplitude and Phase

Dependence of Two-Photon eGFP Bleaching on Femtosecond Pulse Spectral Amplitude and Phase

Two-photon probes for in vivo multicolor microscopy of the structure and signals of brain cells

Shaping and spatiotemporal characterization of sub-10-fs pulses focused by a high-NA objective

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