Multifarious control of two-photon excitation of multiple fluorophores achieved by phase modulation of ultra-broadband laser pulses

Isobe, Keisuke; Suda, Akira; Tanaka, Masahiro; Kannari, Fumihiko; Kawano, Hiroyuki; Mizuno, Hideaki; Miyawaki, Atsushi; Midorikawa, Katsumi

doi:10.1364/oe.17.013737

Cited by 38 publications

(30 citation statements)

References 32 publications

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“…Although two-photon excitation microscopy has been applied to samples loaded with multiple fluorophores for multicolor or FRET imaging, the excitation of one fluorophore relative to the other fluorophores cannot be controlled well in the conventional system that uses a narrowband laser. Recently, a new technology that enables phase modulation of ultrabroadband laser pulses has been reported (Isobe et al 2009). Versatile control of two-photon excitation of CFP and YFP was achieved with their ratio (CFP/YFP) being varied 100-fold.…”

Section: Microscopymentioning

confidence: 99%

Imaging Intracellular Free Ca²⁺ Concentration Using Yellow Cameleons

Miyawaki¹,

Nagai²,

Mizuno³

2013

Cold Spring Harb Protoc

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Green fluorescent protein (GFP)-based fluorescent indicators for Ca 2+ offer significant promise for monitoring Ca 2+ in previously unexplored organisms, tissues, and submicroscopic environments because they are genetically encoded, function without cofactors, can be targeted to any intracellular location, and are bright enough for single-cell imaging. These probes use simple GFP variants, circularly permuted GFP (cpGFP), in which the amino and carboxyl portions have been interchanged and reconnected by short spacers between the original termini, or pairs of GFP variants that permit fluorescence resonance energy transfer (FRET). Yellow cameleons (YCs) use FRET between cyanand yellow-emitting variants of Aequorea GFP (cyan fluorescent protein [CFP] and yellow fluorescent protein [YFP], respectively). YCs are composed of a linear combination of CFP, calmodulin (CaM), a glycylglycine linker, the CaM-binding peptide of myosin light-chain kinase (M13), and YFP. Binding of Ca 2+ to the CaM moiety of the YC initiates an intramolecular interaction between the CaM and the M13 domains, causing the chimeric protein to shift from an extended conformation to a more compact one, thereby increasing the efficiency of FRET from CFP to YFP. This technique is amenable to emission ratioing, which is more quantitative than single-wavelength monitoring. YC3.60 was engineered to enhance its performance as a fluorescent Ca 2+ indicator and here we describe the use of this cameleon to image rapid changes in intracellular free Ca 2+ concentration ([Ca 2+ ] i ) within HeLa cells. FRET imaging is performed using a laser-scanning confocal microscope. MATERIALSIt is essential that you consult the appropriate Material Safety Data Sheets and your institution's Environmental Health and Safety Office for proper handling of equipment and hazardous material used in this protocol. ReagentsCa 2+ -free medium For fast and simultaneous acquisition of cpVenus173 and CFP images from HeLa cells expressing YC3.60, this camera, which comprises three charge-coupled device (CCD) chips (RGB: red, green, and blue) and a prism, can be used. The cpVenus173 and CFP images are captured by the G and B chips, respectively. In addition, to improve spatial resolution along the z-axis, a spinning-disk unit (e.g., CSU21 from Yokogawa Electric Corporation) can be placed in front of the camera.Diode-pumped solid-state laser (430 nm) (Melles Griot) Glass-bottomed dishes (35 mm) (Matsunami Glass Ind., Ltd., D111300) Image acquisition software to control the Ashura 3CCD camera (e.g., . These include intensity of the laser, scanning speed, binning, and sensitivity of the camera.In our experience, the intensity of the laser should be attenuated greatly using neutral-density filters for Ca 2+ imaging on a timescale of subseconds. Photobleaching does not happen with a laser that is sufficiently attenuated.6. Observe fluorescence signals from CFP and cp173Venus by the blue and green channels of the 3CCD camera, respectively. 7. Set 4 × 4 binning for high-sensitive fluorescence ...

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Section: Microscopymentioning

confidence: 99%

Imaging Intracellular Free Ca²⁺ Concentration Using Yellow Cameleons

Miyawaki¹,

Nagai²,

Mizuno³

2013

Cold Spring Harb Protoc

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“…Such approaches have also been extended to applications in fluorescence microscopy [25][26][27][28] (and also other microscopic techniques, e.g. in coherent anti-Stokes Raman scattering or CARS microscopy [29]). …”

Section: Spatiotemporal Controlmentioning

confidence: 99%

Towards controlling molecular motions in fluorescence microscopy and optical trapping: a spatiotemporal approach

Goswami

2011

International Reviews in Physical Chemistry

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This account reviews some recent studies pursued in our group on several control experiments with important applications in (one-photon) confocal and two-photon fluorescence laser-scanning microscopy and optical trapping with laser tweezers. We explore the simultaneous control of internal and external (i.e. centre-of-mass motion) degrees of freedom, which require the coupling of various control parameters to result in the spatiotemporal control. Of particular interest to us is the implementation of such control schemes in living systems. A live cell is a system of a large number of different molecules which combine and interact to generate complex structures and functions. These combinations and interactions of molecules need to be choreographed perfectly in time and space to achieve intended intra-cellular functions. Spatiotemporal control promises to be a versatile tool for dynamical control of spatially manipulated bio-molecules.

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“…Pulse-shaping methods have been used previously in microscopy applications for selective excitation [3][4][5] and imaging of a FRET-based calcium indicator [6]. Here we use two different phase masks designed to selectively excite donor (mCerulean) and acceptor (mCherry) fluorescent proteins in live COS-7 cells.…”

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Pulse-shaping-based two-photon FRET microscopy

Brenner

Cai

Straight

et al. 2013

EPJ Web of Conferences

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Abstract. We present quantitative pulse-shaping-based two-photon fluorescence resonance energy transfer microscopy. We tailor the spectral phase of the excitation pulses to achieve selective excitation of donor and acceptor, demonstrating the method in live cells.Fluorescence Resonance Energy Transfer (FRET) microscopy has been used extensively in biophysics for studying phenomena ranging from protein folding pathways to cellular signalling [1]. A major impediment to obtaining quantitative FRET measurements is the fact that it is often difficult to avoid direct excitation of the acceptor, which produces fluorescence that can be misinterpreted as FRET [2]. In one-photon FRET studies, FRET stoichiometry methods have been developed to resolve this problem, providing quantitative FRET measurements [2]. This approach requires selective excitation of donor and acceptor, and acquisition of separate images from donor and acceptor fluorescence channels. Analogous two-photon FRET stoichiometry has not yet been performed due to the challenges of performing rapid, multicolor two-photon imaging. Typical twophoton fluorescence microscopes utilize ~100 fs titanium sapphire oscillators as the excitation source. With such a source, different fluorophores are excited by tuning the central wavelength. Since tuning is typically a slow process (~seconds), rapid multicolor imaging is difficult unless multiple laser sources are used. An attractive alternative is to use broadband lasers, with bandwidths of >100nm. While such bandwidths enable the simultaneous excitation of multiple fluorophores, the lack of selectivity for separate excitation of donor and acceptor species complicates the implementation of FRET stoichiometry. Here we use pulse-shaping as a method to achieve selective donor and acceptor excitation, resolving the problem of spectral overlap that will enable two-photon FRET stoichiometry.Pulse-shaping methods have been used previously in microscopy applications for selective excitation [3][4][5] and imaging of a FRET-based calcium indicator [6]. Here we use two different phase masks designed to selectively excite donor (mCerulean) and acceptor (mCherry) fluorescent proteins in live COS-7 cells. Rapid switching between phase-masks (~10 ms) enables rapid collection of the images required for FRET stoichiometry. The phase masks were tailored using binary phase shaping [7] to excite the fluorophores in the regions of their two-photon absorption spectra that would lead to optimal contrast. As a result, mCherry was excited via an S 0 -S n transition using a shaped pulse centered at shorter wavelengths than those used to excite mCerulean. A transmissive 4f pulse shaper setup containing a CRi 640 pixel, phase-only spatial light modulator (SLM) was used to shape input EPJ Web of Conferences

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Multifarious control of two-photon excitation of multiple fluorophores achieved by phase modulation of ultra-broadband laser pulses

Cited by 38 publications

References 32 publications

Imaging Intracellular Free Ca²⁺ Concentration Using Yellow Cameleons

Imaging Intracellular Free Ca²⁺ Concentration Using Yellow Cameleons

Towards controlling molecular motions in fluorescence microscopy and optical trapping: a spatiotemporal approach

Pulse-shaping-based two-photon FRET microscopy

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Multifarious control of two-photon excitation of multiple fluorophores achieved by phase modulation of ultra-broadband laser pulses

Cited by 38 publications

References 32 publications

Imaging Intracellular Free Ca2+ Concentration Using Yellow Cameleons

Imaging Intracellular Free Ca2+ Concentration Using Yellow Cameleons

Towards controlling molecular motions in fluorescence microscopy and optical trapping: a spatiotemporal approach

Pulse-shaping-based two-photon FRET microscopy

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Product

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About

Imaging Intracellular Free Ca²⁺ Concentration Using Yellow Cameleons

Imaging Intracellular Free Ca²⁺ Concentration Using Yellow Cameleons