Two-photon microscopy: Imaging in scattering samples and three-dimensionally resolved flash photolysis

Soeller, Christian; Cannell, Mark B.

doi:10.1002/(sici)1097-0029(19991101)47:3<182::aid-jemt4>3.0.co;2-4

Cited by 56 publications

(32 citation statements)

References 32 publications

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“…Soeller and Cannell were also able to produce small repeatable calcium release events using DM-nitrophen in isolated cardiomyocytes, which had spatial and temporal properties very similar to those of naturally occurring Ca 2ϩ sparks. 21 In a subsequent study from the same group, artificial sparks with known underlying SR Ca 2ϩ release fluxes were used to validate various numerical approaches to derive SR Ca 2ϩ release flux underlying Ca 2ϩ sparks and, thus, the number of SR ryanodine receptors contributing to a Ca 2ϩ spark. 9 Two-photon fluorescence photobleaching recovery is a technically related approach to study the three-dimensional mobility of fluorescent molecules with three-dimensional resolution at a micrometer scale.…”

Section: Applications Of Tpe Microscopy Two-photon Photolysis and Twomentioning

confidence: 99%

“…Subcellular resolution Ca 2ϩ imaging in intact myocardium using single-photon confocal microscopy is typically restricted to regions less than Ϸ40 m below the surface. 21,59 The capability of TPLSM to provide optical sectioning with subcellular resolution from deeper within scattering biological specimens than confocal microscopy has recently been exploited to measure [Ca 2ϩ ] i -dependent changes in fluorescence intensity of the Ca 2ϩ indicators rhod-2 and fura-2 at the single cardiomyocyte level in a buffer-perfused mouse heart preparation. 25 Figure 6 demonstrates that TPLSM using a high numerical aperture objective is able to resolve single myocyte [Ca 2ϩ ] i transients at depths of up to 200 m below the epicardial surface.…”

Section: Tpe Fluorescence Imaging Deep Within Scattering Tissuementioning

confidence: 99%

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Two-Photon Microscopy of Cells and Tissue

Rubart

2004

Circulation Research

277

209

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Abstract-Two-photon excitation fluorescence imaging provides thin optical sections from deep within thick, scattering specimens by way of restricting fluorophore excitation (and thus emission) to the focal plane of the microscope. Spatial confinement of two-photon excitation gives rise to several advantages over single-photon confocal microscopy. First, penetration depth of the excitation beam is increased. Second, because out-of-focus fluorescence is never generated, no pinhole is necessary in the detection path of the microscope, resulting in increased fluorescence collection efficiency. Third, two-photon excitation markedly reduces overall photobleaching and photodamage, resulting in extended viability of biological specimens during long-term imaging. Finally, localized excitation can be used for photolysis of caged compounds in femtoliter volumes and for diffusion measurements by two-photon fluorescence photobleaching recovery. This review aims to provide an overview of the use of two-photon excitation microscopy. Selected applications of this technique will illustrate its excellent suitability to assess cellular and subcellular events in intact, strongly scattering tissue. In particular, its capability to resolve differences in calcium dynamics between individual cardiomyocytes deep within intact, buffer-perfused hearts is demonstrated. Potential applications of two-photon laser scanning microscopy as applied to integrative cardiac physiology are pointed out. Key Words: two-photon excitation Ⅲ laser scanning microscopy Ⅲ calcium imaging T wo-photon excitation (TPE) microscopy 1 has evolved as an alternative to conventional single-photon confocal microscopy and has been shown to provide several advantages. These include three-dimensionally resolved fluorescence imaging of living cells deep within thick, strongly scattering samples, and reduced phototoxicity, enabling longterm imaging of photosensitive biological specimens. The inherent three-dimensional resolution of TPE microscopy has been exploited in a number of studies wherein spatial discrimination of fluorescence signals at the micrometer and submicrometer scale within thick biological specimens proved critical. For example, TPE of the calcium-sensitive fluorophore rhod-2 has been used to resolve differences in the kinetics of intracellular calcium ([Ca 2ϩ ] i ) transients in donor myocytes and juxtaposed host cardiomyocytes deep in Langendorff-perfused mouse hearts following intracardiac transplantation of fetal cardiomyocytes and skeletal myoblasts. 2,3 For neuroscientists, TPE microscopy has become an invaluable tool for studying calcium dynamics in thick brain slices and live animals 4,5 and for long-term imaging of neuronal development. 6 The spatial confinement of TPE has also been used for three-dimensional photolysis of caged compounds in femtoliter volumes [7][8][9] or diffusion measurements by two-photon fluorescence photobleaching recov- ery. 10,11 This article describes the basic physical principles of TPE and reviews the advantages and...

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Section: Applications Of Tpe Microscopy Two-photon Photolysis and Twomentioning

confidence: 99%

Section: Tpe Fluorescence Imaging Deep Within Scattering Tissuementioning

confidence: 99%

Two-Photon Microscopy of Cells and Tissue

Rubart

2004

Circulation Research

277

209

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“…In multi-photon imaging, fluorophore excitation is achieved by the non-linear absorption process [16,17]. Specifically, in twophoton microscopy, two near-infrared (near-IR) photons of approximately half the one-photon excitation energy are simultaneously absorbed by the fluorescent molecule [18,19]. This approach can be used to excite intrinsic or extrinsic fluorophores, which are generally excited by ultraviolet (UV) or visible (VIS) light [20].…”

Section: Introductionmentioning

confidence: 99%

Chemical enhancer induced changes in the mechanisms of transdermal delivery of zinc oxide nanoparticles

Kuo

Hsu

et al. 2009

Biomaterials

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“…The use of a pinhole is no longer necessary to achieve optical slicing, and the detection of fluorescence can thus be done in the nondescanned position. This makes instrument design more flexible and increases sensitivity (22).…”

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confidence: 99%

Setup and characterization of a multiphoton FLIM instrument for protein–protein interaction measurements in living cells

Waharte¹,

Spriet²,

Héliot³

2006

Cytometry Pt A

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Background: Fluorescence lifetime microscopy (FLIM) is currently one of the best techniques to perform accurate measurements of interactions in living cells. It is independent of the fluorophore concentration, thus avoiding several common artifacts found in F€ orster Resonance Energy Transfer (FRET) imaging. However, for FLIM to achieve high performance, a rigorous instrumental setup and characterization is needed. Methods: We use known fluorophores to perform characterization experiments in our instrumental setup. This allows us to verify the accuracy of the fluorescence lifetime determination, and test the linearity of the instrument by fluorescence quenching. Results: We develop and validate here a protocol for rigorous characterization of time-domain FLIM instruments. Following this protocol, we show that our system pro-

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Two-photon microscopy: Imaging in scattering samples and three-dimensionally resolved flash photolysis

Cited by 56 publications

References 32 publications

Two-Photon Microscopy of Cells and Tissue

Two-Photon Microscopy of Cells and Tissue

Chemical enhancer induced changes in the mechanisms of transdermal delivery of zinc oxide nanoparticles

Setup and characterization of a multiphoton FLIM instrument for protein–protein interaction measurements in living cells

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