Characterization and optimization of a laser-scanning microscope in the femtosecond regime

Wolleschensky, Ralf; Feurer, Thomas; Sauerbrey, R.; Simon, Ulrich

doi:10.1007/s003400050479

Cited by 56 publications

(44 citation statements)

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“…The maximum value of the two-photon absorption cross-section σ (2) , which indicates the efficiency of two-photon absorption, of FLW was 730 GM (1 GM=1×10 ) at 750 nm (Tani et al, 2012). This value is 9 and 90 times larger than that of the typical mitochondrial selective probes, rhodamine 123 (Wolleschensky et al, 1998) and green fluorescent protein (GFP) (So et al, 2000), respectively. FLW comprises a highly fluorescent fluorene core and cationic pyridinium groups connected by vinylene groups.…”

Section: Introductionmentioning

confidence: 90%

A Biphenyl Type Two-Photon Fluorescence Probe for Monitoring the Mitochondrial Membrane Potential

Moritomo

Yamada

Kojima

et al. 2014

Cell Struct. Funct.

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Section: Introductionmentioning

confidence: 90%

A Biphenyl Type Two-Photon Fluorescence Probe for Monitoring the Mitochondrial Membrane Potential

Moritomo

Yamada

Kojima

et al. 2014

Cell Struct. Funct.

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“…1, the output of a Synergy 20 Femtosource Ti:Sapphire oscillator (40 nm bandwidth) is temporally stretched to 364 fs by a grating compressor (C) to precompensate for dispersion of transmissive optics. [15] The beam is then split at beamsplitter BS1 into the two arms of an interferometer. A delay stage (DS) with 0.017 μm step resolution is used to induce a difference between the optical paths.…”

Section: Methodsmentioning

confidence: 99%

“…By comparing the pulse duration at the input of the microscope and at the focal plane, we can quantify the second-order dispersion parameter to 4416 fs 2 , in line with the values determined by Wolleschensky et al for similar but not identical optical systems. [15] …”

Section: E(t)e(t − τ)mentioning

confidence: 99%

Nano-FROG: Frequency resolved optical gating by a nanometric object

Extermann¹,

Bonacina²,

Courvoisier³

et al. 2008

Opt. Express

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Abstract:We present a technique to characterize ultrashort pulses at the focal plane of a high numerical aperture objective with unprecedented spatial resolution, by performing a FROG measurement with a single nanocrystal as nonlinear medium. This approach can be extended to develop novel phase-sensitive techniques in laser scanning microscopy, probing the microscopic environment by monitoring phase and amplitude distortions of femtosecond laser pulses.

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“…This value is larger than those of typical mitochondria-selective probes, for example, green fluorescent protein (GFP; 8 GM) 8 and rhodamine 123 (80 GM). 9 Although efficient TPA compounds are usually insoluble in the aqueous medium because of their hydrophobic character, the two cationic pyridinium moieties of FLW contribute to the increase of water solubility. FLW was soluble in water at a concentration on the order of 10 ¹4 mol L ¹1 and stained cells without using any organic solvent.…”

Section: ¹1mentioning

confidence: 99%

A Red Fluorescence Two-photon Absorption Probe for Sensitive Imaging of Live Mitochondria

et al. 2014

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We synthesized two-photon absorption probes based on naphthalene and anthracene: 1,1¤-dimethyl-4,4¤-(2,6-naphthylenedi-2,1-ethenediyl)dipyridinium diiodide (NP) and 1,1¤-dimethyl-4,4¤-(2,6-anthrylenedi-2,1-ethenediyl)dipyridinium diiodide (AC). These two probes were successfully accumulated in mitochondria with different fluorescence colors: yellow (NP) and red (AC). Two-photon absorption cross sections (· (2) ) were 712 and 685 GM (1 GM = 10 ¹50 cm 4 s photon ¹1 molecule ¹1), respectively. These values are nine times larger than the · (2) of the commercially available mitochondria-selective probe with efficient two-photon absorption property (rhodamine 123).Two-photon fluorescence microscopy (TPFM) is known as an advanced technology for three-dimensional (3D) imaging of tissues and organs.1 This technique enables 3D imaging of blood flow, 2 neural network, 3 neutrophil movement, 4 local oxygen concentration in the bone, 5 and so forth. In a typical TPFM, the excitation wavelength is ca. 800 nm and the observed fluorescence is between 400 to 550 nm. Transparency for biological tissues in the wavelength region from 400 to 550 nm is insufficient. Therefore, imaging at a point deeper than 1 mm cannot be achieved using the conventional TPFM probe. In contrast, a wavelength region from 600 to 1300 nm is known as the "tissue optical window".6 Biological materials usually have low absorption in this window, so that light penetrates deeper inside the tissue. By employing the probe that can be both excited and emitted in the tissue optical window, deeper observation of tissue and organs by TPFM can be achieved.We have developed an efficient two-photon fluorescence probe: 1,1¤-dimethyl-4,4¤-(9,9¤-diethyl-2,7-fluorenediyl-2,1-ethenediyl)dipyridinium diperchlorate (FLW, Figure 1a). This probe comprises a fluorene core and pyridinium groups. 7 The cationic pyridinium moiety was designed to adsorb onto the negatively charged mitochondrial membrane through electrostatic interaction. The FLW showed a large two-photon absorption (TPA) cross section (· (2) ) with 750 GM (1 GM = 10 ¹50 cm 4 s photon ¹1 molecule ¹1 ) at 730 nm. This value is larger than those of typical mitochondria-selective probes, for example, green fluorescent protein (GFP; 8 GM) 8 and rhodamine 123 (80 GM).9 Although efficient TPA compounds are usually insoluble in the aqueous medium because of their hydrophobic character, the two cationic pyridinium moieties of FLW contribute to the increase of water solubility. FLW was soluble in water at a concentration on the order of 10 ¹4 mol L ¹1 and stained cells without using any organic solvent. This advantage allowed longtime imaging of live cells for more than 24 h because toxicity due to organic solvents was not a concern. However, the fluorescence maximum of FLW was at 545 nm, which is not in the range of the tissue optical window as well as conventional TPFM probes.In this study, we aimed to develop our molecular design for a two-photon fluorescence probe that exhibits both excitation and fluorescence in the tis...

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Characterization and optimization of a laser-scanning microscope in the femtosecond regime

Cited by 56 publications

References 1 publication

A Biphenyl Type Two-Photon Fluorescence Probe for Monitoring the Mitochondrial Membrane Potential

A Biphenyl Type Two-Photon Fluorescence Probe for Monitoring the Mitochondrial Membrane Potential

Nano-FROG: Frequency resolved optical gating by a nanometric object

A Red Fluorescence Two-photon Absorption Probe for Sensitive Imaging of Live Mitochondria

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