Pulsed picosecond 766 nm laser source operating between 1-80 MHz with automatic pump power management

Schönau, Thomas; Siebert, Thorsten; Härtel, Romano; Eckhardt, Th.; Klemme, Dietmar; Lauritsen, Kristian; Erdmann, Rainer

doi:10.1117/12.2003900

Cited by 6 publications

(5 citation statements)

References 6 publications

(3 reference statements)

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“…The results were plotted as shown in Figure 4 (left) and a trend curve was fitted to the data points. [15] At a STED laser intensity of 0 mW, no deactivation will occur and thus a spot diameter of 240 nm is obtained, corresponding to the diffraction limited resolution achievable by confocal microscopy. Further on, the dependence follows the well characterized inverse square-root dependence.…”

Section: Resultsmentioning

confidence: 99%

“…The STED laser with a central emission wavelength of 766 nm and a pulse width around 500 ps (full-width-at-half-maximum, FWHM) was run with variable optical output powers with a repetition rate of either 20 or 40 MHz. [15] The excitation laser with an emission wavelength of 640 nm and a pulse width of less than 90ps (FWHM) was run at a repetition rate of 20 MHz and an optical output power of about 1.9 µW. Both lasers were controlled by a computer controlled PDL 828 "Sepia II" laser driver (PicoQuant GmbH, Germany) equipped with a SOM 828-D oscillator module.…”

Section: Instrumental Set-upmentioning

confidence: 99%

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ns-time resolution for multispecies STED-FLIM and artifact free STED-FCS

Koenig

Reisch

Dowler

et al. 2016

Multiphoton Microscopy in the Biomedical Sciences XVI

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Stimulated Emission Depletion (STED) Microscopy has evolved into a well established method offering optical superresolution below 50 nm. Running both excitation and depletion lasers in picosecond pulsed modes allows for highest optical resolution as well as fully exploiting the photon arrival time information using time-resolved single photon counting (TCSPC). Non-superresolved contributions can be easily dismissed through time-gated detection (gated STED) or a more detailed fluorescence decay analysis (FLIM-STED), both leading to an even further improved imaging resolution. Furthermore, these methods allow for accurate separation of different fluorescent species, especially if subtle differences in the excitation and emission spectra as well as the fluorescence decay are taken into account in parallel. STED can also be used to shrink the observation volume while studying the dynamics of diffusing species in Fluorescence Correlation Spectroscopy (FCS) to overcome averaging issues along long transit paths. A further unique advantage of STED-FCS is that the observation spot diameter can be tuned in a gradual manner enabling, for example, determining the type of hindered diffusion in lipid membrane studies. Our completely pulsed illumination scheme allows realizing an improved STED-FCS data acquisition using pulsed interleaved excitation (PIE). PIE-STED-FCS allows for a straightforward online check whether the STED laser has an influence on the investigated diffusion dynamics.

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Section: Resultsmentioning

confidence: 99%

Section: Instrumental Set-upmentioning

confidence: 99%

ns-time resolution for multispecies STED-FLIM and artifact free STED-FCS

Koenig

Reisch

Dowler

et al. 2016

Multiphoton Microscopy in the Biomedical Sciences XVI

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“…The system was equipped with the easy STED phase plate set to form a depletion beam in donut-shape where the intensity of the depleting beam at the center was around 1% of its maximum, while the excitation beam was unaffected. Excitation and depletion lasers (LDH-640 and VisIR 765, PicoQuant) were operated in pulsed mode. To increase the data quality and resolution of STED-FCS, long accumulation time of measurements (2–4 min) and time-gated approach was applied ,− (for details, see section S1 in the SI).…”

Section: Methodsmentioning

confidence: 99%

Nanoscopic Approach to Quantification of Equilibrium and Rate Constants of Complex Formation at Single-Molecule Level

Zhang

Sisamakis

Sozański

et al. 2017

J. Phys. Chem. Lett.

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Equilibrium and rate constants are key descriptors of complex-formation processes in a variety of chemical and biological reactions. However, these parameters are difficult to quantify, especially in the locally confined, heterogeneous, and dynamically changing living matter. Herein, we address this challenge by combining stimulated emission depletion (STED) nanoscopy with fluorescence correlation spectroscopy (FCS). STED reduces the length-scale of observation to tens of nanometres (2D)/attoliters (3D) and the time-scale to microseconds, with direct, gradual control. This allows one to distinguish diffusional and binding processes of complex-formation, even at reaction rates higher by an order of magnitude than in confocal FCS. We provide analytical autocorrelation formulas for probes undergoing diffusion-reaction processes under STED condition. We support the theoretical analysis of experimental STED-FCS data on a model system of dye-micelle, where we retrieve the equilibrium and rates constants. Our work paves a promising way toward quantitative characterization of molecular interactions in vivo.

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“…Three picosecond pulsed lasers at 595, 640, and 660 nm (LDH series, PicoQuant GmbH, Berlin, Germany) were used for the excitation. Stimulated depletion was performed with a pulsed STED laser at 765 nm (VISIR-STED, PicoQuant GmbH, Berlin, Germany) . System layout can be seen in Figure a.…”

Section: Experimental Sectionmentioning

confidence: 99%

Triple-Color STED Nanoscopy: Sampling Absorption Spectra Differences for Efficient Linear Species Unmixing

et al. 2021

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Stimulated emission depletion (STED) in confocal fluorescence microscopy enables a visualization of biological structures within cells far below the optical diffraction limit. To meet the demand in the field for simultaneous investigations of multiple species within a cell, a couple of different STED techniques have been proposed, each with their own challenges. By systemically exploiting spectral differences in the absorption of fluorescent labels, we present a novel, beneficial approach to multispecies STED nanoscopy. By using three excitation wavelengths in nanosecond pulsed interleaved excitation (PIE) mode, we probe quasi simultaneously multiple species with fluorescent labels having absorption maxima as close as 13 nm. The acquired image is decomposed into its single species contributions by application of a linear unmixing algorithm based on present reference patterns. For multispecies images containing single species regions, we introduce the image correlation map (ICM). Here, the single species regions easily can be identified in order to generate the necessary single species reference patterns. This avoids the otherwise cumbersome and artifact prone preparation and recording of additional reference samples. The power of the proposed imaging scheme persists in species separation quality at high speed shown for up to three species with established reference samples and dyes commonly used for cellular STED imaging.

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Pulsed picosecond 766 nm laser source operating between 1-80 MHz with automatic pump power management

Cited by 6 publications

References 6 publications

ns-time resolution for multispecies STED-FLIM and artifact free STED-FCS

ns-time resolution for multispecies STED-FLIM and artifact free STED-FCS

Nanoscopic Approach to Quantification of Equilibrium and Rate Constants of Complex Formation at Single-Molecule Level

Triple-Color STED Nanoscopy: Sampling Absorption Spectra Differences for Efficient Linear Species Unmixing

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