Time-resolved confocal scanning device for ultrasensitive fluorescence detection

Böhmer, Martín; Pampaloni, Francesco; Wahl, Michael; Rahn, Hans-Jürgen; Erdmann, Rainer; Enderlein, Jörg

doi:10.1063/1.1406926

Cited by 82 publications

(66 citation statements)

References 19 publications

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“…The cells were lysed in Brij-96V lysis buffer (as described above but lacking glycerol). The 2f-FCS experiments were carried out using a setup based on a standard confocal epi-fluorescence microscope (Böhmer et al, 2001). eGFP and eGFP-tagged fusion constructs were excited with a 470 nm laser beam (LDH-P-C-470B).…”

Section: Dual-focus Fluorescence Correlations Spectroscopy (2f-fcs)mentioning

confidence: 99%

The role of the N-terminal domain in dimerization and nucleocytoplasmic shuttling of latent STAT3

Vogt

Domoszlai

Kleshchanok

et al. 2011

Journal of Cell Science

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STAT3 is an important transcription factor involved in immunity and cancer. In response to cytokine stimulation, STAT3 becomes phosphorylated on a single tyrosine residue. Tyrosine-phosphorylated STAT3 accumulates in the nucleus, binds to specific DNA response elements and induces gene expression. Unphosphorylated, latent STAT3 shuttles constitutively between cytoplasm and nucleus. We analysed the importance of previously identified putative nuclear localization sequences (NLS) and nuclear export sequences (NES) for nucleocytoplasmic shuttling of latent STAT3 using STAT3-deficient cells reconstituted with fluorescently labelled STAT3 mutants. Mutation of a putative NLS or NES sequence did not impair nucleocytoplasmic shuttling of latent STAT3. We were also interested in the structural requirements for dimerization of unphosphorylated STAT3 and its relevance for nucleocytoplasmic shuttling. By native gel electrophoresis and dual-focus fluorescence correlation spectroscopy (2f-FCS) we identified the N-terminal domain (amino acids 1–125) to be essential for formation of unphosphorylated STAT3 dimers but not for assembly of tyrosine-phosphorylated STAT3 dimers. In resting cells, the monomeric N-terminal deletion mutant (STAT3-ΔNT) shuttles faster between the cytoplasm and nucleus than the wild-type STAT3, indicating that dimer formation is not required for nucleocytoplasmic shuttling of latent STAT3. STAT3-ΔNT becomes phosphorylated and dimerizes in response to interleukin-6 stimulation but, surprisingly, does not accumulate in the nucleus. These results highlight the importance of the N-terminal domain in the formation of unphosphorylated STAT3 dimers and nuclear accumulation of STAT3 upon phosphorylation.

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Section: Dual-focus Fluorescence Correlations Spectroscopy (2f-fcs)mentioning

confidence: 99%

The role of the N-terminal domain in dimerization and nucleocytoplasmic shuttling of latent STAT3

Vogt

Domoszlai

Kleshchanok

et al. 2011

Journal of Cell Science

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“…Details were described previously. 8 A pulsed diode laser at ϳ470 nm wavelength ͑LDH-P-C 470/ PDL 800B, PicoQuant͒ generating pulses with ϳ70 ps pulse width and 40 MHz repetition rate or a cw Ar + laser ͑Innova 70-5, Coherent͒ operating at 488 nm was used for excitation. The light of the laser was passed through a single-mode optical fiber and subsequently collimated to form a beam with Gaussian beam profile of approximately 2.5 mm beam waist radius.…”

Section: Resultsmentioning

confidence: 99%

Dead-time optimized time-correlated photon counting instrument with synchronized, independent timing channels

Wahl

Rahn²,

Gregor³

et al. 2007

Review of Scientific Instruments

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Time-correlated single photon counting is a powerful method for sensitive time-resolved fluorescence measurements down to the single molecule level. The method is based on the precisely timed registration of single photons of a fluorescence signal. Historically, its primary goal was the determination of fluorescence lifetimes upon optical excitation by a short light pulse. This goal is still important today and therefore has a strong influence on instrument design. However, modifications and extensions of the early designs allow for the recovery of much more information from the detected photons and enable entirely new applications. Here, we present a new instrument that captures single photon events on multiple synchronized channels with picosecond resolution and over virtually unlimited time spans. This is achieved by means of crystal-locked time digitizers with high resolution and very short dead time. Subsequent event processing in programmable logic permits classical histogramming as well as time tagging of individual photons and their streaming to the host computer. Through the latter, any algorithms and methods for the analysis of fluorescence dynamics can be implemented either in real time or offline. Instrument test results from single molecule applications will be presented.

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“…Previous reports show that a SPAD detection architecture can be used in confocal microscopy (Böhmer et al, 2001), fluorescence correlation spectroscopy (FCS) (Maiti et al, 1997;Ray et al, 2010), microarrays (Marangoni et al, 2010), flow cytometry (Li et al, 2012b), 3D acquisition (Aull et al, 2002;Niclass et al, 2008;Niclass et al, 2005), FLIM (Gersbach et al, 2010), single--molecule FRET Michalet et al, 2013;Panzeri et al, 2013), astrophysics (Zappa et al, 2007), astronomy (Nightingale, 1990), quantum mechanics (Rarity and Tapster, 1990), quantum cryptography (Chiangga et al, 1999), diode laser characterization (Cova et al, 1989) and optical fibre testing (Ripamonti et al, 1990).…”

Section: Time Resolved Detection System For Parallelised Tcspcmentioning

confidence: 99%

Multifocal multiphoton microscopy with adaptive optical correction

et al. 2013

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Multiphoton microscopy (MPM) is a remarkably versatile technique in biologicalimaging. MPM provides increased depth over confocal imaging and can be combined with other imaging techniques such as fluorescence lifetime imaging microscopy (FLIM), adding functional information. FLIM read--out is relatively straightforward using time--correlated single photon counting (TCSPC). Fluorescence lifetime detection enhances the power of multiphoton imaging to allow three dimensional, concentration independent, measurements of environmental parameters such as pH, Oxygen tension and Ca 2+ in addtion to the interaction or conformational modification of proteins by Förster resonant energy transfer (FRET); the latter is a particular focus of the Dimbleby research groups at King's CollegeLondon. However, there are significant limitations in both FLIM and MM.Limitations of TCSPC--FLIM include prolonged acquisition times along with signal and resolution degradation as a function of depth. This thesis demonstrates advancements multiphoton fluorescence lifetime imaging through improvements in two principal areas: speed and resolution at depth.In order to improve acquistion rates a multifocal multiphoton microscope (MMM) capable of rapid, parallelized TCSPC--FLIM was developed --MegaFLI. Acquisitions demonstrate rapid 3--dimensional, high temporal resolution FLIM in vivo Zebrafish.Performed by massively parallel excitation/detection the speed is signficantly improved by a factor of 64.In parallel to the MegaFLI project, a second microscope employing adaptive optical correction has been developed. The introduction of Adaptive Optics (AO) serves to improve imaging quality by counteracting the refractive index heterogeneities introduced by the sample, limiting the imaging depth. Incorporated with a single beam scanning FLIM system, a pupil--segmentation AO--TCSPC--FLIM demonstrates improved signal--to--noise ratio (SNR) and resolution, permiting a more accurate determination of fluorescent lifetime in turbid media. 3 DECLARATION OF AUTHORSHIPThe work present is my own work with the exception of TRI2, which was developed The introduction (Chapter 1) concentrates on optical methods developed in the context of multiphoton microscopy (MPM) or more specifically two--photon microscopy (TPM) (Denk et al., 1990). TPM enables long--term imaging of in vivo biological specimens, image generation at increased tissue depth, and higher signal--to--noise images compared to wide--field and confocal approaches (Helmchen and In comparison to confocal microscopy, two--photon microscopy (TPM) offers considerable benefits since excitation is confined to a femtolitre volume in the vicinity of the focal plane, reducing overall photobleaching and phototoxicity of thick samples (Centonze and White, 1998;Denk et al., 1990;Helmchen and Denk, 2005;Williams et al., 2001). Out--of--focus excitation is avoided due to the localization of the excitation making the confocal pinhole obsolete (Amos and Where is the numerical aperture of the obj...

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Time-resolved confocal scanning device for ultrasensitive fluorescence detection

Cited by 82 publications

References 19 publications

The role of the N-terminal domain in dimerization and nucleocytoplasmic shuttling of latent STAT3

The role of the N-terminal domain in dimerization and nucleocytoplasmic shuttling of latent STAT3

Dead-time optimized time-correlated photon counting instrument with synchronized, independent timing channels

Multifocal multiphoton microscopy with adaptive optical correction

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