Simultaneous two‐photon fluorescence correlation spectroscopy and lifetime imaging of dye molecules in submicrometer fluidic structures

Petrášek, Zdeněk; Krishnan, Madhavi; Mönch, Ingolf; Schwille, Petra

doi:10.1002/jemt.20428

Cited by 17 publications

(15 citation statements)

References 27 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The experiments were performed on a homebuilt two-photon laser scanning microscope (14,20) using an UPLAPO 603 W3/IR objective (Olympus, Hercules, CA). The excitation was provided by a tunable Ti:Sapphire laser (Mira 900-F, Coherent, Santa Clara, CA) with the wavelength set to 920 nm, and an average power of 5 mW.…”

Section: Methodsmentioning

confidence: 99%

Characterization of Protein Dynamics in Asymmetric Cell Division by Scanning Fluorescence Correlation Spectroscopy

Petrášek

Hoege

Mashaghi

et al. 2008

Biophysical Journal

Self Cite

View full text Add to dashboard Cite

The development and differentiation of complex organisms from the single fertilized egg is regulated by a variety of processes that all rely on the distribution and interaction of proteins. Despite the tight regulation of these processes with respect to temporal and spatial protein localization, exact quantification of the underlying parameters, such as concentrations and distribution coefficients, has so far been problematic. Recent experiments suggest that fluorescence correlation spectroscopy on a single molecule level in living cells has great promise in revealing these parameters with high precision. The optically challenging situation in multicellular systems such as embryos can be ameliorated by two-photon excitation, where scattering background and cumulative photobleaching is limited. A more severe problem is posed by the large range of molecular mobilities observed at the same time, as standard FCS relies strongly on the presence of mobility-induced fluctuations. In this study, we overcame the limitations of standard FCS. We analyzed in vivo polarity protein PAR-2 from eggs of Caenorhabditis elegans by beam-scanning FCS in the cytosol and on the cortex of C. elegans before asymmetric cell division. The surprising result is that the distribution of PAR-2 is largely uncoupled from the movement of cytoskeletal components of the cortex. These results call for a more systematic future investigation of the different cortical elements, and show that the FCS technique can contribute to answering these questions, by providing a complementary approach that can reveal insights not obtainable by other techniques.

show abstract

Section: Methodsmentioning

confidence: 99%

Characterization of Protein Dynamics in Asymmetric Cell Division by Scanning Fluorescence Correlation Spectroscopy

Petrášek

Hoege

Mashaghi

et al. 2008

Biophysical Journal

Self Cite

View full text Add to dashboard Cite

show abstract

“…lab-on-a chip type experiments. 12,13,65 Incidentally, it may be interesting to see if the distance of a GFP-tagged protein in a cell from a glass surface could be mapped using the GFP fluorescence lifetime change.…”

Section: Gfp Flimmentioning

confidence: 99%

Time-resolved fluorescence microscopy

et al. 2007

View full text Add to dashboard Cite

Fluorescence imaging techniques are powerful tools in the biological and biomedical sciences, because they are minimally invasive and can be applied to live cells and tissues. The fluorescence emission can be characterized not only by its intensity and position, by also by its fluorescence lifetime, polarization and wavelength. Fluorescence Lifetime Imaging (FLIM) in particular has emerged as a key technique to image the environment and interaction of specific proteins in living cells. Using a time-correlated single photon counting (TCSPC)-based FLIM set-up, we find that the fluorescence lifetime of GFP-tagged proteins in cells is a function of the refractive index of the medium the cells are suspended in. In addition, combining Fluorescence Recovery After Photobleaching (FRAP) of fluorescently labeled proteins of different sizes in sol gels with time-resolved fluorescence anisotropy measurements, we demonstrate that we can measure their lateral and rotational diffusion. This allows us to infer the size and connectivity of the pores in the sol gel matrix. Moreover, wide-field photon counting imaging, originally developed for astronomical applications, is a powerful imaging method because of its high sensitivity and excellent signal-to-noise ratio. It has a distinct advantage over CCD-based imaging due to the ability to time the arrival of individual photons. The potential of time-resolved widefield photon counting imaging with a fast CMOS camera applied to luminescence microscopy is demonstrated.

show abstract

“…Fluorescence lifetimes can be measured in the time and frequency domains, and have been used in biological systems to measure ionic concentrations (Ca 2+ , Cl − , O 2 ) or the subcellular localization of various macromolecules [ 140 ]. This technique can also be combined with FCS [ 141 ] to determine the diffusion coefficient of fluorescent molecules within a known volume [ 140 ]. An interesting variation of FCS, scanning FCS (sFCS), does not require a priori knowledge of the measurement volume, and therefore is probably better suited for diffusion estimates in living systems [ 142 ].…”

Section: Sniffers and Imaging Approachesmentioning

confidence: 99%

Determining the Neurotransmitter Concentration Profile at Active Synapses

2009

View full text Add to dashboard Cite

Establishing the temporal and concentration profiles of neurotransmitters during synaptic release is an essential step towards understanding the basic properties of inter-neuronal communication in the central nervous system. A variety of ingenious attempts has been made to gain insights into this process, but the general inaccessibility of central synapses, intrinsic limitations of the techniques used, and natural variety of different synaptic environments have hindered a comprehensive description of this fundamental phenomenon. Here, we describe a number of experimental and theoretical findings that has been instrumental for advancing our knowledge of various features of neurotransmitter release, as well as newly developed tools that could overcome some limits of traditional pharmacological approaches and bring new impetus to the description of the complex mechanisms of synaptic transmission.

show abstract

Simultaneous two‐photon fluorescence correlation spectroscopy and lifetime imaging of dye molecules in submicrometer fluidic structures

Cited by 17 publications

References 27 publications

Characterization of Protein Dynamics in Asymmetric Cell Division by Scanning Fluorescence Correlation Spectroscopy

Characterization of Protein Dynamics in Asymmetric Cell Division by Scanning Fluorescence Correlation Spectroscopy

Time-resolved fluorescence microscopy

Determining the Neurotransmitter Concentration Profile at Active Synapses

Contact Info

Product

Resources

About