Multicolour single molecule imaging on cells using a supercontinuum source

Webb, S; Zanetti-Domingues, Laura C.; Coles, Benjamin C.; Rolfe, Daniel J.; Wareham, Richard J.; Martin-Fernandez, Marisa L.

doi:10.1364/boe.3.000400

Cited by 14 publications

(13 citation statements)

References 16 publications

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“…In contrast to widefield microscopy, an increased signal-to-noise ratio and flexibility of dye excitation and detection are the main advantages of confocal approaches ( Figure 3 ). Nowadays, supercontinuum lasers have been shown to provide a flexible excitation source for fluorescence imaging [ 23 ], and preservation of the biological sample/fluorochrome can also be improved by decreasing the laser power and by increasing the detection of emitted photons. A combination of vacuum-tube and semi-conductor technologies known as hybrid detectors has recently emerged and outperforms other sensors in most applications [ 24 ].…”

Section: Complementary Imaging Approaches For Cell Migration Studimentioning

confidence: 99%

Postnatal Migration of Cerebellar Interneurons

et al. 2017

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Due to its continuing development after birth, the cerebellum represents a unique model for studying the postnatal orchestration of interneuron migration. The combination of fluorescent labeling and ex/in vivo imaging revealed a cellular highway network within cerebellar cortical layers (the external granular layer, the molecular layer, the Purkinje cell layer, and the internal granular layer). During the first two postnatal weeks, saltatory movements, transient stop phases, cell-cell interaction/contact, and degradation of the extracellular matrix mark out the route of cerebellar interneurons, notably granule cells and basket/stellate cells, to their final location. In addition, cortical-layer specific regulatory factors such as neuropeptides (pituitary adenylate cyclase-activating polypeptide (PACAP), somatostatin) or proteins (tissue-type plasminogen activator (tPA), insulin growth factor-1 (IGF-1)) have been shown to inhibit or stimulate the migratory process of interneurons. These factors show further complexity because somatostatin, PACAP, or tPA have opposite or no effect on interneuron migration depending on which layer or cell type they act upon. External factors originating from environmental conditions (light stimuli, pollutants), nutrients or drug of abuse (alcohol) also alter normal cell migration, leading to cerebellar disorders.

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Section: Complementary Imaging Approaches For Cell Migration Studimentioning

confidence: 99%

Postnatal Migration of Cerebellar Interneurons

et al. 2017

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“…Furthermore, it is simpler to integrate a single supercontinuum laser into a microscope than it is to couple multiple single-wavelength lasers. This work extends the use of a supercontinuum source for imaging single molecules from the visible spectrum [29] to the NIR. It would be desirable to image more than two fluorophores within the NIR spectrum, but this has only been achieved by spectral unmixing in software [30] , for which the SNRs required are not found in single molecule microscopy.…”

Section: Discussionmentioning

confidence: 85%

Multicolour Single Molecule Imaging in Cells with Near Infra-Red Dyes

et al. 2012

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BackgroundThe autofluorescence background of biological samples impedes the detection of single molecules when imaging. The most common method of reducing the background is to use evanescent field excitation, which is incompatible with imaging beyond the surface of biological samples. An alternative would be to use probes that can be excited in the near infra-red region of the spectrum, where autofluorescence is low. Such probes could also increase the number of labels that can be imaged in multicolour single molecule microscopes. Despite being widely used in ensemble imaging, there is a currently a shortage of information available for selecting appropriate commercial near infra-red dyes for single molecule work. It is therefore important to characterise available near infra-red dyes relevant to multicolour single molecule imaging.Methodology/Principal FindingsA range of commercially available near infra-red dyes compatible with multi-colour imaging was screened to find the brightest and most photostable candidates. Image series of immobilised samples of the brightest dyes (Alexa 700, IRDye 700DX, Alexa 790 and IRDye 800CW) were analysed to obtain the mean intensity of single dye molecules, their photobleaching rates and long period blinking kinetics. Using the optimum dye pair, we have demonstrated for the first time widefield, multi-colour, near infra-red single molecule imaging using a supercontinuum light source in MCF-7 cells.Conclusions/SignificanceWe have demonstrated that near infra-red dyes can be used to avoid autofluorescence background in samples where restricting the illumination volume of visible light fails or is inappropriate. We have also shown that supercontinuum sources are suited to single molecule multicolour imaging throughout the 470–1000 nm range. Our measurements of near infra-red dye properties will enable others to select optimal dyes for single molecule imaging.

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“…Further, it will be interesting to characterize glycolipid- and domain- specific fluorescently tagged peptides [ 34 ] in comparison to other phase-specific probes. Indeed, the analysis of any given lipid or peptide probe in relation to a second probe or domain marker will be very informative, and can be easily achieved via the use of an emission splitting device, which records two labels simultaneously on adjacent halves of the camera chip [ 35 ]. Given its precision and sensitivity to heterogeneity in diffusion behaviors among populations of particles, TrackArt should be an effective tool for analyzing the effects of drugs and small compounds on membrane fluidity, organization, and dynamics.…”

Section: Future Directionsmentioning

confidence: 99%

TrackArt: the user friendly interface for single molecule tracking data analysis and simulation applied to complex diffusion in mica supported lipid bilayers

Matysik

Kraut

2014

BMC Res Notes

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BackgroundSingle molecule tracking (SMT) analysis of fluorescently tagged lipid and protein probes is an attractive alternative to ensemble averaged methods such as fluorescence correlation spectroscopy (FCS) or fluorescence recovery after photobleaching (FRAP) for measuring diffusion in artificial and plasma membranes. The meaningful estimation of diffusion coefficients and their errors is however not straightforward, and is heavily dependent on sample type, acquisition method, and equipment used. Many approaches require advanced computing and programming skills for their implementation.FindingsHere we present TrackArt software, an accessible graphic interface for simulation and complex analysis of multiple particle paths. Imported trajectories can be filtered to eliminate spurious or corrupted tracks, and are then analyzed using several previously described methodologies, to yield single or multiple diffusion coefficients, their population fractions, and estimated errors. We use TrackArt to analyze the single-molecule diffusion behavior of a sphingolipid analog SM-Atto647N, in mica supported DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine) bilayers. Fitting with a two-component diffusion model confirms the existence of two separate populations of diffusing particles in these bilayers on mica. As a demonstration of the TrackArt workflow, we characterize and discuss the effective activation energies required to increase the diffusion rates of these populations, obtained from Arrhenius plots of temperature-dependent diffusion. Finally, TrackArt provides a simulation module, allowing the user to generate models with multiple particle trajectories, diffusing with different characteristics. Maps of domains, acting as impermeable or permeable obstacles for particles diffusing with given rate constants and diffusion coefficients, can be simulated or imported from an image. Importantly, this allows one to use simulated data with a known diffusion behavior as a comparison for results acquired using particular algorithms on actual, “natural” samples whose diffusion behavior is to be extracted. It can also serve as a tool for demonstrating diffusion principles.ConclusionsTrackArt is an open source, platform-independent, Matlab-based graphical user interface, and is easy to use even for those unfamiliar with the Matlab programming environment. TrackArt can be used for accurate simulation and analysis of complex diffusion data, such as diffusion in lipid bilayers, providing publication-quality formatted results.

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Multicolour single molecule imaging on cells using a supercontinuum source

Cited by 14 publications

References 16 publications

Postnatal Migration of Cerebellar Interneurons

Postnatal Migration of Cerebellar Interneurons

Multicolour Single Molecule Imaging in Cells with Near Infra-Red Dyes

TrackArt: the user friendly interface for single molecule tracking data analysis and simulation applied to complex diffusion in mica supported lipid bilayers

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