Journal: Nature Communications

Article Title: Measurement of nanoscale three-dimensional diffusion in the interior of living cells by STED-FCS

doi: 10.1038/s41467-017-00117-2

Figure Lengend Snippet: Schematic principle of the SPLIT-FLCS method. a A pulsed laser excitation source is coupled with a CW-STED beam to exploit the variations in the fluorescence dynamics observed in the nanosecond time scale ( t < T ). b The spatial variations of fluorescence lifetime within the effective observation volume of the microscope can be modeled as a gradient in the radial direction, with the lifetime value being the highest at the center and decreasing towards the periphery. A molecule transiting within the observation volume will emit photons with different fluorescence lifetime according to the radial position. c The temporal fingerprints associated to different spatial positions can be used to weight the photons based on their time of arrival in the nanosecond time scale. For instance, the decay associated to the region defined by radial position r < r 1 can be used to generate a statistical filter that, by over-weighting the late-arriving photons, sort out only the intensity emitted from a smaller effective observation volume. d Correlation of the weighted photons on the time scale relevant for molecular diffusion ( t > 1 μs >> T ) results in a filtered ACF associated to fluctuations on a smaller effective observation volume, namely an ACF which is narrower and has larger amplitude

Article Snippet: The fluorescence light was collected by the same objective lens, de-scanned, and passed through the dichroic mirrors as well as through a fluorescence band pass filter (ET Bandpass 525/50 nm, AHF analysentechnik) before being focused (focal length 60 mm, AC254-060-A-ML, Thorlabs) into a fiber pigtailed single-photon avalanche diode (PDM Series, Micro Photon Devices, Bolzano, Italy).

Techniques: Fluorescence, Microscopy, Diffusion-based Assay