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Optimized processing and analysis of conventional confocal microscopy generated scanning FCS data.

ABSTRACT: Scanning Fluorescence Correlation Spectroscopy (scanning FCS) is a variant of conventional point FCS that allows molecular diffusion at multiple locations to be measured simultaneously. It enables disclosure of potential spatial heterogeneity in molecular diffusion dynamics and also the acquisition of a large amount of FCS data at the same time, providing large statistical accuracy. Here, we optimize the processing and analysis of these large-scale acquired sets of FCS data. On one hand we present FoCuS-scan, scanning FCS software that provides an end-to-end solution for processing and analysing scanning data acquired on commercial turnkey confocal systems. On the other hand, we provide a thorough characterisation of large-scale scanning FCS data over its intended time-scales and applications and propose a unique solution for the bias and variance observed when studying slowly diffusing species. Our manuscript enables researchers to straightforwardly utilise scanning FCS as a powerful technique for measuring diffusion across a broad range of physiologically relevant length scales without specialised hardware or expensive software.

SUBMITTER: Waithe D

PROVIDER: S-EPMC6026296 | biostudies-literature | 2018 May

REPOSITORIES: biostudies-literature

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Optimized processing and analysis of conventional confocal microscopy generated scanning FCS data.

Waithe Dominic D Schneider Falk F Chojnacki Jakub J Clausen Mathias P MP Shrestha Dilip D de la Serna Jorge Bernardino JB Eggeling Christian C

Methods (San Diego, Calif.) 20170928

Scanning Fluorescence Correlation Spectroscopy (scanning FCS) is a variant of conventional point FCS that allows molecular diffusion at multiple locations to be measured simultaneously. It enables disclosure of potential spatial heterogeneity in molecular diffusion dynamics and also the acquisition of a large amount of FCS data at the same time, providing large statistical accuracy. Here, we optimize the processing and analysis of these large-scale acquired sets of FCS data. On one hand we prese ...[more]

PMID: 28963070

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Project description:Objectives:To validate novel non-contacting Confocal-Laser-Scanning-Microscopy (CLSM) methodology with conventional Contacting Profilometry (CP) measures investigating brushing or dab-on of stannous-fluoride dentifrice on early aggressive dentine erosion. Methods:75 polished human dentine samples were prepared and eroded in agitated 6% citric acid then randomly allocated into 5 intervention groups; artificial saliva control (1); controlled use of a pressure sensitive counter-rotating oscillatory powered toothbrush with sodium-fluoride NaF (2) or stannous-fluoride SnF2 (3), and dab-on application of NaF (4) or SnF2 (5). Samples underwent three cycles of intervention and 2-min agitated 6 % citric acid challenges. CLSM images were taken and 3D reconstructions produced of step height using a developed software algorithm. In addition, 20 % samples were randomised and profiled using CP to measure step height and surface roughness. Vickers's diamond micro-hardness testing was carried out on all samples. Results:Comparing CLSM and CP; Pearson correlation was 0.77 and Intra-class correlation 0.81 (p = 0.01). There were no significant statistical differences in step height between groups using both CLSM and CP. From baseline, SnF2 brushing (3) increased micro-hardness more than control (1) (p = 0.03), NaF (4) and SnF2 dab-on (5) (p ? 0.001), and increased surface roughness more than control (p = 0.02), NaF brushing (2) and NaF dab-on (4) (p ? 0.017). Dab-on of SnF2 (5) produced rougher surfaces than control (1) (p = 0.014) and reduced hardness compared with NaF brushing (p = 0.04). Conclusions:Good agreement and correlation exists between CLSM and CP measures in dentine. There were no significant differences in surface loss after interventions between groups. Compared with control, SnF2 application increased dentine surface roughness and SnF2 controlled powered brushing application increased dentine hardness, likely caused by exposure of uneroded dentine. Clinical significance:Isosurfaces produced using CLSM can be used to represent dentine step height loss. They show good correlation and agreement with conventional CP measures, following early aggressive erosion-abrasion cycles of dentine. The CLSM and computer algorithm therefore provides an accurate, standalone and non-contacting three-dimensional measurement of early dentine wear. Stannous-fluoride brushing, and dab-on application offer no benefits following early aggressive erosion in dentine. To reduce dentine wear, limiting erosive challenges and avoiding brushing post-erosion is advised.

Dataset Information

Optimized processing and analysis of conventional confocal microscopy generated scanning FCS data.

Publications

Optimized processing and analysis of conventional confocal microscopy generated scanning FCS data.

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