RESUMO
Single-molecule Förster Resonance Energy Transfer (smFRET) is a powerful technique capable of resolving both relative and absolute distances within and between structurally dynamic biomolecules. High instrument costs, and a lack of open-source hardware and acquisition software have limited smFRET's broad application by non-specialists. Here, we present the smfBox, a cost-effective confocal smFRET platform, providing detailed build instructions, open-source acquisition software, and full validation, thereby democratising smFRET for the wider scientific community.
RESUMO
Previous studies have used analysis of Ca(2+) sparks extensively to investigate both normal and pathological Ca(2+) regulation in cardiac myocytes. The great majority of these studies used line-scan confocal imaging. In part, this is because the development of open-source software for automatic detection of Ca(2+) sparks in line-scan images has greatly simplified data analysis. A disadvantage of line-scan imaging is that data are collected from a single row of pixels, representing only a small fraction of the cell, and in many instances x-y confocal imaging is preferable. However, the limited availability of software for Ca(2+) spark analysis in two-dimensional x-y image stacks presents an obstacle to its wider application. This study describes the development and characterization of software to enable automatic detection and analysis of Ca(2+) sparks within x-y image stacks, implemented as a plugin within the open-source image analysis platform ImageJ. The program includes methods to enable precise identification of cells within confocal fluorescence images, compensation for changes in background fluorescence, and options that allow exclusion of events based on spatial characteristics.
