At Imperial College London, we have developed a range of instruments for FLIM, including microscopes, endoscopes and optical tomography systems, and we have developed open source software tools for FLIM data acquisition and analysis. For practical FLIM with rapid image acquisition and low phototoxicity that is compatible with live cell imaging, we have focussed on wide-field time gated FLIM using a gated optical intensifier (GOI). This approach provides robust FLIM data acquisition that is sufficiently rapid to enable automated multiwell plate FLIM for high content analysis, including FLIM/FRET assays and label-free readouts of cellular autofluorescence to report changes in cell metabolism. We have also implemented FLIM in laser scanning microscopes utilising time-correlated single photon counting (TCSPC), including microscopes combining TCSPC with polarisation-resolved detection for studies complementing FLIM/FRET data with time-resolved fluorescence anisotropy measurements and laser scanning confocal FLIM endomicroscopy for in vivo FLIM/FRET studies to quantify intracellular chemotherapeutic drug-target binding.
Our open source FLIM data analysis software, FLIMfit, is applicable to both wide-field time-gated FLIM and TCSPC data and provides global analysis capabilities.
For FLIM data acquisition, TCSPC FLIM is available with a range of commercial microscopes or can be implemented as an commercially available upgrade with proprietary software. Wide-field time-gated FLIM can be implemented using our open source FLIM software to control the instrument and the data acquisition. This is available as a MicroManager plug-in for manual FLIM microscopy, as a module for our new MicroManager-based HCA platform or FLIM HCA can be implemented using our standalone FLIM HCA MicroManager plug-in that is supported by detailed instructions and hardware component lists in this JoVE paper.
Oblique Plane Microscopy (OPM) is a type of light-sheet fluorescence microscopy that uses a single microscope objective to generate the illumination light sheet and detect the resulting fluorescence. The light sheet is tilted with respect to the optical axis and correction optics are placed before the camera to enable the system to image the tilted plane that is illuminated by the illumination light sheet. As OPM only requires a single microscope objective near to the sample, it is compatible with conventional commercial microscope frames and sample mounting techniques, including arrays of samples in 96 and 384-well plates. Our recent publications on this work can be found here.
FLIM data analysis software: FLIMfit, our open source MATLAB-based software that provides a range of fitting techniques including global analysis. This will run on a standard personal computer or laptop but we recommend investing in at least 64 GB RAM and a reasonable multicore processor if you intend to analyse large FLIM data sets. The FLIMfit software package is available as a client for the OMERO platform and can also be used as a stand-alone MATLAB application.
Wide-field time-gated FLIM data acquisition: our open source software, openFLIM-GOI, is a MicroManager plug-in and runs on a standard personal computer requiring ~8 GB RAM with USB and serial ports to interface with the equipment components.
FLIM high content analysis: our openFLIM-HCA software for automated multiwell plate wide-field time-gated FLIM (instrument control and data acquisition) using wide-field time-gated FLIM of samples arrayed in a 96 well plate is written in MicroManager.
The technology developed in this project will contribute to the openScopes initiative, that aims to provide the know-how and open source software tools to implement a suite of open source instrumentation for light microscopy and related modalities that are intended to enable researchers to implement, maintain and upgrade advanced optical imaging capabilities at relatively low cost, including in less affluent research communities where technical support may not be available. These capabilities include FLIM, optical projection tomography, high content analysis and super-resolved microscopy. In general they can be implemented as upgrades to existing commercial microscope frames or they can be constructed around the new, cost-effective, modular openFrame platform, for which CAD files may be downloaded for laboratories to fabricate their own components or they can be purchased commercially.
openFLIM hardware: For a list of components to assemble a wide-field time-gated FLIM microscope system or an optical sectioning wide-field FLIM microscope utilising a Nipkow spinning disc confocal scanner, please download the openFLIM hardware list.