Modular open-source microscopy: openFrame

In the Photonics Group of the Physics Department at Imperial College London, we are developing an open microscopy hardware platform that aims to enable researchers to rapidly implement modular, upgradeable, and easily maintained instruments with a range of add-on open source modules, such as excitation coupling systems for fluorescence microscopy, as well as commercial components. This approach is increasingly based around a core modular microscope frame (openFrame, developed in partnership with Cairn Research Ltd). We intend this microscopy platform to be controlled using µManager and will use open-source software wherever possible. We aim to share software well as the CAD designs and component lists so that other researchers can easily replicate the instrumentation or adapt it to their requirements.
You can find further information about this open microscopy platform at our Imperial College London biophotonics webpages or at www.opensScopes.com. While most of the instruments being developed within this Accelerator will be realised using open-source modules implemented on proprietary microscope frames, the M3M HCA microscope will be implemented as an openFrame-based instrument.

openFrame schematic

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.

Label-free segmentation

A key goal of this CRUK Accelerator is to study individual cell fate in 3D cell cultures, e.g., in response to chemotherapy, and to understand the heterogeneity in the response of cells to such interventions. To this end, it is necessary to resolve single cells and to track them over time. Whiles this can be undertaken using fluorescent labels of cellular structures, such labels can be toxic to the cells and the fluorescence process is itself phototoxic, which can limit the extent to which cell scan be imaged during time-lapse experiments. We are therefore exploring label-free approaches to image single cells with a view to track individual cell trajectories and to generate segmentation masks to be able to allocate detected fluorescence photons to specific cells.
As a first step, within the Photonics Group at Imperial, we have developed a novel single-shot, semi-quantitative phase contrast method called polarisation-resolved differential phase contrast microscopy (pDPC) that utilises a novel polarisation-sensitive camera. pDPC can be implemented using low phototoxicity near infrared radiation and be readily integrated with fluorescence microscopes.

openHCA

As part of their openScopes initiative, the Photonics Group at Imperial is developing low-cost, modular automated microscopes for high content analysis that are controlled by open-source software like µManager. At the heart of any HCA is an automated microscope with motorised x-y stage to move the field of view across an array of samples – typically in a multiwell plate format – with motorised z-drive for focussing and acquisition of z-stacks for 3D imaging. All the CRUK Accelerator instruments are fully motorised to enable HCA and we are developing open-source software to control the image data acquisition and are working on open-source image data analysis pipelines. Almost any imaging modality can be automated for HCA and we have already demonstrated FLIM HCA, OPM HCA, which are being refined as part of this CRUK Accelerator project, as well as super-resolved microscopy (SRM) HCA.
For unsupervised imaging of sample arrays, it is essential to implement an “autofocus” that can automatically determine the extent to which the instrument is out of focus and then correct this, typically by axially translating the microscope objective lens or the sample stage. This is necessary because the microscope can drift out of focus over time, e.g., due to thermal changes or mechanical relaxation of components, and because the focus condition of the microscope can change when the sample array is translated to image the next field of view. We have developed an open source optical autofocus system that can be implemented on proprietary microscope frames or on an openFrame instrument.



As part of our mission in this CRUK Accelerator to make advanced imaging capabilities more accessible, we are developing the M3M HCA instrument to be fully open-source, incorporating our open optical autofocus and µManager data acquisition software. We will also test novel low-cost, high performance cooled CMOS cameras and aim to integrate semi-quantitative phase contrast with multiphoton multibeam multiwell plate fluorescence imaging.

Open source software tools

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.

Open instrumentation

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.