We present a robust, low-cost single-shot implementation of differential phase microscopy utilising a polarisation-sensitive camera to simultaneously acquire 4 images from which the phase gradients and quantitative phase image can be calculated.
We present a robust, low-cost neural network-based optical autofocus system that can operate over a range of ±100μm with submicron precision, enabling automated high-content super-resolved imaging with a 1.3 NA objective lens.
Super-resolved microscopy techniques have overcome the diffraction limit to provide image resolutions approaching the scale of fluorescent labels. However, many of these techniques require significant experimental resources and expertise and impose long image data acquisition times, making it difficult to acquire super-resolved data from sufficiently large sample numbers to overcome intrinsic biological variation. We have worked to make stimulated emission depletion (STED) microscopy and single molecule localisation microscopy (SMLM) more straightforward to implement and more practical to image larger numbers of cells. Here we present work in progress developing easySLM STED and easySTORM, including a new modular microscope frame that we believe can make it easier to prototype microscopy techniques and to implement and maintain them in lower resourced settings.
Fluorescence lifetime imaging (FLIM) provides a means to contrast different molecular species and to map variations in the local fluorophore molecular environment, including to read out Förster resonant energy transfer (FRET), e.g. to assay protein interactions or genetically expressed FRET biosensors. We have implemented wide-field time-gated FLIM in a modular open automated microscopy platform for high content analysis (HCA). To demonstrate its relevance to drug discovery, we have demonstrated the capability of our openFLIM HCA platform to assay interactions of low copy number endogenous proteins in yeast cells labelled with fluorescent proteins. We have also demonstrated the capability of multiwell plate FLIM assays to provide readouts of a FRET biosensor in 2-D and 3-D cell cultures.
Among super-resolved microscopy (SRM) methods, single molecule localisation microscopy techniques, such as photo-activated localisation microscopy (PALM) [1] and stochastic optical reconstruction microscopy (STORM) [2], enable imaging beyond the classical diffraction limit to gain new insights in subcellular biological processes with relatively simple instrumentation. This has led to a number of low-cost instruments, e.g. for STORM microscopy [3-6], which can benefit from an array of software tools for the single molecule localisation microscopy (SMLM) data analysis [7]. Our low-cost “easySTORM” approach [4] implements dSTORM [8] with multimode diode lasers and optical fibres to provide STORM images with fields of view up to ~125 μm diameter using μManager [9] to control the image data acquisition and ThunderSTORM [10] to analyse the SMLM data. We and others [11,12] are motivated to develop automated SMLM for high content analysis (HCA) that enable rapid imaging of sample arrays, allows statistical analysis of samples that may vary in terms of labelling and biological heterogeneity and enable moderate throughput screening applications.
Multispectral fluorescence lifetime imaging (FLIM) using two photon microscopy as a non-invasive technique for the
diagnosis of skin lesions is described. Skin contains fluorophores including elastin, keratin, collagen, FAD and NADH.
This endogenous contrast allows tissue to be imaged without the addition of exogenous agents and allows the in vivo
state of cells and tissues to be studied. A modified DermaInspect® multiphoton tomography system was used to excite
autofluorescence at 760 nm in vivo and on freshly excised ex vivo tissue. This instrument simultaneously acquires
fluorescence lifetime images in four spectral channels between 360-655 nm using time-correlated single photon counting
and can also provide hyperspectral images. The multispectral fluorescence lifetime images were spatially segmented and
binned to determine lifetimes for each cell by fitting to a double exponential lifetime model. A comparative analysis
between the cellular lifetimes from different diagnoses demonstrates significant diagnostic potential.
To aid the in vivo diagnosis of skin lesions, we present the design and implementation of a 4 channel FLIM
detector and a hyperspectral imaging detector into a clinically licensed commercial two-photon tomograph. We
have also implemented image segmentation algorithms to facilitate the automated processing of the large
volumes of data produced. The first detector is based on multispectral time correlated single photon counting,
providing four channel fluorescence lifetime images. The second detector is a prism-based CCD hyperspectral
imager. These detectors provide the capability to extract the relative content and state of autofluorescence
compounds present in biological tissue.
We describe an optically-sectioned FLIM multiwell plate reader that combines Nipkow microscopy with
wide-field time-gated FLIM, and its application to high content analysis of FRET. The system acquires
sectioned FLIM images in <10 s/well, requiring only ~11 minutes to read a 96 well plate of live cells
expressing fluorescent protein. It has been applied to study the formation of immature HIV virus like
particles (VLPs) in live cells by monitoring Gag-Gag protein interactions using FLIM FRET of HIV-1 Gag
transfected with CFP or YFP. VLP formation results in FRET between closely packed Gag proteins, as
confirmed by our FLIM analysis that includes automatic image segmentation.
Fluorescence intensity imaging and fluorescence lifetime imaging microscopy (FLIM) using two photon microscopy
(TPM) have been used to study tissue autofluorescence in ex vivo skin cancer samples. A commercially available system
(DermaInspect®) was modified to collect fluorescence intensity and lifetimes in two spectral channels using time
correlated single photon counting and depth-resolved steady state measurements of the fluorescence emission spectrum.
Uniquely, image segmentation has been used to allow fluorescence lifetimes to be calculated for each cell. An analysis
of lifetime values obtained from a range of pigmented and non-pigmented lesions will be presented.
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