Aim of the PhD Project:
- Kidney disease is highly complex, typically requiring light microscopy, immunohistology and electron microscopy (EM) for diagnosis.
- EM is not available to much of the world’s population
- We aim to replace EM with low-cost dSTORM microscopy for diagnosis of kidney diseases, including structural abnormalities of basement membrane and immune complex disease
Project Description / Background:
EM is useful in the histopathology of ~50% of native kidney biopsies and essential for the diagnosis of ~20%, making the use of EM prudent for all kidney biopsies in developed countries. However, the high instrumentation cost and expert staff required for EM histopathology mean that it is not available to many less well-resourced communities. EM is required for the diagnosis of kidney diseases associated with structural abnormalities of the basement membrane (e.g. Alport’s syndrome), fibrillar disease (e.g. Fibrillary glomerulopathy and immunotactoid glomerulopathy) and rare genetic diseases such as Fabry disease or LCAT deficiency. It is also used to document morphological changes in podocytes, and to document shape and position of immune complex and/or complement fragment deposition relative to the glomerular basement membrane (GBM).
We propose to explore the replacement of EM with super-resolved microscopy (SRM) following earlier work using SIM  and STORM  to study renal podocyte substructure and protein organisation in the GBM. This prior work demonstrates the potential of this project but was realised with expensive commercial SRM instrumentation and the STORM work was undertaken as a research project with mouse tissue (and non-clinically approved antibodies). We propose to develop a low-cost approach that could be accessible by low and middle-income countries (LMIC) clinicians to this by adapting our low-cost super resolving microscopy technique, “easySTORM”  to image clinically relevant proteins, such as IgG in GBM, with clinically validated antibodies and to develop low-cost instrumentation and practical protocols to work with existing biopsy samples such as those embedded in paraffin.
easySTORM provides a low-cost implementation of dSTORM  using cheap multimode diode lasers and multimode fibres to provide sufficiently high excitation powers for SRM of fields of view exceeding 100 μm diameter. Recognising that the microscope frame is the most significant cost of the resulting SRM instrument, we are developing a new modular inverted microscope platform designed to be manufactured on standard machining tools with a component cost <£3000. This can be easily configured for imaging immunolabelled samples and light microscopy and can be combined with low cost CMOS cameras (<£1000). With the addition of a motorised x-y stage (~£6000) and piezo-driven focus (£6000), we propose that this instrument can be converted into an automated slide scanner for normal and super-resolved imaging. Thus, we aim to develop a low-cost modular microscope system, particularly targeting kidney disease, that could be applied more widely for clinical histopathology and research. This instrument would use open source software, particularly μManager for instrument control and ImageJ/Icy for analysis with image data to be stored in an OMERO database. As well as sharing all the relevant software, we would publish detailed parts lists and instructions to enable other researchers, particularly in LMIC, to assemble and/or maintain their instruments – thus avoiding the significant cost and delays associated with maintaining commercial instruments.
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