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Developing a microscope system for simultaneous imaging of cells in multiple Z-dimensions

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  • Full or part time
    Prof Robin May
    Prof D Hodson
  • Application Deadline
    No more applications being accepted
  • Funded PhD Project (UK Students Only)
    Funded PhD Project (UK Students Only)

Project Description

Research interests/description of main research theme:
AIMS
This project aims to develop and test a novel microscope that will enable truly simultaneous imaging of several axial ‘slices’ (Z-sections) through a live sample, something that is not currently possible on existing commercial microscopes.

BACKGROUND
Movement is a defining feature of biological systems, but one that leads to a fundamental trade-off between sample movement and image resolution, especially when imaging in three dimensions. Most three-dimensional live imaging techniques project a two-dimensional image plane that is then moved up and down in the Z-dimension by adjusting the sample or focal plane. Technical advances such as piezo objective motors can achieve rapid image slicing in the Z-plane but no truly simultaneous three-dimensional imaging system currently exists. This is a particular problem when an object is to be reconstructed in 3D but is moving whilst Z-planes are being imaged, or if a fluorophore is redistributing rapidly between two membranes (e.g. during vesicle trafficking).

HYPOTHESIS
These problems could be solved if a microscope was able to generate three or more genuinely simultaneous images, displaced in Z by a known distance (e.g. to generate an image of the top and bottom of a cell at the same time). Here we propose to build and test a system to do exactly this.

EXPERIMENTAL METHODS & RESEARCH PLAN
We will start by building a microscope system that generates three simultaneous Z-plane images from an object at fixed Z-separations of 3 μm (see figure). Light emerging from the illuminated sample will be split into three paths using an OptoSplit III. One light path will have no additional lensing, whilst the other two will have convex and concave lenses in the light path to refocus at +3μm and -3μm respectively and collected (as three split images) on an sCMOS camera. We will initially test this approach using Brownian motion of fluorescent polystyrene beads of varying sizes and using back calibration to demonstrate correct estimation of bead sizes in 3D.
We will then proceed to test the system on live cell biological phenomena by exploiting macrophage phagocytosis as our paradigm; a process that has been characterized in great molecular detail by ourselves and others. We will expose GFP-actin expressing macrophages labelled with cytoplasmic membrane dyes to opsonised fluorescent beads and then produce simultaneous Z-plane images of the host membrane extending rapidly over the bound particle.

Lastly, we will test the use of deformable lenses aiming to increase the versatility of this system. Such lenses have been extensively developed by the mobile telephone industry and are thus both reliable and inexpensive. By replacing the lenses in the Sim-Z lightpath by tuneable deformable lenses, it is possible to vary the focal displacement of the three Z-planes. Thus one could generate a system in which three Z images could be captured simultaneously at any range of focal depth permitted by the deformable lenses.

Person Specification
Applicants should have a strong background in either a) microscopy/cell biology or b) optical physics/engineering , or related fields. Additional expertise in image analysis and processing would be a strong advantage. Enthusiasm and commitment to working in a multidisciplinary team of microbiologists, immunologists and microscopists is critical. Candidates should hold or realistically expect to obtain at least an Upper Second Class Honours Degree in either the life sciences/medicine or the physical sciences.

How good is research at University of Birmingham in Biological Sciences?

FTE Category A staff submitted: 42.80

Research output data provided by the Research Excellence Framework (REF)

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