Developing Novel Time-Resolved Optical Methods for High Throughput Measurement of Cells

Supervisor: Dr Gordon Hedley

School: Chemistry


This summer project would pursue proof-of-concept development of a new method to measure cells (both in simple counting terms but also more complex cytometry). This will be achieved by harnessing time-resolved (down to picosecond) multichannel confocal fluorescence microscopy.Most cytometry techniques utilise widefield imaging microscopy. Flow speeds and cell counting rates are limited by imaging CCD frame rates. In this project cells would instead be detected on multiple point detectors (single photon avalanche photodiodes) using picosecond event timing electronics that allows multiple detectors to be used in concert to “image” the same confocal volumesimultaneously, thus allowing near continuous temporal coverage of it, and thus much higher achievable cell count rates.To begin with and to simplify and develop the experimental methodology,fluorescent beads rather than cells willbe used, dissolved in water and held on a solution sample holder on an existing time-resolved fluorescence microscope.A sequential science package will be pursued:

•At first, the simple free diffusion of the beads in water will allow assessmentof fluorescentcount ratesthat are achievable. Effective variation of the “measurable throughput”of beads can be achieved with simple diffusion just by changing the concentration of them in the solution. The questionsto be answered are:what is the limit of distinguishably for separate“objects”(a single bead passing through the laserfocus)?And can this limit be altered by using multiple detectors (to provide gap-free temporalcoverage)?

•In the second phase the same measurements would now be made but in a microfluidic sample chamber, using a syringe to flow solution containingthe beads across the focal volume. The key questions to answer are: how does this compare to the diffusion results already measured? What arethe highest achievable flow rates?If a mixture of two beads with different coloured dyes in them is usedcanthey be distinguished (using a dichroic beamsplitter on two detectors)?

•In the final phase, a switch to fixedstained cells would be made and the proof-of-principle work brought tofruition by assessing how well this could work in a real system. Onward work would then be to assess the cytometry of the cells rather than simple distinguishability, and this would sit well in alonger-termoutlook and funding proposals.