Particle Tracking for biological and medical applications
In addition to 3D and 4D imaging the diffraction grating multi-plane imaging technique has been successfully applied to particle tracking. An initial feasibility study (EPSRC grant EP/E01500X/1, in collaboration with the HW Nano-Optics Group) demonstrated that an image quality metric called Image Sharpness (IS) can be used to provide unambiguous, accurate, depth measurement. IS was originally used in Astronomy to aid in the focussing of telescopes. It's a single number, which is maximised for an in-focus, unaberrated image[1].
We have developed an algorithm, based on IS and Maximum Likelihood Estimation, which allows us to extract accurate 3D position information from fluorescent particles contained within the focal volume imaged by the grating. We aim to use this to perform particle tracking of fluorescently tagged bio-particles in vivo or in vitro. We have performed initial testing of this method using laser illuminated nano-holes (210nm dia.) in a metal film to simulate biological particles. Using piezo positioning stages we are able to move these 'particles' by known amounts to test the accuracy of our particle tracking algorithm. Experimental testing has shown that this method can produce unambiguous particle position information in x,y and z with 12nm accuracy over the focal volume of the grating (a depth range of 2.4μm in this example) [2] . We aim to eventually be capable of tracking fluorescent particles moving at Brownian motion speeds which, for example, would be useful for tracking and mapping the trajectories of viruses as they attack cells [3].
It would also be possible to combine our wavefront sensing expertise with these bio-imaging measurements. Using the same data as the particle tracking algorithm (i.e. without further experimental steps) we can obtain wavefront sensing results which allow us to characterise the aberrations of our optical system as well as measure specimen induced aberrations. Some static system aberrations (e.g. Spherical Aberration) could be pre-compensated by altering the design of the diffraction grating, while dynamic aberrations could perhaps be corrected using Adaptive Optics. Characterisation and subsequent rejection of the non-defocus specimen-induced aberrations would also help to increase the accuracy of our particle tracking measurements.
References and suggestions for further reading...
We have also produced a training document designed to be both a tutorial for the complete beginner and a handy reference guide for those already familiar with the diffraction grating multiplane imaging technique. To request a PDF copy of "Introduction to Diffraction Gratings and their Application in 3D Imaging"[4] simply fill our our publication request form.
1. R. A. Muller and A. Buffington, Real-Time Correction of Atmospherically Degraded Telescope Images through Image Sharpening, JOSA A, 64, 1200-1210 (1974).
2. P.A.Dalgarno, H.I.C.Dalgarno, A. Putoud, et al., Three dimensional biological imaging and nano particle tracking, submitted to Optics Express, currently under review.
3. G. Seisenberger, M.U. Ried, T. Endress, et al., Real-Time Single Molecule Imaging of the Infection Pathway of an Adeno-Associated Virus, Science, 294(5548): p. 1929-1932, (2001).
4. H.I.C. Dalgarno, Introduction to Diffraction Gratings and their Application in 3D Imaging, training document, 2008.
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Our Research
Find information on our current research projects, and our research interests both past and present...
Life Sciences Interface
- Introduction
- Real-time 3D imaging
- 3D Live-cell imaging
- Particle Tracking
- Sperm Motility
- Bioimage Gallery