Dr Alex Corbett

Research Interests and Experience

 High_speed_imaging

High speed 3D microscopy

One of the main challenges of imaging networks of excitable cells is the speed with which imaging information is required. To measure the passing of an action potential at a single point requires a temporal resolution of less than a millisecond. Tracking electrical activity across multiple cells requires correspondingly higher sampling rates. In addition to this, the cells of interest will typically be distributed across a relatively large three-dimensional volume.

In order to meet these imaging challenges, new approaches to acquiring the desired information are needed. In this project, recently funded by the Next Generation Optical Microscopy initiative, we are looking to combine several complimentary imaging technologies that will allow imaging information to be quickly and efficiently recovered from arbitrary points within a large (100µm cube) tissue volume.

 

Key collaborators:

-          Dr. Gil Bub, Dept. Physiology Anatomy and Genetics, Oxford University, UK

-          Dr. Ed Mann, Dept. Physiology Anatomy and Genetics, Oxford University, UK

-          Dr. Roger Light, Dept. of Engineering, Nottingham University, UK

-          Dr. Kevin Webb, Dept. of Engineering, Nottingham University, UK

-          Prof. Tony Wilson, Dept. Engineering Science, Oxford University, UK

 

3D calibration specimen

Volumetric calibration tools using laser writing

In high resolution optical microscopy, there is an increasing need for quantitative structural information. For example, rather than saying that heart cell volumes are larger in diseased tissue than in the control, there is a need to obtain reliable values for the precise cell volumes in both tissues. To achieve this requires the size and shape of the imaging volume to be known exactly. For most light microscopes, flat specimens with a known surface feature size can be used to calibrate the lateral (XY) scale. The scale in Z is then determined by the calibration of the motorised stage used to translate the specimen. In remote focussing microscopes, the stage remains fixed, and the excitation is swept over the specimen. An alternative means of calibrating the Z-scale is then required. Laser writing of features into fluorescent specimens could be one means of obtaining this calibration. As well as being easily portable and robust, these specimens allow the Z-scale to be calibrated continuously over a range of depths, rather than using a single point to point measurement of fixed size, as in most SIP charts used today.

 

Key collaborators:

-          Dr. Patrick Salter, Dept. Engineering Science, Oxford University, UK

-          Dr. Simon Tuohy, Dept. Engineering Science, Oxford University, UK

-          Prof. Tony Wilson, Dept. Engineering Science, Oxford University, UK


Publications:

Alex Corbett, Rebecca Burton, Patrick Salter, Simon Tuohy, Martin Booth, Tony Wilson, and Gil Bub. “Quantifying distortions in two-photon remote focussing microscope images using a volumetric calibration specimen” Frontiers in Physiology, Cardiac Electrophysiology subtopic (2014, in press)

 

 cardiac_imaging

Oblique plane imaging of cardiac tissue   

The muscle tissue of the heart is made up of many cardiomyocyte cells. In healthy tissue, these rectangular cells stack together like bricks in a wall. When these cells contract together they are able to generate macroscopic changes in the size of the chambers of the heart. Of particular interest to physiologists is the spacing between sub-cellular features (sarcomeres). The sarcomere length (SL) determines the contractility of individual cells and is believed to vary between disease models. Direct imaging of sarcomeres in the arrested intact rodent heart is made difficult by the small size (2µm) and small natural variability (<10%) in SL. This requires precise measurements to avoid artificially broadening the distribution of SL measurements. One possible measurement error arises from the unknown inclination angle of the cell with respect to the focal plane of the microscope (α). Not accounting for tissue orientation would broaden the range of measured SL values, blurring out distinctions between disease models in the process. Finally, residual bulk motion of the tissue requires the image data to be acquired very quickly to temporally freeze tissue motion.

By imaging individual cells in two oblique planes it is possible to obtain two orthogonal views of the cell. As individual cell images are small (100x200 pixels) they can be acquired quickly (<200ms) and avoid movement artefacts. The orthogonal views of the cell allow the value of α to be uniquely recovered and reduce the broadening of the SL measurements by 23%. This then allows fine distinctions in SL between disease models to be determined.


Key collaborators:

-          Dr. Gil Bub, Dept. Physiology Anatomy and Genetics, Oxford University, UK

-          Dr. Ed Mann, Dept. Physiology Anatomy and Genetics, Oxford University, UK

-          Prof. Tony Wilson, Dept. Engineering Science, Oxford University, UK

 

Publications:

Edward Botcherby*, Alex Corbett*, Rebecca A. B. Burton*, Chris W. Smith, Christian Bollensdorff, Martin J. Booth, Peter Kohl, Tony Wilson, and Gil Bub. “Fast Measurement of Sarcomere Length and Cell Orientation in Langendorff-Perfused Hearts Using Remote Focusing Microscopy.” Circulation Research 113 (7): 863–70 (2013)  *Joint first author

 

SHG_imaging

Monitoring transmembrane potential with second harmonic generation dyes

One of the great advantages of optical microscopy over other medical imaging modalities is the sheer variety of molecular reporters. In addition to label-free methods for recovering information about the chemical environment, dye molecules can be introduced to identify changes in temperature, mobility, acidity and even the local electric field.

This project investigated the use of a novel class of porphyrin dyes to identify changes in the transmembrane potential. Optically monitoring the action potential of excitable cells, rather than the downstream surge in calcium ions, is of particular interest to neuroscientists and cardiologists. Porphyrin dyes can be engineered to have a high degree of hyperpolarisability. When excited by a powerful electric field from a laser, the large hyperpolarisability results in a significant amount of non-linear scattering called second harmonic generation (SHG). The amount of SHG produced by the dye is determined by the local electric field. When the dye is embedded in the membrane of an excitable cell, SHG can be used to monitor the electric field across the membrane and identify changes in action potential.

One of the key features of a successful voltage-sensitive dye is the way it orientates itself in the cell membrane. In a related project, it was possible to combine the information from several different emission mechanisms (single photon fluorescence, two photon fluorescence and SHG) to place constraints on the orientation of different dye molecules with respect to the membrane surface.


Key collaborators:

-          Prof. Harry Anderson, Dept. of Chemistry, Oxford University, UK

-          Prof. Hagan Bayley, Dept. Dept. of Chemistry, Oxford University, UK

-          Dr. James Reeve, Dept. Dept. of Chemistry, Oxford University, UK

-          Prof. Tony Wilson, Dept. Engineering Science, Oxford University, UK

 

Publications:

J. E. Reeve, A. D. Corbett, T. Wilson, H. Bayley and H. L. Anderson, “Electric field sensitive porphyrins” Angewandte Chemie 125(34):9214–18 (August 2013)

J. E. Reeve, A. D. Corbett, I. Boczarow, T. Wilson, H. Bayley and H. L. Anderson, “Probing the orientational distribution of dyes in membranes through multiphoton microscopy” Biophysical Journal 103(5) 907-917 (Sept 2012)

 

Previous Research

 Ocular aberration detection

Adaptive Optics for Retinal imaging

The ability to image the light sensitive tissue at back of the eye is limited by the quality of the cornea and crystalline lens. For the purposes of vision, these biological lenses are often adequate to form an image the outside world onto the retina. However, when using these lenses to image the back of the eye the lens quality is not sufficient to allow us to see the fine structures which provide key indicators of retinal health. To image fine retinal structures requires accurate compensation of the lens imperfections. As the eye is constantly undergoing small changes which degrade the retinal image, real time compensation is required through adaptive optics.

There are several techniques which can be used to characterise the aberrations of the human eye. The challenge is to obtain a high precision correction of ocular aberrations at a rate which is fast enough to allow imaging of the retinal tissue region of interest. Holography is one method which allows for very rapid detection of the kind of large aberrations seen in the human eye. Studying ocular aberrations for a population of subjects, it is possible to exploit some of the properties of the statistics to optimise the detection of aberrations both for the individual and for the population as a whole. Computer modelling of the semi-coherent light scattered back from the retina can also be used to further inform the sensor design.

 

Key collaborators:

-          Prof. Tim Wilkinson, Dept. of Engineering, Cambridge University, UK

-          Dr. Luis Diaz-Santana, Division of Optometry and Visual Science, City University, UK

-          Dr. Diego Gil Leyva, Dept. of Engineering, Cambridge University, UK

 

Publications:

A. D. Corbett, T. D. Wilkinson, J. J. Zhong, and L. Diaz-Santana, "Designing a Holographic Modal Wave Front Sensor for the Detection of Static Ocular Aberrations", Feature Issue "Advances in Retinal Imaging", J. Opt. Soc. Am. A. (Dec 2006)

A. D. Corbett, D. Gil  Leyva,  L.  Diaz-Santana, T. D. Wilkinson,  J. J. Zhong, “Characterising a holographic modal phase mask for the detection of ocular aberrations”, Proc. SPIE 6018, 5th International Workshop on Adaptive Optics for Industry and Medicine (June 2006)

J. J. Zhong ; D.  Gil Leyva ; A. D. Corbett ; L.  Diaz-Santana ; T. D. Wilkinson, “Mirror-mode sensing with a holographic modal wavefront sensor”, Proc. SPIE 6018, 5th International Workshop on Adaptive Optics for Industry and Medicine (June 2006)