How Multidisciplinarity can
Advance Lifescience
Understanding
Combining Optics, Image Processing,Computational Simulation to Aid
Biological Understanding
John Girkin
Department of Physics
University of Durham, UKj.m.girkin@durham.ac.uk© University of Durham 2023

Durham University© University of Durham 2023

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Durham, 3rd oldest English University
More than just a set for Harry Potter!
Small and research intensive
Members from 160 countries
Strength in multidisciplinary approach© University of Durham 2023

The City of Durham© University of Durham 2023

Outline• Philosophy• Mechanotransduction by Geometric
Modification• Summary© University of Durham 2023

Multidisciplinarity• Get the original question right• Open discussion accepting lack ofknowledge- No Stupid questions• “Transfer” technology/methods• Choose the right tool for the task inhand• Don’t over complicate!

© University of Durham 2023

Biology and the Physical Sciences• The Philosophical Approach

The technology/methodology should help the Biology© University of Durham 2023

Biology and the Physical Sciences

Multi-scale considerations
1 x 1027 in scale© University of Durham 2023

Combining micro-optics, image
analysis and computer simulation
to understand vascularendothelia sensing© University of Durham 2023

The Endothelium
Monolayer of cells
Innermost layer in all vasculature
Fully connected over whole body
10x more endothelial cells in you thanneurones
Controls almost everything…
• Regulates blood vessel formation(angiogenesis)• Prevents blood clotting• Regulates vascular permeability• Determines the extent of smoothmuscle proliferation.
• Controls arterial tone (regulate bloodflow)
The Circulatory, Respiratory, and Digestive Systems
http://www.uic.edu/classes/bios/bios100/lecturesf04am/lect20.htm•All without involving the nervoussystem• How?

© University of Durham 2023

Introduction to IP3R mediated Calcium waves
ER is a Ca2+ reservoir – [Ca]ER ~500µM
Cytoslic Ca2+ strongly buffered[Ca]Cyt ~ 70nM
[B]Cyt ~ 1mM• Locally elevated Ca2+ or IP3 opens anIP3R– Local source of cytosolic Ca2+ with current J0– First IP3R often opened by IP3 from gap
junction– “Diffusing” Ca2+ wavefront triggers acascade of IP3R openings© University of Durham 2023

Endothelium© University of Durham 2023

© University of Durham 2023

© University of Durham 2023

© University of Durham 2023

© University of Durham 2023

© University of Durham 2023

Visualising the Endothelium from inside
intact arteries• Desire– camera in a pressurised artery– Cellular Resolution on curved surface– wide field of view and high speed– Operating in fluorescence“Micro-endoscope for in vivo widefield high spatial resolution fluorescent imaging” C D
Saunter, S Semprini, C Buckley, J Mullins, J M Girkin, Biomedical Optics Express, 3, 1274,
2012© University of Durham 2023

The Technology

0.75 mm diameter
Prism gives 90° view to flow axis
Can use any conventional camera© University of Durham 2023

The Technology0.75 mm diameter
Prism gives 90° view to flow axis
Can use any conventional camera© University of Durham 2023

In Use© University of Durham 2023

ACh Dose Response
Large field of view (~200 endothelial cells)“Clusters of specialized detector cells provide sensitive and high fidelity receptor signaling in the intact
endothelium, C. Wilson, C. D. Saunter, J. M. Girkin, and J. G. McCarron, ” FASEB J., vol. 30, pp. 1–14, 2016.

© University of Durham 2023

Data Analysis:- Time Synchronisation

Align peak rise of each signal© University of Durham 2023

ACh Dose Response: More cells respond &magnitude of response increases

Ca2+ rise originated from an IP3-sensitive Ca2+ store as
signal went when blocked, caffeine no effect not RyR© University of Durham 2023

Increased pressure decreases activation ofendothelial cells

60 mmHg

160 mmHg“Pressure-dependent regulation of Ca 2+ signaling in the vascular endothelium,” C. Wilson, C. D.
© Universityof Durham
2023
Saunter, J. M. Girkin, and J. G. McCarron,J. Physiol.,vol. 593, no.
24, pp. 5231–5253, 2015.

Increased pressure decreases activation ofendothelial cells

Ca2+ signals are abolished bycyclopiazonic acid and 2-APB

60 mmHg

160 mmHg“Pressure-dependent regulation of Ca 2+ signaling in the vascular endothelium,” C. Wilson, C. D.
© Universityof Durham
2023
Saunter, J. M. Girkin, and J. G. McCarron,
J. Physiol.,vol. 593, no.
24, pp. 5231–5253, 2015.

Data Analysis:
Signally via Spacing of Receptors?

                                                                                !                       "        #    $        %       

• Complex but repeatable patterns seen
• Calcium computing?
              "  $               

© University of Durham 2023

   

Simple
SignalPathway  Time© University of Durham 2023

Ring Oscillator = Clock or Memory?
) *   (  $  +, Time-         © University of Durham 2023

How does the cell sense pressure?
• Calcium release from ER to cytosol by IP3R isreduced at higher lumen pressures• The IP3R receptor…

– Is tiny - tetramer of ~300 kDa molecules• Pressure differential small• Thermal noise large– Is deeply embedded within a cell– Has no known mechano-sensor© University of Durham 2023

Working hypothesis•

Artery expands with pressure•

Cells stretched in width•

Conservation of volume– Cell must thin in radialdirection•

Thin cells limit Ca2+ diffusion
within IP3R microdomains– Increased [Ca2+]– Decreased conc. gradientfrom store– Decreased injection
current
Lawton, P. F. et al. VasoTracker, a Low-Cost and Open Source Pressure Myograph
System for Vascular Physiology. Front. Physiol. 10, 99 (2019).

© University of Durham 2023

Computer Modelling• Diffusion model of Ca2+ and buffer• Used literature figures• Solved by simulation of coupled equations• Model single IP3R and micro-domain cluster• Change height of ER to maintain constantvolume© University of Durham 2023

Individual vs clustered IP3RClusterIP3R© University of Durham 2023

Modelling Results© University of Durham 2023

[Ca2+] at a cluster under thinning© University of Durham 2023

Fixed Tissue Imaging: Supporting Evidence
10µm

5µm

“Pressure-dependent regulation of Ca 2+ signaling in the vascular endothelium, C. Wilson, C. D. Saunter, J. M.
Girkin, and J. G. McCarron, ” J. Physiol., vol. 593, no. 24, pp. 5231–5253, 2015.

© University of Durham 2023

Summary• Now looking at signally through tosmooth muscle cells• Remember your core science skills• Ask questions and discuss openly• Vascular Pressure Sensing possible
without direct sensors• Simple Models often work
Lawton, P. F. et al. Multi-plane remote refocusing epifluorescence microscopy toimage dynamic Ca 2 + events. Biomed. Opt. Express 10, 5611–5624 (2019).

© University of Durham 2023

Acknowledgements• University of Strathclyde– Dr Calum Wilson, Prof John McCarron,
Dr Charlotte Buckley• University of Durham– Dr Chris Saunter, Dr Penny Lawton© University of Durham 2023

Thank You for YourAttention© University of Durham 2023