Lens Free Live Cell Imaging Ian Pykett, Vassilios Albanis

1. Lens Free Live Cell Imaging Ian Pykett, Vassilios Albanis

2. The Phase Focus Virtual Lens™ • A method for imaging and microscopy that transfers the task of image formation from physical components (lens) to a software algorithm • A platform technology – applicable in principle to the entire electromagnetic spectrum • Initial market focus: optical, electron and X-ray microscopy

3. Benefits of the Virtual Lens • Benefits applicable at all wavelengths: • “Lensless” – no high performance focussing devices required • Imaging of arbitrarily large fields of view within extended specimens • Post-acquisition multi-focal-plane reconstructions though the specimen thickness • Routine acquisition of quantitative phase data • Resolution limited in principle only by wavelength • The generic product (a processor-embedded Virtual Lens) can be readily interfaced with existing instrumentation

4. Technology • Hardware Step: • Moving illumination • Creates multiple “diffraction” patterns from the specimen • Software Step: • Iterative phase retrieval algorithm: the Virtual Lens, a.k.a. Ptychographical Iterative Engine (PIE) • From the diffraction patterns, reconstructs two separate images, from any (or every) focal plane within the specimen • Absorption image (traditional “brightfield” image) …how much the specimen absorbs the light • Quantitative phase image …how much the specimen changes the phase of the light

5. Diffraction Patterns Technology

6. Technology Diffraction Patterns

7. Technology • Iterative Phase RetrievalAlgorithm (Virtual Lens) J. M. Rodenburg, A. C. Hurst, A. G. Cullis. Transmission Microscopy Without Lenses for Objects of Unlimited Size”. Ultramicroscopy 107: 227-231, 2007. J. M. Rodenburg, A. C. Hurst, A. G. Cullis, B. R. Dobson, F. Pfieffer, O. Bunk, C. David, K. Jefimovs, I. Johnson. Hard-X-Ray Lensless Imaging of Extended Objects. Phys. Rev. Lttrs. 98:034801-1 – 034801-4 (2007) J. M. Rodenburg, A. M. Maiden. An improved ptychographical phase retrieval algorithm for diffractive imaging. Ultramicroscopy (2009), doi:10.1016/j.ultramic.2009.05.012 (in press)

8. Technology Eggs: lily anther Fly’s wing Brightness: absorption. Colour: phase

9. Retrospective focussing • Multi-focal-plane reconstructions from single acquisition

10. Specimen characterisation 25.4 mm • Thickness measurement Phase image

11. Specimen characterisation • Quantitative Refractive Index Measurement Specimen Frog’s egg section Preparation Standard microtome Stain None Field of view 2 mm x 2 mm Acquisition Single pass   mode Resolution ~10 µm Wavelength 633 nm

12. Applications: Stand-alone prototype

13. Applications: Stand-alone prototype • Initial conventional low magnification “scout scan” defines region of interest for fully-automated diffraction imaging • Illumination currentlyrastered via specimenstage translation • Acquisition timecurrently ~2 s per spot • In future, via (e.g.)galvanometerlaserscannner • Parallel processing option • Currently ~5 s per iteration(500 x 500 array)

14. Applications: contact lenses • Analysis of contact lenses suspended in hydration solution • Exemplar for hydrogels; biofilms; etc. • Competing methods (conventional microscopy; atomic force microscopy) are destructive, of limited field of view, uninformative, or can be used only with non-hydrated (e.g.: freeze-dried) lenses Absorption (conventional brightfield) image Phase image Phase discontinuity(“phase island”):Demarcation ofhydrophobic area?

15. Applications: life sciences • Non-contact microscopy of immersed cells • High contrast without stains • Retrospective focussing • Specimen characterisation (refractive index; thickness) • E.g.: Changes in cellular refractive index are early harbingers of apoptosis • Wide field of view (typically 400µ2 – 2mm2) • Field of view independent of resolution • Large working distance(30mm+)

16. Cell imaging Fibroblasts Absorption image Phase image Cell type Keratinocytes Cell System Single layer adherent Medium Phosphate buffered    solution Cell age ~ 2 days Vessel 8-well plate Stain None Working 30mm   distance Field of view 1 mm x 1 mm Acquisition Single pass   mode Resolution ~1 µm Wavelength 405 nm Keratinocyte colonies

17. Cell imaging Absorption image Phase image Cell type Human dermal     fibroblasts Cell System Single layer adherent Medium Phosphate buffered    solution Cell age ~2 days Vessel Sealed 35 mm plastic    petri dish Stain None Working 30 mm   distance Field of view 1 mm x 1 mm Acquisition Single pass   mode Resolution ~1 µm Wavelength 405 nm Absorption (conventional brightfield) image Phase image

18. Live cell imaging Absorption image Phase image Cell type Human epithelial Cell System Multilayer adherent Medium Epidermal Growth    Factor Cell age 5 - 7 days Vessel T-25 flask Stain None Working 30 mm   distance Field of view 1 mm x 1 mm Acquisition Single pass   mode Resolution ~1 µm Wavelength 405 nm Absorption (conventional brightfield) image Phase image

19. Cell segmentation Phase image Cell type    Human metastatic       melanoma Cell System    Single adherent Medium    Phosphate buffered       solution Cell age    5 - 7 days Vessel    T-25 flask Stain    None Working    30mm   distance Field of view    1 mm x 1 mm Acquisition    Single pass   mode Resolution    ~1µm Wavelength    405 nm Post-processing  Matlab   application Segmentation  Standard threshold   technique Segmented image

20. Cell imaging applications strategy • Confirm the unique combination of benefits of the Virtual Lens for high content screening applications: • Goal: Automated in situ live cell imaging to report on cell death, proliferation, and cell cycle dynamics on a 3D basis over prolonged periods of time

21. Acknowledgements • University of Sheffield • John Rodenburg(Phase Focus CSO) • Andrew Maiden • Sheila McNeil • Louise Smith • University of York • Peter O’Toole • Cardiff University • Nick White • Rachel Errington • University of Manchester • Sandra Downes • Michael Read

22. Redefining microscopyand imaging