Phase Focus Limited 0 Part of the group of companies Lens Free Live Cell Imaging Ian Pykett, Vassilios Albanis

Phase Focus Limited 1 Part of the group of companies 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

Phase Focus Limited 2 Part of the group of companies 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

Phase Focus Limited 3 Part of the group of companies 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

Phase Focus Limited 4 Part of the group of companies Technology Diffraction Patterns

Phase Focus Limited 5 Part of the group of companies Technology Diffraction Patterns

Phase Focus Limited 6 Part of the group of companies Technology Iterative Phase Retrieval Algorithm (Virtual Lens) J. M. Rodenburg, A. C. Hurst, A. G. Cullis. Transmission Microscopy Without Lenses for Objects of Unlimited Size”. Ultramicroscopy 107: , 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: – (2007) J. M. Rodenburg, A. M. Maiden. An improved ptychographical phase retrieval algorithm for diffractive imaging. Ultramicroscopy (2009), doi: /j.ultramic (in press)

Phase Focus Limited 7 Part of the group of companies Technology Eggs: lily antherFly’s wing Brightness: absorption. Colour: phase

Phase Focus Limited 8 Part of the group of companies Retrospective focussing Multi-focal-plane reconstructions from single acquisition

Phase Focus Limited 9 Part of the group of companies Specimen characterisation Thickness measurement 25.4 mm Phase image

Phase Focus Limited 10 Part of the group of companies Specimen characterisation Quantitative Refractive Index Measurement SpecimenFrog’s egg section PreparationStandard microtome StainNone Field of view2 mm x 2 mm Acquisition Single pass mode Resolution~10 µm Wavelength633 nm

Phase Focus Limited 11 Part of the group of companies Applications: Stand-alone prototype

Phase Focus Limited 12 Part of the group of companies Applications: Stand-alone prototype Initial conventional low magnification “scout scan” defines region of interest for fully-automated diffraction imaging Illumination currently rastered via specimen stage translation Acquisition time currently ~2 s per spot In future, via (e.g.) galvanometer laser scannner Parallel processing option Currently ~5 s per iteration (500 x 500 array)

Phase Focus Limited 13 Part of the group of companies Applications: contact lenses Phase image Absorption (conventional brightfield) image Phase discontinuity (“phase island”): Demarcation of hydrophobic area? 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

Phase Focus Limited 14 Part of the group of companies Applications: life sciences 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 400µ 2 – 2mm 2 ) Field of view independent of resolution Large working distance (30mm+) Non-contact microscopy of immersed cells

Phase Focus Limited 15 Part of the group of companies Cell imaging Absorption image Phase image Fibroblasts Keratinocyte colonies Cell typeKeratinocytes Cell SystemSingle layer adherent MediumPhosphate buffered solution Cell age~ 2 days Vessel8-well plate StainNone Working30mm distance Field of view1 mm x 1 mm Acquisition Single pass mode Resolution~1 µm Wavelength405 nm

Phase Focus Limited 16 Part of the group of companies Cell imaging Absorption (conventional brightfield) image Phase image Cell typeHuman dermal fibroblasts Cell SystemSingle layer adherent MediumPhosphate buffered solution Cell age~2 days VesselSealed 35 mm plastic petri dish StainNone Working30 mm distance Field of view1 mm x 1 mm Acquisition Single pass mode Resolution~1 µm Wavelength405 nm Absorption image Phase image

Phase Focus Limited 17 Part of the group of companies Live cell imaging Absorption (conventional brightfield) image Phase image Cell typeHuman epithelial Cell SystemMultilayer adherent MediumEpidermal Growth Factor Cell age5 - 7 days VesselT-25 flask StainNone Working30 mm distance Field of view1 mm x 1 mm Acquisition Single pass mode Resolution~1 µm Wavelength405 nm Absorption image Phase image

Phase Focus Limited 18 Part of the group of companies Cell segmentation Cell type Human metastatic melanoma Cell System Single adherent Medium Phosphate buffered solution Cell age 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 Phase image Segmented image

Phase Focus Limited 19 Part of the group of companies 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

Phase Focus Limited 20 Part of the group of companies 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 U niversity of Manchester Sandra Downes Michael Read

Phase Focus Limited 21 Part of the group of companies Redefining microscopy and imaging