Phase Focus Limited1 Part of the group of companies

Lens Free Live Cell Imaging

Ian Pykett, Vassilios Albanis

Phase Focus Limited2 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 Limited3 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

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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 Limited5 Part of the group of companies

Technology

Diffraction Patterns

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Technology

Diffraction Patterns

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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: 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)

Phase Focus Limited8 Part of the group of companies

Technology

Eggs: lily anther Fly’s wing

Brightness: absorption. Colour: phase

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Retrospective focussing

• Multi-focal-plane reconstructions from single acquisition

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Specimen characterisation

• Thickness measurement25.4 mm

Phase image

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Specimen characterisation

• Quantitative Refractive Index Measurement

Specimen Frog’s egg sectionPreparation Standard microtomeStain NoneField of view 2 mm x 2 mmAcquisition Single pass   modeResolution ~10 µmWavelength 633 nm

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Applications: Stand-alone prototype

Phase Focus Limited13 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)

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Applications: contact lenses

Phase imageAbsorption

(conventional brightfield) image

Phase discontinuity(“phase island”):Demarcation ofhydrophobic 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

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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

400µ2 – 2mm2)

• Field of view independent of

resolution

• Large working distance

(30mm+)

• Non-contact microscopy of immersed cells

Phase Focus Limited16 Part of the group of companies

Cell imaging

Absorption image Phase image

Fibroblasts

Keratinocyte colonies

Cell type KeratinocytesCell System Single layer adherentMedium Phosphate buffered

   solutionCell age ~ 2 daysVessel 8-well plateStain None

Working 30mm   distanceField of view 1 mm x 1 mmAcquisition Single pass   modeResolution ~1 µmWavelength 405 nm

Phase Focus Limited17 Part of the group of companies

Cell imaging

Absorption (conventional brightfield) image Phase image

Cell type Human dermal    fibroblasts

Cell System Single layer adherentMedium Phosphate buffered

   solutionCell age ~2 daysVessel Sealed 35 mm plastic

   petri dishStain None

Working 30 mm   distanceField of view 1 mm x 1 mmAcquisition Single pass   modeResolution ~1 µmWavelength 405 nm

Absorption image Phase image

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Live cell imaging

Absorption (conventional brightfield) image Phase image

Cell type Human epithelialCell System Multilayer adherentMedium Epidermal Growth

   FactorCell age 5 - 7 daysVessel T-25 flaskStain None

Working 30 mm   distanceField of view 1 mm x 1 mmAcquisition Single pass   modeResolution ~1 µmWavelength 405 nm

Absorption image Phase image

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Cell segmentation

Cell type    Human metastatic      melanoma

Cell System    Single adherentMedium    Phosphate buffered

      solutionCell age    5 - 7 daysVessel    T-25 flaskStain    None

Working    30mm   distanceField of view    1 mm x 1 mmAcquisition    Single pass   modeResolution    ~1µmWavelength    405 nm

Post-processing  Matlab   applicationSegmentation  Standard threshold   technique

Phase image Segmented image

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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

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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

Phase Focus Limited22 Part of the group of companies

Redefining microscopyand imaging