phase focus limited 0 part of the group of companies lens free live cell imaging ian pykett,...
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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
Phase Focus Limited4 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 Limited5 Part of the group of companies
Technology
Diffraction Patterns
Phase Focus Limited6 Part of the group of companies
Technology
Diffraction Patterns
Phase Focus Limited7 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: 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
Phase Focus Limited9 Part of the group of companies
Retrospective focussing
• Multi-focal-plane reconstructions from single acquisition
Phase Focus Limited10 Part of the group of companies
Specimen characterisation
• Thickness measurement25.4 mm
Phase image
Phase Focus Limited11 Part of the group of companies
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
Phase Focus Limited12 Part of the group of companies
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)
Phase Focus Limited14 Part of the group of companies
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
Phase Focus Limited15 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
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
Phase Focus Limited18 Part of the group of companies
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
Phase Focus Limited19 Part of the group of companies
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
Phase Focus Limited20 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 Limited21 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
• University of Manchester
• Sandra Downes
• Michael Read
Phase Focus Limited22 Part of the group of companies
Redefining microscopyand imaging