SPIE Photonics West February 2015 Coherence-controlled holographic microscopy for live-cell QPI Tomáš Slabý, Aneta Křížová, Martin Lošťák, Jana Čolláková, Pavel Kolman, Zbyněk Dostál, Lukáš Kvasnica, Martin Antoš, Pavel Veselý, Radim Chmelík Institute of Physical Engineering, Faculty of Mechanical Engineering, Brno University of Technology Central European Institute of Technology, Brno University of Technology TESCAN Brno, s.r.o.
Coherence-controlled holographic microscopy (CCHM) CCHM is a technique for quantitative phase imaging (QPI) label-free no staining low light power densities low phototoxicity simple image segmentation quantitative cell dry-mass density measurements 1,2 based on off-axis holographic configuration single-shot technique fast acquisition (no scanning) adapted for incoherent illumination (halogen lamp) strong suppression of coherent noise (speckles) & parasitic interferences lateral resolution of convetional optical microscopes no image artifacts 1 R. Barer: Interference microscopy and mass determination. Nature 169, 1952, 366-367. 2 H. Davies, M. Wilkins: Interference microscopy and mass determination. Nature 169, 1952, 541.
Coherence-controlled holographic microscope (CCHM) based on off-axis holographic configuration adapted for incoherent illumination T. Slabý et al., Optics Express 21 (2013) 14747 CCHM prototype at Brno University of Technology
Coherence control Imaging properties of CCHM can be controlled by adjusting the degree of coherence spatial coherence is controlled by aperture diaphragm temporal coherence is controlled by bandpass filters Incoherent illumination strong suppression of coherence noise (speckles) & parasitic interferences improved lateral resolution up to factor of 2 coherence-gate effect Coherent illumination numerical refocusing in larger range
Coherence control elimination of speckles & parasitic interferences improved lateral resolution Resolution target, objective 10x/0.25 Incoherent halogen lamp + 650(10) nm filter Coherent HeNe laser (633 nm) T. Slabý et al., Optics Express 21 (2013) 14747
Coherence-gate effect specimen diffuser induced by incoherent illumination eliminates contribution of light scattered in out-offocus planes Observation of copper foil with rectangular holes covered with a diffuser T. Slabý et al., Optics Express 21 (2013) 14747 10x/0.25 650(10) nm filter brightfield CCHM amplitude CCHM phase
Increasing concentration of phospholipids Coherence-gated QPI in turbid medium Zernike PhC 0 % CCHM phase 0.15 % rat embryo fibroblasts K2 in flow chamber in emulsion of phospholipids with increasing concentration 0.3 % 1.5 % Zernike phase contrast is inapplicable at higher concentrations CCHM provides good contrast at higher concentrations and does not suffer from halo effect 20x/0.4 40x/0.65, 650(10) nm filter
Coherence-gated QPI in turbid medium human breast cancer cells MCF7 reaction to treatment by active phospholipid emulsion (0.5 %) observed in flow chamber 3 phase [rad] 20x/0.4, halogen lamp + 650(10) nm filter, total time 90 minutes 0
Coherence-gated QPI in turbid medium human breast cancer cells MCF7 reaction to treatment by active phospholipid emulsion (0.5 %) observed in flow chamber 3 phase [rad] 20x/0.4, halogen lamp + 650(10) nm filter, total time 90 minutes 0
Coherence-gated QPI in turbid medium colorectal cancer cells DLD1 reaction to treatment by active phospholipid emulsion (0.15 %) observed in flow chamber 20x/0.5, halogen lamp + 650(10) nm filter, total time 6 hours
Coherence-gated QPI in collagen gel human breast cancer cells MCF7 in collagen gel basic biological test of cancer cell invasivity recorded mechanism of cell motion, not detectable by common methods 20x/0.5, halogen lamp + 650(10) nm filter, total time 100 min
Dynamic phase differences method that makes slight changes more visible and enables dynamics evaluation and quantification mitosis of rat sarcoma cell K2 Phase t 0 Phase t 0 +τ Subtraction Area with increased mass New area with increased mass Area with decreased mass Abandoned area with decreased mass
Multimodal imaging human prostate cancer cells PC3 apoptosis detection combined holography and fluorescence imaging phase image (holography) Annexin V (fluorescence) 20x/0.5, halogen lamp + 650(10) nm filter In collaboration with RNDr. Jan Balvan, Faculty of Medicine, Masaryk University, Czech Republic
Multimodal imaging human prostate cancer cells PC3 combined holography and fluorescence imaging enables to clearly distinguish oncosis from apoptosis and to stratify the progression of oncosis In collaboration with RNDr. Jan Balvan, Faculty of Medicine, Masaryk University, Czech Republic
Conclusions Coherence-controlled holographic microscopy (CCHM) QPI with incoherent illumination Disadvantages more complicated set-up & alignment ( automated) Advantages speckle-free real-time QPI without image artifacts lateral resolution of convetional optical microscopes coherence-gate effect
Acknowledgements Experimental Biophotonics Group & Centre for Innovative Microscopy Radim Chmelík Zbyněk Dostál Martin Antoš Aneta Křížová Hana Uhlířová Veronika Jůzová Matěj Týč Michala Slabá Jiří Komrska Pavel Kolman Martin Lošťák Tomáš Zikmund Jana Čolláková Pavel Veselý Věra Kollarová Lukáš Kvasnica Petr Bouchal TESCAN Brno, s.r.o. Filip Lopour Josef Pokorný Václav Procházka Marek Minář This work was supported by Ministry of Industry and Trade of the Czech Republic (FR-TI4/660) CEITEC Central European Institute of Technology (CZ.1.05/1.1.00/02.0068) from European Regional Development Fund
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