| 1. |
- Bertilson, Michael, et al.
(författare)
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Laboratory Soft X-Ray Cryo Tomography
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- X-rays allow quantitative high-spatial-resolution three-dimensional (3D) imaging of intact unstained cells. Such 3D imaging is provided by soft x-ray lens-based methods (water-window cryo tomography) and hard x-ray lens-less methods (coherent diffraction imaging) are emerging. However, both methods rely on high-brightness synchrotron-radiation sources, which limit the accessibility of a wider scientific community. Here we show 3D water-window cryo tomography with a laboratory-source-based microscope arrangement. The system relies on a λ=2.48-nm liquid-jet laser-plasma source, normal- incidence multilayer condenser optics, 30-nm zone-plate optics, and a cryo sample chamber. We demonstrate imaging of intact unstained yeast, protozoan parasites and mammalian cells. 3D images show noise-limited features close to ~100 nm and intra-cellular structure is classified based on the local absorption coefficient. A comprehensive theoretical model of the tomographic imaging system allows optimization of system parameters and a quantitative estimate of the 3D imaging accuracy. The model includes issues such as non-geometric projections of the thick samples and stray light, and is applicable to laboratory as well as synchrotron-based x-ray microscopes. The model shows that laboratory x-ray cryo tomography will allow quantitative 3D imaging with ~30-nm (half-period) resolution over a full 5 µm object.
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| 2. |
- Bertilson, Michael, et al.
(författare)
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Laboratory soft-x-ray microscope for cryotomography of biological specimens
- 2011
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Ingår i: Optics Letters. - 0146-9592 .- 1539-4794. ; 36:14, s. 2728-2730
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Tidskriftsartikel (refereegranskat)abstract
- Soft-x-ray cryotomography allows quantitative and high-resolution three-dimensional imaging of intact unstained cells. To date, the method relies on synchrotron-radiation sources, which limits accessibility for researchers. Here we present a laboratory water-window microscope for cryotomography. It is based on a lambda = 2.48nm liquid-jet laser-plasma source, a normal-incidence multilayer condenser, a 30nm zone-plate objective, and a cryotilt sample holder. We demonstrate high-resolution imaging, as well as quantitative tomographic imaging, of frozen intact cells. The reconstructed tomogram of the intracellular local absorption coefficient shows details down to similar to 100nm.
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| 3. |
- Christakou, Athanasia, et al.
(författare)
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Characterization of natural killer cell immune surveillance against solid liver tumors
- 2015
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Ingår i: MicroTAS 2015 - 19th International Conference on Miniaturized Systems for Chemistry and Life Sciences. - : Chemical and Biological Microsystems Society. - 9780979806483 ; , s. 915-917
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Konferensbidrag (refereegranskat)abstract
- We demonstrate a method for investigating natural killer (NK) cell aggression against ultrasound-assisted human hepatocellular carcinoma (HCC) HepG2 solid tumors in a multi-well microdevice. We quantify the activity of human primary IL-2 activated NK cells against HepG2 tumors for up to five days and we present the correlation between NK cell numbers versus average tumor volume and final tumor outcome (growth or defeat). We suggest future applications on formation of tumors originated from primary tumors cells and other tumor components as well as primary NK originating from the patient for use in personalized immunotherapy.
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| 4. |
- Christakou, Athanasia. E., et al.
(författare)
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Aggregation and long-term positioning of cells by ultrasound in a multi-well microchip for high-resolution imaging of the natural killer cell immune synapse
- 2011
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Ingår i: 15th International Conference on Miniaturized Systems for Chemistry and Life Sciences 2011, MicroTAS 2011. - 9781618395955 ; , s. 329-331
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Konferensbidrag (refereegranskat)abstract
- In this study we investigate the ability of Natural Killer (NK) cells to form ultrasound-mediated intercellular contacts with target cells in a multi-well microdevice by high-resolution confocal-microscopy imaging of inhibitory immune synapses. Furthermore, we compare the NK-Target cell cluster migration with and without ultrasound actuation. Experiments indicate that clusters of cells are positioned and maintained centered in the wells for 17 hours when they are exposed continuously to ultrasound. Our system can be used for both screening high numbers of events in low resolution and also for high resolution imaging of long term cell-cell interactions.
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| 5. |
- Christakou, Athanasia E., et al.
(författare)
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Characterization of natural killer cells' cytotoxic heterogeneity using an array of sono-cages
- 2012
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Ingår i: Proceedings of the 16th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2012. - : Chemical and Biological Microsystems Society. - 9780979806452 ; , s. 1555-1557
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Konferensbidrag (refereegranskat)abstract
- Using a multi-well device as an array of sono-cages for single cell analysis, we quantify for the first time the heterogeneity of natural killer (NK) cells' cytotoxic response against cancer cells. We report a fraction of inactive NK cells within the tested population (36%), as well as the existence of few 'serial killers' that eliminate up to six targets during 4 hours. We also characterize the multi-well acoustic device in terms of trapping efficiency at different actuation voltages, using adherent and non-adherent cell lines. We show that the acoustic forces applied on the cells can be compared to forces of biological processes (i.e. cell adherence).
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| 6. |
- Christakou, Athanasia E., et al.
(författare)
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Live cell imaging in a micro-array of acoustic traps facilitates quantification of natural killer cell heterogeneity
- 2013
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Ingår i: Integrative Biology. - : Oxford University Press (OUP). - 1757-9694 .- 1757-9708. ; 5:4, s. 712-719
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Tidskriftsartikel (refereegranskat)abstract
- Natural killer (NK) cells kill virus-infected or cancer cells through the release of cytotoxic granules into a tight intercellular contact. NK cell populations comprise individual cells with varying sensitivity to distinct input signals, leading to disparate responses. To resolve this NK cell heterogeneity, we have designed a novel assay based on ultrasound-assisted cell-cell aggregation in a multiwell chip allowing high-resolution time-lapse imaging of one hundred NK-target cell interactions in parallel. Studying human NK cells' ability to kill MHC class I deficient tumor cells, we show that approximately two thirds of the NK cells display cytotoxicity, with some NK cells being particularly active, killing up to six target cells during the assay. We also report that simultaneous interaction with several susceptible target cells increases the cytotoxic responsiveness of NK cells, which could be coupled to a previously unknown regulatory mechanism with implications for NK-mediated tumor elimination.
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| 7. |
- Christakou, Athanasia E., et al.
(författare)
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Solid tumor spheroid formation by temperature-controlled high voltage ultrasound in a multi-well microdevice
- 2014
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Ingår i: 18th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2014. - : Chemical and Biological Microsystems SocietyChemical and Biological Microsystems Society. - 9780979806476 ; , s. 573-575
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Konferensbidrag (refereegranskat)abstract
- In the present work we demonstrate effective 3D growth of human hepatocellular carcinoma (HCC) HepG2 cell spheroids in parallel in a multi-well microdevice actuated with high voltage ultrasound in a temperature-controlled system. We compare the spheroid formation during continuous ultrasound exposure for one week where we formed spheroids in 59% of the wells, with the spheroid formation without ultrasound actuation, where we obtained 0% spheroids. Furthermore, we present an application of the tumor spheroids for investigating natural killer (NK) cells behavior against solid tumors.
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| 8. |
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| 9. |
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| 10. |
- Christakou, Athanasia, et al.
(författare)
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Ultrasonic three-dimensional on-chip cell culture for dynamic studies of tumor immune surveillance by natural killer cells
- 2015
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Ingår i: Lab on a Chip. - : Royal Society of Chemistry. - 1473-0197 .- 1473-0189. ; 15:15, s. 3222-31
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Tidskriftsartikel (refereegranskat)abstract
- We demonstrate a simple method for three-dimensional (3D) cell culture controlled by ultrasonic standing waves in a multi-well microplate. The method gently arranges cells in a suspension into a single aggregate in each well of the microplate and, by this, nucleates 3D tissue-like cell growth for culture times between two and seven days. The microplate device is compatible with both high-resolution optical microscopy and maintenance in a standard cell incubator. The result is a scaffold- and coating-free method for 3D cell culture that can be used for controlling the cellular architecture, as well as the cellular and molecular composition of the microenvironment in and around the formed cell structures. We demonstrate the parallel production of one hundred synthetic 3D solid tumors comprising up to thousands of human hepatocellular carcinoma (HCC) HepG2 cells, we characterize the tumor structure by high-resolution optical microscopy, and we monitor the functional behavior of natural killer (NK) cells migrating, docking and interacting with the tumor model during culture. Our results show that the method can be used for determining the collective ability of a given number of NK cells to defeat a solid tumor having a certain size, shape and composition. The ultrasound-based method itself is generic and can meet any demand from applications where it is advantageous to monitor cell culture from production to analysis of 3D tissue or tumor models using microscopy in one single microplate device.
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