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xhtmlA novel imaging system providing high-content high-throughput multi-dimensional analysis of microscopic biological structure inside non-adherent living cells.
AbstractThree-dimensional (3D) fluorescence imaging microscopy of individual living cells is an essential tool for cellular biology, pathology and the study of infection and virulence therein. However, today, one critical constraint of established techniques is that samples must be stabilised by attachment to an optically transparent surface, thereby completely precluding their use for non-adherent cell types. The severity of this limitation becomes clear when one considers that for basic- and biomedical-research, cell-based assay and cell-diagnostic applications some of the most important targets are non-adherent cells, for example, stem cells, systemic cancer cells and lymphocytes. We therefore propose a completely new 3D imaging strategy targeted specifically at live, non-adherent cells. The core technology combines proprietary state of the art hardware for suspended cell manipulation with super high-speed dynamic imaging methods. Around this, our consortium draws together unique expertise from three companies and three academic teams providing the necessary critical-mass to industrialize this methodology as 1) a routine research-bench tool, and 2) a high-throughput, high-content imaging device AUTOMATION (AUtomated TOMographic Analysis staTION). Towards these goals completed pilot studies already demonstrate the feasibility for hardware and mathematical development, which will pave the way towards prototype design, and construction. As such, AUTOMATION envisages an altogether new micro-imaging technology; enabling hands-off, rapid, quantitative 3D reconstruction, as never before realised.
Current state-of-the-art: Dynamic multi-dimensional imaging inside single living cells.Today, burning questions in biology require the extraction of quantitative information hidden within the dynamics of genome, transcriptosome, and proteome activity inside living tissues. Clearly, in this context, techniques for multi-dimensional imaging microscopy inside isolated individual cells are certainly among the most important of available tools because they allow specific molecular events to be recorded through time and space in situ. Thus, multi-dimensional refers specifically to combinations of: three (3D, volume), four (4D, time), and five (5D, multi-channel, -colour, -mode, and -parameter) dimensional measurement. However, the technologies to achieve such measurements are themselves complex, time-consuming, and sufficiently exigent to exclude visualisation of paradigms that do not fully lend themselves to the constraints imposed by the experimental set-up. Currently, most all commercialised 3D fluorescence imaging microscopes use z-stack axial sampling based on the assumption that the imaging field is an optical section. This premise is based upon the fact that the microscopic imaging field may be considered a volume of definite geometry: the focal depth wherein the focal plane lies. The dimensions of the focal depth are determined mainly by the angular light collecting characteristics of the objective, and in general (for high-resolution applications) is between 200-500nm, well below the size of a eukaryotic cell. Because of these dimensions our in-focus view of a cell can be considered a quasi-optical section, and it is this effect (notwithstanding the complications of out-of-focus-light) that enables "even" a so-called conventional microscope to be used effectively for 3D imaging. Thus, a sample volume is imaged repeatedly while mechanically shifting the microscope objective focal plane in small axial steps and acquiring an image at each new focal position until the object of interest is fully sampled. The resulting planar axial "z-stack" image series can then be processed and rendered in 3D using computer-software. Today, considerable effort and ingenuity is directed towards improving this approach, and in particular novel engineering and mathematical innovations. Many of these efforts are aimed primarily at overcoming the aberrations caused by the necessary movement of the optic system, which in itself compromises quantitative spatial processing and reconstruction. However, until now, few developments have recognised, or effectively addressed the more intrinsic problem that multi-dimensional fluorescence imaging requires that samples be adherently fixed to an immobile surface.
The novel core technology innovation underlying AUTOMATION.Our core technology uses a novel method for live cell micro-manipulation under dielectric field control enabling individual, non-adherent cells to be immobilised in suspension, and rotated smoothly around a fixed focal plane, through any chosen vector. The basic hardware comprises a three-dimensional dielectrophoretic field cage comprising micro-electrodes fabricated photolithographically on optically transparent glass substrates and assembled face to face, at a distance of ~200 µm. Cells suspended in buffered medium can be perfused into the cage using ultra-low speed micro-fludics control, and can be trapped and manipulated inside the cage using high-frequency polarization of di-electrics, creating forces in the range of pico-newtons repelling particles from regions of high field strength toward electric field minima. This principle permits stable and accurate positioning of micro-objects (i.e. a living cell) within micrometer dimensions (note: the field cage "centre" can be modified during fabrication to accommodate objects with diameters between 1-200 µm across). Fig.1 (left to right) shows a low magnification photo of the field-cage, a high-magnification detail of a cage wherein a living cell is trapped, and finally a schematic of the electrode geometry.
Fig.1- The "Cell Rotator" dielectrophoretic field cage
The live cell-trapping/rotator device offers an exquisite degree of control over suspended cell (or other micro-object) position. Importantly, it enables for cells to be rotated relative to a fixed imaging field. Based upon this ability, our innovative approach uses the "optical section" assumption (already providing the foundation to other existing 3D imaging techniques), but with one key difference that changes completely the sampling paradigm. Thus, in place of changing the focus of the objective relative to the cell, we maintain all optical imaging components immobile, and instead move the cell itself through a fixed axial vector (i.e. around the "ideal" focal plane). As the cell rotation progresses, snapshot images are recorded, and passing through 360 ° the cell is observed from multiple angles of view using a high-speed camera. The net result of this new 3D sampling method is best observed in movies (see movie and figs.2-4).
The first example (fig.2) shows a living human red blood cell infected with plasmodium parasite stained with DAPI. In the fluorescence images the dividing parasite nucleus is clearly observed from different view-points, and demonstrates definitively how in the dimensions of the focal depth, rotational imaging immediately increases the information content and quality inasmuch as the view point determines how many nuclei are actually observed (i.e. the number of bright points).
Fig.2- Living plasmodium parasite nuclei labelled with DAPI inside a living red blood cell.
Multi-colour imaging is important for co-localization studies and because our rotator imaging paradigm does not involve moving the optical plane, it is devoid of axial chromatic aberrations. Fig.3 illustrates one example of dual-colour measurements using this approach. An H2-histone (green) expressing living lymphocyte, infected with Theileria parva was transfected with a JNK dominant negative vector known to induce apoptosis. The cell was co-labelled with annexinV (red) and visualised inside the cell-rotator device, and selected multiple angles of view, captured in just a few seconds are shown, giving a priori an extensive appreciation of multi-colour 3D structure from different view points.
Fig.3- Living lymphocyte expressing H2-histone (green) and labelled with AnnexinV (red).
Rotational stability is critical to the premise of our project, and in the final example (fig.4), we demonstrate that the rotator device is able to maintain reproducible rotation. A living HeLa cell (made non-adherent by trypsinisation) and stably expressing DsRed-labelled nuclear lamin, was trapped inside the dielectrophoretic cage and rotated around the x,y plane at a constant speed during 200s. An image series was recorded (one image per 204 ms), from which three example images are shown, separated by approximately 90°. In addition, the graph representation shows a measure of the rotation stability achieved using detection of intensity changes in two regions of interest through which a conspicuous fluorescent accumulation passes once during each full 360° rotation, and provides an internal-fiducial marker. Fourier analysis of the amplitude fluctuation indicates periodicity, whereby a full rotation occurred in just 5 seconds, and during this time was mapped by ~25 images (angular displacement of just 15° between images). Pertinently, this value reflects the angular shift used in single cell fluorescence tomography applications based on adhesion to glass capillaries, and repeated positional z-stack sampling following precise angular rotation of the specimen. This comparison invites the idea that cell-rotation image datasets can similarly be processed by image alignment followed by a novel tomographic reconstruction approach.
Fig.4 Nuclear DsRed-lamin inside living HeLa cell during stable rotation imaging.
