Live cell sorting

Fluorescence-activated cell sorting (FACS) applying flow cytometry to separate cells on a molecular basis is a widespread method. We demonstrated that both fluorescent and unlabelled live cells in a Petri dish observed with a microscope can be automatically recognized by computer vision and picked up by a computer-controlled micropipette fixed to the objective lens. The maximum number of cells picked up from a Petri dish was about 1,000 limited by our current sorting speed of 1 cell per second. This method can be routinely applied as a FACS down to the single cell level with a very high selectivity, and opens the way for automated single cell manipulations controlled by computer vision. Sorting resolution, i.e., the minimum distance between two cells from which one could be selectively removed was 50-70 micrometers, same as the micropipette aperture we used in the specific experiment. The device will be able to collect single cells for further cultivation, cloning, RNA or protein preparation, and to sort also fixed cells after immunocytochemical preparation. Detection of cells with a characteristic feature in their microscopic image similarly to face recognition in digital photos and subsequent automated manipulation will be feasible.


sorting fluorescent live cells with the micropipette

Phase contrast and fluorescent images of cell cultures before and after sorting. (a,b,c,d) NE-4C, (e, f, g, h) 3T3, ( i, j, k, l) astroglia-microglia cell cultures. (a, c, e, g, i, k) before, (b, d, f, h, j, l) after sorting. Aperture of the micropipette in action is shown in the insets of (d, h, l). A portion of NE-4C cells and all microglia cells were labelled by GFP. A portion of 3T3 cells were stained by DiI. Sorting process removed these fluorescent cells detected by software. Cells detected in phase contrast and in fluorescent images before sorting are indicated by white frames. Straight lines between cells in (c) show the path of the micropipette: three cells out of the path were excluded from sorting due to a neighbour closer than the sorting resolution of 50 μm, same as the inner diameter of the micropipette. We did not need to detect astroglia cells in phase contrast images (i) because GFP-labelled microglia cells could be removed even from the very close proximity of strongly adherent astroglia cells without perturbing them. Frames in (i) show microglia cells detected in panel k. Scale bars: 100 μm.


Fluorescence-activated cell sorting briefly

Since the invention of the fluorescence-activated cell sorter (FACS) [1] in the 1960s the method gained widespread application both in research and medical diagnosis [2]. Several new developments appeared in the last decades including lab-on-a-chip versions of miniature FACS devices, called μFACS [3,4,5,6]. Cells move along with the fluid flow either in a microfabricated channel or in a nozzle with a diameter of 50-400 μm driven by a pressure of 10,000-400,000 Pa resulting in a flow velocity of 10 m/s [7]. The sort rate in a FACS can be 10,000 cells per second or more. In a μFACS it also exceeds 1,000 cells per second using piezoelectric actuation [6]. Although limited spatial resolution has been demonstrated in latest innovations [8] the fluorescent or scattered light of cells is normally detected without imaging the cells. There are several fluorescence-activated sorting mechanisms, among which the most successful is the electrostatic deflection of charged droplets containing single cells sprayed out from a nozzle. All these solutions are based on flow cytometry and turn to be difficult to apply if the number of cells is low not to mention single cell manipulations.


Automated cell manipulations on a microscope briefly

An inverted fluorescent microscope equipped with a digital camera is also capable of the automatic detection of live fluorescent cells [9] in a culture dish using appropriate image analysing software. Such fluorescent cytometry is straightforward, and applied in specific fluorescent scanners or plate readers. The manipulation of cell cultures in a Petri dish or culture plate is, however, more demanding, especially on the single cell level. A recent innovation, called CellCelectorTM can select and collect cells from culture dishes [10] using a micropipette. The micropipette is positioned by a robotic arm above the cell colony detected previously on the microscope stage, and picks up the colony or fraction of the colony. Subsequently the robotic arm moves the micropipette above an other culture dish transferring the cells into that. The application of the robotic arm results in a low sort rate. Although the ability of this technique for isolating cell colonies was demonstrated, single cell sorting with a reasonable speed and efficiency seems to be uneasy applying this method. Semi-automated microinjection of adherent cells has been introduced [11] using the customary arrangement with a micropipette oriented diagonally relative to the optical axis and positioned by a motorized micromanipulator. Nevertheless such specialized complex devices are not very cost effective and it is hard to make extensive use of them. Image-controlled automated single cell manipulations, such as cloning, sorting or microinjection are still missing from the toolbox of most cell biologists. We propose a simple accessory and method to overcome the technical difficulties of automated single cell manipulations on a microscope. If the goal is efficient single cell sorting by a micropipette, its positioning accuracy and delay are crucial parameters. In our case these are not limited by an extra robotic arm or micromanipulator. We argue that its simplicity, the precise 3D positioning of the micropipette and its relatively high sorting frequency make the device we use more suitable for automated single cell manipulations and sorting than previous techniques. Although the very high sort rate achieved by flow cytometry cannot be obtained by our approach, the more sophisticated cell recognition is expected to induce extensive applications.


Flow field of the CellSorter micropipette

  • Mild flow conditions to minimize biological stress
  • Laminar flow assures reliable and gentle single cell handling
  • Excellent pressure characteristics for efficient sorting
flow field of the micropipette

(a) Fluorescent beads at the bottom of the Petri dish moving with the flow generated by the micropipette shown in the centre. Moving beads appear as radial lines, beads adhered to the surface appear as dots. Vacuum: -6,000 Pa, exposition time: 10 ms, bead size: 2 μm, scale bar: 100 μm. (b) Side view of the simulated velocity and pressure of flow generated by a vertical micropipette in the proximity of a horizontal plane with the same geometry and flow rate as applied in the experiment. Panel size: 0.4 x 0.2 mm. Velocity and pressure is indicated by streamlines and colour code, respectively. The darker colour means the lower pressure from 0 to -40,600 Pa. (c) Radial flow velocity as a function of radial distance measured from the centre of the micropipette. Experimental (□) and simulated (♦) data calculated from the displacement of beads shown in (a) and from the model outlined in (b) at a height of 1 μ above the surface, respectively. (d) Pressure at 1 μm (▲) and 5 μm (∆)above the surface, respectively, as a function of radial distance calculated from the simulation. (e) Shear stress of the flow presented similarly to (d).


References

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  10. Schneider, A., Spitkovsky, D., Riess, P., Molcanyi, M., Kamisetti, N., Maegele, M., Hescheler, J., Schaefer, U. 2008 "The good into the pot, the bad into the crop!"--a new technology to free stem cells from feeder cells. PLoS ONE 3 (11), e3788.
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