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. 2006 Nov 1;91(9):3465-73.
doi: 10.1529/biophysj.106.084079. Epub 2006 Aug 4.

Laser-guided Assembly of Heterotypic Three-Dimensional Living Cell Microarrays

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Free PMC article

Laser-guided Assembly of Heterotypic Three-Dimensional Living Cell Microarrays

G M Akselrod et al. Biophys J. .
Free PMC article

Abstract

We have assembled three-dimensional heterotypic networks of living cells in hydrogel without loss of viability using arrays of time-multiplexed, holographic optical traps. The hierarchical control of the cell positions is achieved with, to our knowledge, unprecedented submicron precision, resulting in arrays with an intercell separation <400 nm. In particular, we have assembled networks of Swiss 3T3 fibroblasts surrounded by a ring of bacteria. We have also demonstrated the ability to manipulate hundreds of Pseudomonas aeruginosa simultaneously into two- and three-dimensional arrays with a time-averaged power <2 mW per trap. This is the first time to our knowledge that living cell arrays of such complexity have been synthesized, and it represents a milestone in synthetic biology and tissue engineering.

Figures

FIGURE 1
FIGURE 1
Schematic diagram of a time-shared holographic optical trapping apparatus. Trap arrays are formed using a high NA objective in a commercial optical microscope in conjunction with two AODs and an SLM to produce a time-shared (3D) array of optical traps. The plane of the SLM, a, is imaged into the microscope OEA, a*, and the corresponding planes b and c are imaged into the focal region of the microscope. The same microscope that is used to produce the cell traps is also used for viewing (via the blue beam). The inset in the lower left shows an example of a 2D 5 × 5 array of P. aeruginosa formed using this apparatus and subsequently embedded in hydrogel. The distances are AODs–L1 = 165 mm; L1–L2 = 650 mm; L2–SLM = 332 mm; SLM–L3 = 421 mm; L3–L4 = 1400 mm; and L4–OEA = 493 mm, where the focal lengths are L1 = 150 mm, L2 = 500 mm, L3 = 1000 mm, and L4 = 400 mm.
FIGURE 2
FIGURE 2
Optical micrographs showing 2D microarrays of P. aeruginosa bacteria. (a) A transmission micrograph of a 21 × 21 2D microarray of P. aeruginosa formed with a 100×-, 1.25-NA oil immersion (Zeiss Plan-Apo) objective at λ = 900 nm using <2 mW per trap. (b) A false-color isosurfaces were generated from volumetric data obtained from deconvolved confocal images of a 5 × 5 microarray of P. aeruginosa assembled with a 100×-, 1.3-NA oil immersion (Zeiss Plan-Apo) objective at λ = 514 nm using <2 mW per trap, and embedded in hydrogel. The average center-to-center distance is 1.52 ± 0.06 μm and the average space between each bacterium is 354 ± 134 nm. (c) A 3D representation of (b).
FIGURE 3
FIGURE 3
A 3 × 3 × 3 3D array of P. aeruginosa bacteria. (a) A transmission micrograph of three overlapping 3 × 3 arrays of P. aeruginosa, shifted by 3 μm from each other along the optical (z) axis and embedded in hydrogel. The arrays are formed with a 100×-, 1.3-NA oil immersion (Zeiss Plan-Apo) objective using 514 nm light with <1 mW per trap. The corner vertex of each of the arrays is highlighted by a blue, green, and red circle in the figure. The focus is coplanar with the middle of the three arrays so that the underfocused top array (blue) appears bright and the overfocused bottom array (red) appears to be dark relative to the center array (green). The three arrays are shifted by 4 μm along both the x and y axes to facilitate imaging. (b) A false-color isosurface, reconstructed from volumetric data obtained from a series of confocal images, showing the offset along the x and y axes with the xy-projection. (c) Reconstructed (false color) isosurface xz-projection, illustrating the 3-μm separation along the z axis. (d) A false-color isosurface perspective reconstruction illustrating the top (blue), middle (green), and bottom (red) arrays separated by 2 μm.
FIGURE 4
FIGURE 4
Heterotypic microarray of Swiss 3T3 mouse fibroblast and P. aeruginosa bacteria. (a) Swiss 3T3 mouse fibroblasts trapped in a 3 × 3 2D array formed at λ = 514 nm using a 40× objective with 20 mW per trap. (b) A false-color isosurface reconstruction obtained from a confocal image of a Swiss 3T3 cell trapped with 100× objective using 9–2 mW beams at λ = 900 nm, surrounded by a ring of 16 P. aeruginosa with each bacterium trapped using a single 2-mW beam. This image was obtained by exciting SYTO 9 labels with 488 nm. (c) The same microarray as in b rotated to reflect the 3D aspects of the array. (d and e) Viability assay of the same heterotypic microarray showing an image obtained by exciting propidium iodide labels with 488 nm. The lack of red fluorescence in d indicates viability, but after killing the cells with ethanol the fluorescence is intensely red (e).
FIGURE 5
FIGURE 5
A 5 × 5 2D array of E. coli bacteria incorporating the receiver plasmid pFNK-203. (a) A transmission micrograph of a 5 × 5 array of E. coli embedded in hydrogel. The array is formed with a 100×-, 1.3-NA oil immersion (Zeiss Plan-Apo) objective using λ = 900 nm using <2 mW per trap. (b) A green fluorescent image of the same array obtained using 470 nm excitation, after inducing the production of GFP within the E. coli with 500 nm of AHL 43 h after gelling. Every element of the array is fluorescing green. These images indicate metabolic activity and cell viability up to 43 h after fixing the array in hydrogel.

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