Impact of release dynamics of laser-irradiated polymer micropallets on the viability of selected adherent cells

Abstract

We use time-resolved interferometry, fluorescence assays and computational fluid dynamics (CFD) simulations to examine the viability of confluent adherent cell monolayers to selection via laser microbeam release of photoresist polymer micropallets. We demonstrate the importance of laser microbeam pulse energy and focal volume position relative to the glass-pallet interface in governing the threshold energies for pallet release as well as the pallet release dynamics. Measurements using time-resolved interferometry show that increases in laser pulse energy result in increasing pallet release velocities that can approach 10 m s(-1) through aqueous media. CFD simulations reveal that the pallet motion results in cellular exposure to transient hydrodynamic shear stress amplitudes that can exceed 100 kPa on microsecond timescales, and which produces reduced cell viability. Moreover, CFD simulation results show that the maximum shear stress on the pallet surface varies spatially, with the largest shear stresses occurring on the pallet periphery. Cell viability of confluent cell monolayers on the pallet surface confirms that the use of larger pulse energies results in increased rates of necrosis for those cells situated away from the pallet centre, while cells situated at the pallet centre remain viable. Nevertheless, experiments that examine the viability of these cell monolayers following pallet release show that proper choices for laser microbeam pulse energy and focal volume position lead to the routine achievement of cell viability in excess of 90 per cent. These laser microbeam parameters result in maximum pallet release velocities below 6 m s(-1) and cellular exposure of transient hydrodynamic shear stresses below 20 kPa. Collectively, these results provide a mechanistic understanding that relates pallet release dynamics and associated transient shear stresses with subsequent cellular viability. This provides a quantitative, mechanistic basis for determining optimal operating conditions for laser microbeam-based pallet release systems for the isolation and selection of adherent cells.

Figure 1.

(a) Light and (b) electron microscopy of 1002F pallet arrays. The pallet elements are 50 µm tall and have lateral dimensions of 100 × 100 µm. Adjacent pallet elements are separated by 20 µm to form the array. Titanium markings deposited onto the underlying glass support and used for accurate focusing appear as black ‘dots’ adjacent to the corner of each pallet element in the light microscopy image.

Figure 2.

Schematic of pallet release energy threshold measurement.

Figure 3.

Schematic of the time-resolved heterodyne interferometer. AOM, acousto-optic modulator; PBS, polarizing beam splitter; QWP, quarter-wave plate; HWP, half-wave plate; NPBS, non-polarizing beam splitter; BP, band pass filter; PD, photodiode; power, power output of RF driver (high power); Ref, reference output of RF driver (low power); CH1, channel 1 of oscilloscope and CH2, channel 2 of oscilloscope.

Figure 4.

Central region of the dynamic mesh used for the CFD simulation. The underlying glass support occupies the region Z ≤ 0 while the fluid is present in regions occupied by the mesh. The overall computation domain is 1.5 × 1.5 mm. The mesh resolution is 1 µm in regions immediately adjacent to the pallet and increases gradually to 10 µm at the outer boundaries of the computational domain.

Figure 5.

Pallet release probability for different focal volume positions. Squares with solid line, h = 2 µm; circles with dashed line, h = 6 µm; diamonds with dotted line, h = 10 µm.

Figure 6.

Representative pallet (a) displacement, (b) velocity and (c) acceleration for E_p = 3.5 µJ and h = 6 µm obtained from the interferometry data. (a) A denotes the initial upward pallet motion owing to the plasma-induced shock wave, B denotes either permanent deformation of the pallet or elastic recoil owing to the initial shock wave excitation and C denotes the initiation of the pallet release. The breakdown of these traces at times larger than approximately 4.5 µs is from the loss of the interferometer signal owing to rotational motion of the pallet.

Figure 7.

Release time, maximum release velocity and maximum release acceleration as a function of microbeam pulse energy and focal volume position. Squares, h = 2 µm; triangles, h = 6 µm; diamonds, h = 10 µm.

Figure 8.

(a) Co-stained calcein AM (green) and Hoechst 33342 (red) confluent HeLa cells on pallets prior to release; (b–d) pallets after release using pulse energies of (b) 2.5 µJ, (c) 3.0 µJ and (d) 3.5 µJ. Axial position of focal volume was h = 6 µm. As described in the text, live cells will posses a green cytoplasm with an orange nucleus while dead cells will appear with only a red nucleus without any green staining.

Figure 9.

Graphical depiction of variation in cell viability rates on the pallet surface as a function of pulse energy. MM/NN refers to the number of viable cells counted out of the total number of cells residing in the respective regions.

Figure 10.

Variation in cell viability rates with pulse energy and cellular position on the pallet surface. Increases in pulse energy and/or cell position away from the pallet centre will increase cellular exposure to hydrodynamic shear stress during release and reduces cell viability. Green bars, 2.5 µJ; blue bars, 3.0 µJ; pink bars, 3.5 µJ.

Figure 11.

(a) Measured pallet release velocities for E_p = 2.5, 3.0 and 3.5 µJ at focal volume position h = 6 µm. Diamonds with dotted line, E_p = 3.5 µJ; squares with dashed lines, E_p = 3.0 µJ; circles with solid line, E_p = 2.5 µJ. (b) Predicted time-resolved shear-stress distributions for E_p = 3.5 µJ and focal volume position h = 6 µm at various pallet surface locations.

Figure 12.

Spatial distributions of maximum shear stress for pulse energies of E_p = 2.5, 3.0 and 3.5 µJ at focal volume position h = 6 µm. Diamonds with dotted line, E_p = 3.5 µJ; squares with dashed lines, E_p = 3.0 µJ; circles with solid line, E_p = 2.5 µJ.