2.1.

Cell Culture and Incubation

HELA cells were routinely cultured in Dulbecco's modified eagle's medium (DMEM) supplemented with 5% fetal bovine serum. The cultures were maintained at 37 in a 5% CO₂ humidified atmosphere. For cell adhesion and detachment research, cells were trypsinized, plated on cover slips, and used after 2 h of culturing. DMEM and fetal bovine serum were purchased from Lonza (Maryland); Trypsin was purchased from Mediatech (Manassas, Virginia). All other reagents were purchased from Fisher Scientific except those specifically mentioned. Cells were incubated with PI (5 μm) 10 min before laser manipulation experiments.

2.2.

Near-Infrared Laser Manipulation and Cell Imaging Setup

A schematic of the setup for detachment, transportation, and orientation is shown in Fig. 1. A 785 nm Ti:Sapphire laser (Maitai HP, Newport Spectra-Physics Inc.) beam was expanded (BE), and coupled to a low NA (0.5) 20× microscope objective on a Nikon Ti-U inverted microscope platform. The dichroic mirror 1 (DM1) combined the laser beam and the visible light from the excitation source. The laser beam was made astigmatic by use of a cylindrical lens (RCL), which was rotated for orienting the elliptic beam profile. At the sample plane, the spot size was ∼2 μm × 10 μm. The dichroic mirror 2 (DM2) reflects the excitation lamp light and transmits the emitted fluorescence to the CCD camera. Bright field and fluorescence images were captured using a cooled CCD and processed using IMAGEJ software. The power of the laser beam at the focal plane was estimated by multiplying the transmission factor of the objective with the power measured with a power meter (PM100D, Thorlabs Inc.) at the back of the objective.

Fig. 1

Experimental setup for laser-assisted detachment, transportation, and orientation. L: Ti: Sapphire laser; BE: beam expander; S: shutter; P: polarizer; RCL: rotatable cylindrical lens; Fl.: fluorescence excitation source; Ex: excitation filter; Em: emission filter; MO: microscope objective; CL: condenser lens; DM1 and DM2: dichroic mirrors; M: mirror; HL: halogen lamp.

2.3.

Principle

Figure 2 shows the schematic of cellular detachment and follow-up manipulation processes. When the cell is irradiated continuously by a near-infrared (NIR) laser beam with power levels on the orders of magnitude below plasma formation threshold, the mechanism of detachment is primarily due to radiative forces induced by the laser beam,¹⁵ instead of other disruptive forces used in single-pulse laser catapulting experiments.^{16, 17} The detachment [Fig. 2a] force required for bond breaking could consist of two major components: i. Photothermal interaction and absorption force (F _ab); ii. Photomechanical interaction and scattering force (F _sc). Using single cell force spectroscopy,¹⁸ the rupture force of integrin-binding with substrate has been measured to be ∼30 pN. In the case of early binding events, depending on the binding sites, the required rupture force can be several hundreds of pN. The scattering force by laser beam power (P) at the cell membrane-substrate interface can be estimated using the equation F _sc = Q _s. n ₁ P/c, where Q _s is dependent¹⁹ on relative refractive index (n ₂/n ₁). Using refractive index of cell (n ₁) to be 1.4 and that of surrounding media to be 1.33, Q _s will be ∼0.1, which corresponds to scattering force F _sc of ∼100 pN for a laser power level of 200 mW. This may be sufficient to break the focal adhesion sites and detach the early-adhering cells with few binding sites. Further, the required rupture force may be reduced due to an increase in temperature created by the laser beam at cell-substrate interface. For strongly-adhering cells, the laser beam can be made to operate in a pulsed condition leading to higher axial (e.g., scattering) force.^{20, 21} Subsequent to detachment, the cells (if not placed at the center of the laser beam) can slide toward the center of the beam as a result of gradient force in the horizontal direction [Fig. 2b]. In order to minimize the potential energy, the flattened cell flips into the vertical plane [Fig. 2c]. This tilting maximizes the interaction energy with the laser beam, leading to higher axial transport (absorption and scattering) forces by laser beam which can completely break the remaining bonds (if any).

Fig. 2

A schematic process of the cellular manipulations. (a) Detachment; (b) Transverse movement; (c) Flipping; (d) Levitation; (e) Orientation; (f) Transportation; (g) Release.

In the absence of tight focusing, the cell can be levitated at a plane higher than the focal plane [Fig. 2d]. Stable axial position of levitation is determined by balance of the sum of buoyant and radiation forces in vertical up-direction against the downward gravitation force [Fig. 2d]. Figure 2e shows the rotation of the cylindrical lens resulting in rotation of the astigmatic (elliptic) beam profile. This results in transfer of orbital angular momentum^{22, 23} to the vertically tilted cell, leading to orientation of the cell around the optical axis [Fig. 2e]. Transportation of the cell, in its new orientation, is carried out by moving the laser beam or moving the stage to the area of interest [Fig. 2f]. Finally, the cell can be brought back to the targeted area on the substrate by reducing the laser beam power [Fig. 2g]. The reoriented cell now attaches to the substrate at the desired distance and orientation with respect to other cell(s).

3.1.

Bond Breaking and Flipping of Cells

When the selected cell was irradiated at a laser power of 90 mW focused to a spot radius of ∼1 μm by a 0.5 NA objective (intensity ∼3.6×10⁶ W/cm²), the processes described in Figs. 2a, 2b, 2c was found to occur. After bond breakage, the cell moved toward the center of the beam and got oriented by the laser beam without complete detachment and levitation (2 out of 5 cells). Figure 3 shows manipulation of a cell, irradiated by a 785 nm laser microbeam for 20 s. 4.58 s after laser irradiation, the cell moved in the horizontal plane [Fig. 3b]. Nine seconds after irradiation, the cell was seen to be flipped into a vertical plane on the Petri dish [Figs. 3c and 3d] and maintained this orientation until the laser beam was switched off. With a laser power lower than 70 mW, the cell was not able to flip even after irradiation for a longer duration.

Fig. 3

Flipping of cell by 785 nm laser beam at a power of 110 mW. (a) Before irradiation of cell. (b) 4.58 s, (c) 9.08 s, and (d) 130 s of laser irradiation.

3.2.

Levitation of the Detached Cells

Figure 4 (Video 1) shows complete detachment and levitation of a targeted cell [marked by a circle, Fig. 4a] cell irradiated by a 785 nm laser at power of 141 mW. Figure 4b shows propidium iodide fluorescence before laser irradiation. The targeted cell first feels the force of attraction toward the center of the focused laser beam (marked by the arrow) as shown in Figs. 4c and 4d. This can be attributed to the transverse gradient force exerted by the laser beam. During process of detachment, the cell experienced torque around the horizontal axis leading to flipping of the cell in the vertical plane [Fig. 4e]. After 4.17 s of laser irradiation, the cell was completely detached and levitated up in the media to a height of ∼110 μm [Fig. 4f]. By translating and orienting the astigmatic laser beam (2 μm × 10 μm), the cell was transported to the desired location and reoriented anticlockwise at a higher plane [Fig. 4g]. Decreasing the laser beam power led to bringing the cell down to the substrate [Figs. 4h, 4i, 4j 97.00, 111.67, and 121.58 s, respectively]. The reattached cell on the substrate is shown in Fig. 4k. Propidium iodide exclusion in the targeted cell after laser manipulation [Fig. 4l] shows that the viability of the laser manipulated cell is not compromised. No fluorescence emission from the cell demonstrates that the cell membrane is intact after detachment.

Fig. 4

Cell detachment, levitation, and orientation by the NIR laser beam at 141 mW. (a) Targeted cell (marked by a circle) before irradiation (0 s); (b) Propidium iodide fluorescence before irradiation; [(c) and (d)] Cell movement to the left toward the center of the laser spot (1.17 s, 2.83 s); (e) Flipping of the cell in a vertical plane (3.87 s); (f) Levitation of the cell at a higher plane (4.17 s). (g) Anticlockwise orientation of the cell at a higher plane (12.75 s). [(h)–(j)] Bringing the cell down to the substrate (97.00, 111.67, and 121.58 s, respectively). (k) Cell reattached to the bottom. (l) Propidium iodide staining of cells after laser manipulation. All images are in the same magnification. Scale bar: 50 μm. (AVI, 1.8 MB).

3.3.

Dependence of Height of Levitation on Laser Power

76.93% of cells were detached by different powers of laser beam (20 out of 26). The effect of laser power on the distance of levitation was studied by varying the laser power up to ∼240 mW. The height of the cell was measured by first switching off the laser beam and then moving the microscope objective upward to refocus the cell. The translation of the objective was measured from the rotational displacement (number of divisions) of the focusing knob. As shown in Fig. 5, the height of levitation (flying distance) increased from 138 ± 12 μm (laser power of 111 mW) to 152 ± 21 μm when the laser power is increased to 149 mW. At a laser power of 232 mW, the flying distance was 176 ± 48 μm. It may be noted that the levitation distance was found to vary linearly with laser power (Fig. 5). This can be attributed to the fact that the levitating scattering force depends linearly on incident laser power.

Fig. 5

Effect of laser power on cell levitation height. The error bars represent standard error of the mean, n = 4. The straight line represents the linear fit to the data.

3.4.

Orientation, Transportation, and Repositioning of the Detached Cell

Figure 6 shows laser assisted multiple cell detachment–attachment cycles with controlled orientation, transportation, and repositioning of the targeted cell. PI fluorescence and a bright field image of cells before laser irradiation is shown in Figs. 6a and 6b, respectively. Figure 6c shows the first detachment and levitation of the targeted cell [marked by a circle in Fig. 6b] using laser beam power of 138 mW. By lowering the laser power, the cell was brought back to the Petri dish [Fig. 6d]. Figure 6e shows the second detachment of the same cell after an adhesion period of 5 min. Transportation of the levitated cell and the second attachment close to another cell was carried out by translation of the stage [Fig. 6f]. Figures 6g and 6h show the third detachment and attachment of the cell, respectively. After the fourth detachment, reorientation of the cell was carried out by rotation of the cylindrical lens as described in Fig. 2e. The major axis of the cylindrical lens and orientation of the levitated cell is marked by a double-sided arrow in Figs. 6e and 6i. Attachment of the cell for the fourth time was carried out by lowering the laser beam power [Fig. 6j]. Each intermediate adhesion period was ∼5 min and the detachment period was ∼1 min. After four attachment–detachment cycles lasting for ∼30 min, the cell was still viable as confirmed by PI exclusion assay. Figure 6k shows no detectable PI fluorescence of cells at the end of laser manipulation. Further, the cell was found to retain its shape even after multiple detachments and manipulation using the laser beam. Compared to the first detachment of the cell, the second detachment required significantly lower irradiation time (or dose) (p < 0.05, n = 4 for laser powers between 111 to 149 mW). This is indicative of the fact that the force required for breaking early adhesion bonds between the cells and the Petri dish is significantly lower than that of long-term adhesion.

Fig. 6

Laser assisted multiple cell detachment–attachment cycles with controlled orientation, transportation, and repositioning of the targeted cell. (a) Propidium iodide fluorescence of cells before laser irradiation (–50 s); (b) Bright field image before irradiation (0 s); (c) Levitation of detached cell (15 s); (d) Reattachment of the cell on the Petri dish (60 s); (e) Second-detachment of cell (400 s); (f) Transportation and second attachment of the cell (460 s); (g) Third detachment of the cell (800 s); (h) Third attachment of the cell (850 s); (i) Fourth detachment and reorientation of the cell (1200 s); (j) Fourth attachment of the cell (1270 s); (k) Propidium iodide fluorescence of cells after multiple attachment–detachment cycles (1700 s). All images are in same magnification. Scale bar: 50 μm.

As shown in Fig. 6, the NIR laser beam is able to detach the cell multiple times from the Petri dish. Comparing the second detachment of cells with the first time, the irradiation time required for detachment decreased significantly when laser powers were low (p < 0.05, n = 4 when laser powers were 111 and 149 mW, Fig. 7). This indicates that the second detachment requires a lower laser dose. These results suggest to us that the adhesion force between the cells and the Petri dish during the second attachment is not as strong as the adhesion before laser irradiation. Further, the time required for the second cell detachment was found not to depend significantly on the laser power.

Fig. 7

Effect of different laser power on time required for first and second detachment (n = 4). The cell was allowed to adhere for 5 min after the first detachment.