2.1.

Yeast Strains and Growth Conditions

Saccharomyces cerevisiae (Saint Georges S101, Bio Springer, France) was used for all experiments. Cells were grown in YPG medium (glucose $10\mathrm{g}∕\mathrm{L}$ , pH 5.6) at $25°\mathrm{C}$ under shaking $\left(120\mathrm{rpm}\right)$ , harvested in the midexponential phase (concentration: $5\times {10}^5\text{cells}∕\mathrm{ml}$ ), and transferred to a growth chamber (see in the following), where they were left to settle and attach to the surface for $5\min$ . Subsequently, the growth chamber was flushed with fresh YPG medium to remove any cells not attached to the surface, which could diffuse into the field of view and interfere with measurements.

The growth chamber used for the experiments consisted of a cover glass slide (Menzel-Gläser #1; $0.15\mathrm{mm}$ ; size, $24\times 40\mathrm{mm}$ ) and a perfusion chamber (CoverWell, Grace-BioLab). The cover glass slides were first cleaned in 70% ethanol, 1% HCl, and then washed with demineralized water and left to dry. The cover glass slides were coated with a $0.8\mathrm{mg}∕\mathrm{ml}$ solution of concanavalin A ( $0.5\mathrm{ml}$ per cover glass) and left to dry. Once dry, the CoverWell perfusion chamber was attached to the coated cover glass slides, and sealed with adhesive tape. The presence of Concanavalin A did not affect the growth rate of the cells (data not shown).

2.2.

BioPhotonics Workstation

The biophotonics workstation¹⁵ (BWS) used in this work is illustrated schematically in Fig. 1 . It employs an optical mapping from a beam modulation module to obtain reconfigurable patterns corresponding to two independently addressable regions which are relayed to a BS, creating the two opposing beam patterns that are sent into the sample. The addressable regions occupied approximately half the field of view of the camera. The spatial addressing of the expanded laser source is done in real time through a high-speed computer-controlled SLM that is integrated in the beam modulation module (patent pending). Through a computer interface, the operator can simply select, trap, and move the desired objects with a mouse. The trap sizes can also easily be changed in diameter. The pattern created in the trapping region in the chamber was homogenous across the $x\text{-}y$ plane, and the trapping power is not dependent on the number of traps created. The generated array of counterpropagating trapping beams is then easily aligned using a computer-guided alignment procedure.¹⁶

Fig. 1

Biophotonics workstation. The beam from the laser source is sent to the beam modulation and is split into two beam arrays that are, respectively, diverted to the upper and lower objective by the beamsplitter (BS) and four mirrors. In this study, the separation between objectives 1 and 2 is $1.2\mathrm{cm}$ . M1 and M2 are measurement points for laser power.

The laser used in the experiments is a ytterbium fiber laser with a central wavelength of $1070\mathrm{nm}$ (IPG photonics, type: YLM-20-SC). It has a maximal output power of $P_{\text{output}}\left(\max \right)=20\mathrm{W}$ . The camera used for top-view observation is a JAI CV-A10GE $(800\times 600\text{pixels})$ . Two identical objectives were used. These were long-working-distance air objectives optimized for IR (Olympus, LMPlan $50\times \mathrm{IR}$ , numerical aperture 0.55), which generated a large field of view and the long working distance of $6\mathrm{mm}$ leaves space for integrating other enabling tools for probing the trapped particles, such as linear and nonlinear microscopy or microspectroscopy.¹⁷ However, one is not restricted to using long-working-distance (LWD) objectives and they can easily be replaced with high-numerical-aperture (NA) objectives offering a higher resolution, at the cost of a smaller field of view.

To measure the transmittance we used a dual-objective transmittance technique¹⁸ whereby the laser is sent through oppositely aligned facing objectives. This technique was used because it required the least amount of adjustment of the setup. The power $\left(P_1\right)$ of the beam was measured at M1 and $P_2$ was measured at M2 after passing through the objectives (see Fig. 1). The transmittance coefficient of a single objective $\left({\tau }_{\mathrm{obj}}\right)$ is then the square root of $P_2∕P_1$ . The transmittance through a single objective $\left({\tau }_{\mathrm{obj}}\right)$ was measured to be ${\tau }_{\mathrm{obj}}=0.75$ . The transmittance coefficient at $1064\mathrm{nm}$ of the cover glass $\left({\tau }_{\mathrm{cov}}\right)$ was specified by the manufacturer at ${\tau }_{\mathrm{cov}}=0.92$ . We assume that the shift in wavelength to $1070\mathrm{nm}$ does not change the value of ${\tau }_{\mathrm{cov}}$ significantly.

2.3.

Flux Calculation

Because of the technique used in the BWS, the laser power is evenly distributed across the trapping area. This is different from single-beam optical tweezers, which focus the laser to a diffraction limited spot. To calculate the flux at the specimen plane we measured the power $\left({\Phi }_{\mathrm{obj}}\right)$ just before the objectives at M1, as seen in Fig. 1, at multiple trap sizes. This gave us the conversion factor $\left(Y_{\mathrm{conv}}\right)$ $\left(\mu {\mathrm{m}}^{-2}\right)$ accounting for both attenuation and scaling of the laser beam though the setup. The flux in the specimen plane $\left(E_{\text{specimen}}\right)$ is then given by

2.4.

Determination of Cell Area Index

Once the growth chamber was flushed and sealed, it was put under the microscope. After visual inspection, a number of cells was found within close proximity to each other and as close to the same stage in the cell cycle as possible. The stage in the cell cycle was determined by the size of an emerging bud. Images were taken at the start of irradiation at $t=0$ , and consequently every $2\min$ . Image analysis was performed on images selected at $30\text{-}\min$ intervals.

To measure the cell growth as a function of time, the area occupied by all cells within a region of interest (ROI) was extracted from every image. The ROI was defined as the cells that were all originating from the same mother cell. The area occupied by all cells within an ROI was given by the number of pixels (NoP) that it occupied in a picture after image processing and thresholding, as seen in Fig. 2 . Figure 2 shows the growth of cells within three ROIs during $3\mathrm{h}$ . ROI 1 was not irradiated (control) and ROIs 2 and 3 were exposed to $5\mathrm{mW}$ for $10\min$ .

Fig. 2

(a) Original and (b) thresholded images of a single growth experiment. In the thresholded images, the area occupied by the ROI is represented by a white color. The cells within ROI 1 are the control cells. The cells within ROIs 2 and 3 were irradiated for $10\min$ at a power of $5\mathrm{mW}$ . The black arrows show the appearance of two consecutive buds from the same mother cell.

Due to the variations in the size of the cells at the start of the experiments, the NoP values were not directly comparable. Thus, the NoP for every data point was normalized by dividing with the area of the ROI at time $t=0$ . This quantity was defined as the cell area index (CAI) and is simply given by

Using the CAI it was possible to calculate the maximum specific growth rate $\left({\mu }_{\max }\right)$ by fitting an exponential function, which also gave the doubling time $t_d=\ln 2∕{\mu }_{\max }$ of the cell population within an ROI.

Using the area occupied by the cells for growth determination limited the time during which the cell growth could be followed accurately. We found that $3\text{to}4\mathrm{h}$ was the maximum time that the measured area of the ROIs could describe exponential growth. After this period, the cells would begin to bud vertically and the area did not increase exponentially. If ROIs began to form buds vertically with the time frame of the experiment, the ROIs were omitted from the analysis. For ROIs exposed to continuous irradiation, 15 to 20 ROIs were recorded for every power value with a total of 35 control ROIs. For ROIs exposed to laser pulses, a minimum of 25 ROIs were recorded for every power value with a total of 40 control ROIs.

2.5.

Calculation of Generation Time

To obtain growth kinetic data for the individual cells, we measured the generation time of the individual cells. The generation time was defined as the period between two consecutive buds from the same mother cell (Fig. 2). As images were taken at $2\text{-}\min$ intervals, the temporal resolution of the time between two buds was $\pm 2\min$ . For all experiments, the generation times of both irradiated cells and control cells were obtained. For cells exposed to continuous irradiation, 20 to 30 cells were recorded for every power value with a total of 40 control cells. For cells exposed to laser pulses, 30 to 35 cells were recorded for every radiant flux value with a total number of 41 control cells.

2.6.

Image Processing

All images were processed using LabVIEW 8.2 Vision software. A sharpening convolution kernel was applied first to enhance the contrast between the cell wall and background. Then local thresholding with background correction was applied. This left only the cell wall in the binary image. Due to the budding of daughter cells, the cell wall did not always form a complete circle after thresholding. Using the binary morphology (BM) operations “dilate” the cell walls were expanded with a number of iterations until they formed a complete circle. The BM operation “fill hole” was then used to fill the inside of the cell. To return the cells to normal size, the BM “erode” was then applied for same number of iterations as the BM operation “dilate.” Finally, to remove dust and cells touching the side, BM operations “remove small particles” and “remove border objects” were applied.

3.1.

Continuous Irradiation

The control cells exhibited exponential growth with a doubling time of $97\min$ (data not shown). As seen clearly in Fig. 3 , all cells exposed to continuous laser light were inhibited in their growth and did not show exponential growth. After $1.5\mathrm{h}$ , the cells subjected to the lowest power $\left(0.7\mathrm{mW}\right)$ were also significantly inhibited, but their growth rate increased slightly with time. The cells exposed to $1.3\mathrm{mW}$ were inhibited after $60\min$ and had a linear CAI increase, suggesting that the mother cells produced only a single daughter cell. The cells exposed to $2\mathrm{mW}$ were visibly inhibited after $60\min$ and the ROIs exposed to $2.6\mathrm{mW}$ were inhibited after $30\min$ . For both the 2- and $2.6\text{-}\mathrm{mW}$ -exposed cells, the growth rate continuously declined and was close to zero after $3\mathrm{h}$ . However, the $2\text{-}\mathrm{mW}$ -exposed cells leveled out at a higher CAI.

Fig. 3

Effect of continuous irradiation on CAI of S. cerevisiae: controls cells (◆), $0.7\mathrm{mW}$ (×), $1.3\mathrm{mW}$ (▲), $2.0\mathrm{mW}$ (◼), and $2.6\mathrm{mW}$ (●). Data are $\text{means}\pm \text{standard}$ error of the mean (SEM). Every data point in the control and irradiated ROI series is an average of between 35 and 15 to 20 measurements, respectively.

The variation of the generation times of individual mother cells increased with laser power (Fig. 4 ). However, absolute values for generation times were not attainable, as only cells that divided within the time frame of $5\mathrm{h}$ could be included in the measurement. The number of successful cell divisions within the $5\text{-}\mathrm{h}$ period showed a continuous decline as a function of laser power with 100% cell division at $0.7\mathrm{mW}$ , 80% at $1.3\mathrm{mW}$ , 45% at $2.0\mathrm{mW}$ , and 0% cell division at $2.6\mathrm{mW}$ (data not shown). These results suggest that the measured generation times, and their variations, were increasingly underestimated with increasing laser power.

Fig. 4

Effect of continuous irradiation on percentage cell division of S. cerevisiae within the $5\text{-}\mathrm{h}$ duration of the experiments. Control cells were not irradiated. Data points are $\text{means}\pm \mathrm{SEM}$ of 20 to 30 single cells.

3.2.

Pulsed Irradiation

To test whether the cell growth is affected only by the total dose received, we subjected cells to the same amount of energy but with different power; i.e., the cells subjected to 1, 5, and $10\mathrm{mW}$ were irradiated for 50, 10, and $5\min$ , respectively, after which cell growth was monitored. Thus, the total dose delivered to an average size cell was $D=3\mathrm{J}$ . From Fig. 5 we observed that, although the total dose received was the same, the difference in the power through the cells clearly affected the CAI increase of the cells; i.e., the CAIs of 1-, 5-, and $10\text{-}\mathrm{mW}$ exposures after $3\mathrm{h}$ were 89, 79, and 68%, respectively, of the control CAI.

Fig. 5

Plot of log CAI as a function of time of pulsed irradiated S. cerevisiae cells. Control cells (◆) were not irradiated, $1\mathrm{mW}$ for $50\min$ (×), $5\mathrm{mW}$ for $10\min$ (▲), and $10\mathrm{mW}$ for $5\min$ (◼). The solid line is the exponential fit to the control data points starting a $t=0$ . The dashed lines are the exponential fits to the irradiated ROIs. The data points and the exponential fits for the 1-, 5-, and $10\text{-}\mathrm{mW}$ exposures start after 50, 10, and $5\min$ , respectively, which indicate the end of the irradiation period. Data points are $\text{means}\pm \mathrm{SEM}$ of minimum 25 ROIs.

For all three pulses, the exposed cells recovered from the initial pulse and resumed exponential growth after the exposure period (Fig. 5, Table 1 ). To quantitatively examine how the cells recovered from the irradiation, exponential curves were fitted at the end of the irradiation period for the three different radiant flux series, and ${\mu }_{\max }$ and $t_d$ were calculated from the CAI plots in Fig. 5. In the series with cells exposed to $1\mathrm{mW}$ for $50\min$ and $5\mathrm{mW}$ for $10\min$ , the exponential fits were good with $R^2>0.99$ and the two series had similar $t_d$ values of 115 and $119\min$ , respectively, corresponding to approx. 120% of the control cells. Cells exposed to $10\mathrm{mW}$ for $5\min$ showed an even larger $t_d$ value of $146\min$ , corresponding to 150% of the control cells; however, the fit was slightly worse than for cells exposed to 1 and $5\mathrm{mW}$ .

Table 1

Growth data of pulsed irradiated S. cerevisiae cells.

Note that because the cells were growing during exposure to the irradiation, the ROIs exposed to a low power for a long time received a larger dose than the ROIs exposed to a high power for a short time, simply due to the increase in area. Using the data points in Fig. 5 to calculate the area under the curve by simple triangulation gives a total dose increase of 18% for ROIs exposed to $1\mathrm{mW}$ . For the cells exposed to 5 and $10\mathrm{mW}$ , the total dose increases are 1.7 and 0.7%, respectively. However, the consequence of correcting for the different total doses received by the cells would result in an even larger difference in growth between the exposed cells.

The generation times of pulse-irradiated individual mother cells within the ROIs were also recorded for the three different intensities (1, 5, and $10\mathrm{mW}$ ) and the control cells (Table 1). The generation time of the cells was severely affected by the total dose, as it increased to 124 to 151% of the control cells. The cells exposed to $10\mathrm{mW}$ for $5\min$ had the largest increase (151%), while the cells exposed to $1\mathrm{mW}$ for $50\min$ were the least inhibited with a generation time increase of 124% as compared with the control.

As seen in Table 1, the generation time was significantly shorter than the $t_d$ value of the control cells. However, this was not the case for the irradiated cells, where the generation time and the $t_d$ were virtually similar.