1.

Introduction

In this study, picosecond and femtosecond laser systems are used in combination with a tubulin cyan fusion protein (CFP) to elucidate the dynamics of single microtubules in living cells. This study further considers the suggestion that ultra-short femtosecond laser pulses produce a more precise ablation zone.¹

Near-infrared femtosecond lasers were first used by Konig and Riemann for cellular nanosurgery to ablate regions within the nucleus of Chinese hamster ovary (CHO) cells.² Further work demonstrated the ability of femtosecond laser pulses to produce regions of damage 100 to $200\mathrm{nm}$ in diameter in extracellular isolated and dried chromosomes. This was well below the diffraction limit for the focused 800-nm laser.³ Other studies utilizing femtosecond lasers as nanoscale ablation tools include targeting of mitochondria,⁴ mitotic spindles in yeast,⁵ single microtubules,¹ and single stress fibers.⁶

It has been suggested that with its short pulse duration, femtosecond lasers are capable of efficient ablation dependent on plasma formation by multiphoton absorption and ionization. This is due to the lower energy threshold for optical breakdown as compared to longer pulsed lasers. Thus under optimal conditions, it is thought that femtosecond laser ablation events are confined only within the focal point of the laser.⁷

Of particular interest is the claim that femtosecond lasers are superior to other pulsed laser systems for the nanosurgery of cells.¹ There is sufficient literature demonstrating that nanosecond 532-nm, ^{8, 9, 10, 11} picosecond UV 355-nm,¹² nanosecond UV 266-nm,¹³ picosecond 532-nm,¹⁴ and nonlaser continuous wave UV ^{1, 13, 15, 16} sources can all be used for subcellular surgery, especially as related to cytoskeletal microtubules and cell division.

We demonstrate that both the near-IR femtosecond laser and the picosecond frequency-doubled 532-nm $\mathrm{Nd}:\mathrm{YAG}$ laser can ablate submicron regions of single microtubules in living cells. Furthermore, our microtubule dynamics experiments demonstrate that depolymerization rates of single laser-cut microtubules are faster for microtubules located above or below the nucleus, as opposed to those located in the peripheral cytoplasm. These results are discussed with respect to other laser nanosurgery studies on microtubules as well as the extensive literature on nonlaser UV microbeam irradiation of microtubules, and observations on microtubule growth and shrinkage rates in nonirradiated cells.

2.

Results

2.1.

Single Cyan Fusion Protein Microtubule Cutting

It was possible to cut single microtubules with the femtosecond laser and determine the postirradiation rate of depolymerization for up to sixty seconds following the laser exposure [Figs. 1a, 1b, 1c, 1d, 1e, 1f, 1g, 1h ]. Depolymerization was defined as a decrease in the length of the microtubules, as tubulin monomers are released from the ends of the microtubule polymers. Varying depolymerization rates were observed within the microtubule population, as depicted by the difference in microtubule depolymerization between Figs. 1a, 1b, 1c, 1d and Figs. 1d, 1e, 1f, 1g, 1h.

Fig. 1

Laser exposure and effects on single microtubules. (a), (e), and (i) Fluorescence images before laser exposure. (b), (f), and (j) Images captured immediately after laser exposure show a loss of fluorescence in the targeted region. (c), (d), (g), and (h) Time periods of 30 and $60\mathrm{s}$ following laser exposure above irradiance and energy levels of $25.6\times {10}^5\mathrm{W}∕{\mathrm{cm}}^2$ ; note changes in the targeted microtubule length by depolymerization. (k), and (l) At time periods of 30- and 60-s postlaser exposures with irradiance and energy levels below $20\times {10}^5\mathrm{W}∕{\mathrm{cm}}^2$ ; note no subsequent depolymerization follows laser exposure.

By varying laser irradiance $(\mathrm{W}∕{\mathrm{cm}}^2)$ distinct categories of microtubule responses and their threshold values were observed (Table 1 and Fig. 2 ). First, immediate depolymerization of the targeted microtubule occurred at an irradiance of $24\times {10}^5\mathrm{W}∕{\mathrm{cm}}^2$ where three of eight (40%) irradiated microtubules depolymerized. When the irradiance was reduced to $20\times {10}^5\mathrm{W}∕{\mathrm{cm}}^2$ one of ten (10%) depolymerized. An increase in Irradiance to $26\times {10}^5\mathrm{W}∕{\mathrm{cm}}^2$ always resulted in microtubule depolymerization [Figs. 1a, 1b, 1c, 1d, 1e, 1f, 1g, 1h]. Second, a loss in fluorescence at the targeted region with no subsequent depolymerization following laser exposure was indicative of photobleaching of the fluorescent CFP molecule [Figs. 1i, 1j, 1k, 1l] (see tubulin antibody staining in next section). Photobleaching of the targeted microtubule was consistently observed between irradiance levels of $12\times {10}^5$ and $20\times {10}^5\mathrm{W}∕{\mathrm{cm}}^2$ . To confirm this threshold value, the irradiance was decreased to $8\times {10}^5$ and $4\times {10}^5\mathrm{W}∕{\mathrm{cm}}^2$ , where no change in microtubule fluorescence was detected. (Fig. 2). Thus, under the conditions of this study, the photobleaching threshold is between $12\times {10}^5$ and $20\times {10}^5\mathrm{W}∕{\mathrm{cm}}^2$ .

Fig. 2

Irradiation of a microtubule bundle at irradiance levels below ablation threshold. (a) Image prior to laser exposure. (b), (c), (d) Crosshairs depict exact location of laser exposure for $12\times {10}^5$ , $8\times {10}^5$ , and $4\times {10}^5\mathrm{W}∕{\mathrm{cm}}^2$ . (e) Image following ablation shows a slight loss in fluorescence in the region corresponding to the $12\times {10}^5\mathrm{W}∕{\mathrm{cm}}^2$ . (f) A contrast-enhanced image clearly shows a change in fluorescence at $12\times {10}^5\mathrm{W}∕{\mathrm{cm}}^2$ but no effect on fluorescence for $8\times {10}^5$ $\mathrm{W}∕{\mathrm{cm}}^2$ or below.

Table 1

Irradiance and energy density values for photobleaching and cutting thresholds by the femtosecond laser. Settings below the photobleaching thresholds result in no change to the targeted microtubule. At the cutting threshold, 50% of targeted microtubules are successfully ablated. Settings above the supra-cutting threshold always result in successful ablation of the targeted microtubule.

Irradiance (W∕cm2)	Energy density (J∕cm2)	Threshold
$20\times {10}^5$	$3.0\times {10}^4$	Photobleaching
$24\times {10}^5$	$3.6\times {10}^5$	Ablation
$26\times {10}^5$	$4\times {10}^5$	Supra-ablation

2.2.

Immunofluorescence of Cut Cyan Fusion Protein Microtubules

To determine if a loss of fluorescence was indicative of microtubule cleavage as opposed to photobleaching, single irradiated microtubules were fixed and stained with fluorescently conjugated antibodies to tubulin immediately following laser exposure. Figure 3 compares a photobleached microtubule [Figs. 3a, 3b, 3c] and a cleaved microtubule [Figs. 3d, 3e, 3f]. For the photobleached microtubule, there was a loss of tubulin fluorescence immediately after irradiation, but positive tubulin immunofluorescence staining in the irradiated region. For the cleaved microtubule, the 0.47 μm diameter cut exhibited a loss of fluorescence immediately following irradiation, and immuno-stained negatively in that region [Figs. 3d, 3e, 3f]

Fig. 3

Single irradiated microtubules at different laser exposure parameters resulting in an absence or presence of immunofluorescence staining. (a) and (d) Prelaser exposure images of single microtubules. (b) and (e) Images immediately after laser exposure, demonstrating a loss in fluorescence at the targeted region. Immunofluorescence staining shows the presence of staining at $6.7\times {10}^5\mathrm{W}∕{\mathrm{cm}}^2$ , but an absence of staining (c) at $26\times {10}^5\mathrm{W}∕{\mathrm{cm}}^2$ . (f) Loss of staining confirms a successful ablation event.

2.3.

Microtubules Dynamics

2.3.1.

Femtosecond cutting of cyan fusion protein microtubules

Depolymerization rates were calculated for newly formed plus ends of cut microtubules and for control nonablated microtubules that were observed to depolymerize spontaneously. Figure 4 illustrates depolymerization of both cut microtubules and control microtubules in the same region of a cell. The mean rate of induced plus end depolymerization was $3.54\pm 3.14\mathrm{\mu }\mathrm{m}∕\min$ , while the mean rate of spontaneous depolymerization of noncut control microtubules was $5.78\pm 4.50\mathrm{\mu }\mathrm{m}∕\min$ (Fig. 5 ). Using a rank-sum statistical test, it was determined that new plus ends created by laser cutting depolymerized at rates slower than the rates of spontaneously depolymerizing microtubules $(\mathrm{p}=0.002)$ .

Fig. 4

Depolymerization of a single microtubule (white) following laser cutting observed among untargeted, control microtubules (black). Depolymerization rates were calculated for microtubules in the adjacent nonlaser-exposed region over a 60-s time period.

Fig. 5

Distribution of depolymerization rates for control microtubules and newly formed plus ends. (a) and (c) Distribution of mean depolymerization rates of untargeted, control microtubules. (b), (d), and (e) Distribution of mean depolymerization rates of newly formed plus ends of microtubules resulting from laser irradiation. Two data sets (a) and (c) represent varying experimental conditions between experiments with the same call line. Data correspond to experiments targeting (a) and (d) CFP-tubulin with the femtosecond laser, (c) and (b) CFP tubulin with the picosecond laser, and (e) CFP-tubulin microtubules residing near the nucleus ablated with the picosecond laser. Each histogram includes information about the parameters, mean depolymerization rate, number of microtubules, and number of cells observed.

2.3.2.

Picosecond cutting of cyan fusion protein microtubules

To determine if the pulse duration or wavelength of the laser had an effect on the depolymerization rates at plus ends, a picosecond laser emitting at $532\mathrm{nm}$ was used to ablate CFP microtubules. The average rate of depolymerization for ablated plus ends was $3.95\pm 2.54\mathrm{\mu }\mathrm{m}∕\min$ and $3.72\pm 1.84\mathrm{\mu }\mathrm{m}∕\min$ for control microtubules (Fig. 5). There was no significant difference between cut and control depolymerization rates $(\mathrm{p}=0.704)$ .

To determine if the position of the microtubule with respect to the nucleus had an effect on microtubule depolymerization rates, single microtubules residing above or below the nucleus were targeted with the picosecond laser (Fig. 6 ). The average rate for plus end depolymerization was $13.66\pm 8.84\mathrm{\mu }\mathrm{m}∕\min$ [Fig. 5d]. Plus-end depolymerization rates above or below the nucleus were significantly different $(p\ll 0.01)$ than microtubules cut at the periphery of the cell $(3.54\pm 3.14\mathrm{\mu }\mathrm{m}∕\min )$ .

Fig. 6

Laser irradiation of microtubules positioned near the microtubule organizing center, above or below the nucleus. (a) Preablation image compared to (b) post ablation image reveals the location of the target prior to laser exposure. (c) $15\mathrm{s}$ and (d) $30\mathrm{s}$ after ablation, showing the depolymerization of the targeted microtubule (black arrows). Depolymerization of a previously ablated microtubule in the same field of view (white arrows). (e) through (h) Contrast-enhanced images clearly showing the depolymerization of microtubules across the nucleus region.

3.

Discussion

We have demonstrated that a near-IR $\left(800\mathrm{nm}\right)$ femtosecond laser can be used to perform precise subcellular nanosurgery on individual microtubules in live cells. Severed microtubules can be followed and analyzed with respect to depolymerization (shrinkage) dynamics. However, when compared to the 532-nm picosecond $\mathrm{Nd}:\mathrm{YAG}$ laser,¹⁴ the 532-nm nanosecond laser,⁸ and the 355-nm picosecond $\mathrm{Nd}:\mathrm{YAG}$ laser,¹² we find that there is no significant advantage of the femtosecond laser, and there may, in fact, be some disadvantages.

It is noteworthy that the average rates of depolymerization after the 800-nm femtosecond laser cuts were different from the control noncut depolymerization rates $(\mathrm{p}=0.002)$ , whereas there was no difference in the average rates of depolymerization between the 532-nm picosecond-cut microtubules and the depolymerization rates of nonirradiated control microtubules $(\mathrm{p}=0.704)$ (Fig. 5). The shorter pulse femtosecond laser had a peak irradiance of $157\mathrm{GW}∕{\mathrm{cm}}^2$ in the focused spot, whereas the picosecond laser had a peak irradiance of $7\mathrm{GW}∕{\mathrm{cm}}^2$ (Table 2 ). This twenty-fold difference in peak irradiance could have played a role in the observed differences between the picosecond and femtosecond laser effects on microtubule depolymerization rates. Calculations in a previous picosecond laser study¹⁴ strongly suggested a multiphoton-induced ionization and plasma formation mechanism of ablation. It is likely that similar mechanisms are occurring in the femtosecond laser ablations reported here. This would be consistent with other femtosecond laser studies.^{3, 7} However, the difference in the depolymerization rates following exposure to the two lasers bears further examination with respect to biological and physical mechanisms.

Fluorescent images from postirradiation and immunofluorescence staining revealed an ablation diameter of $0.47\mathrm{\mu }\mathrm{m}$ , which is smaller than the $0.7\text{-}\mathrm{\mu }\mathrm{m}$ calculated focal spot of the laser (see Sec. 4). To determine the ability of the femtosecond laser to make precise cuts in cellular structures within living cells, EM analysis of picosecond ablations demonstrated its ability to ablate regions $0.34\mathrm{\mu }\mathrm{m}$ in diameter, which was below the $0.47\text{-}\mathrm{\mu }\mathrm{m}$ focal diameter of the laser.¹⁴ Thus both laser systems appear to be able to ablate regions of cellular structure less than the theoretical size of the laser focal point. This may be due to the fact that a Gaussian profile laser beam is focused to a Gaussian focal spot, which may be above lesion threshold only at the center of the Gaussian.^{17, 18}

Our results on microtubule depolymerization following cutting must be viewed in the context of the many published in-vitro (test tube) and in vivo (live cell) studies on single microtubule dynamics. First, we have determined the ablation (cutting) threshold for individual microtubules. (Table 1). This is important both for repeatability by other investigators as well as for understanding the physical mechanism(s) of the ablation process.^{14, 18} Furthermore, a loss of fluorescence in the irradiated region of a microtubule may be due either to photobleaching of the fluorescent fusion protein (CFP, YFP, GFP, etc.), or to an actual ablation/cut of the microtubule. Thus it was important to fix and stain cells with fluorescent antitubulin and relocate the irradiated microtubule (Fig. 3). In this way ablation thresholds were determined. The ablation threshold for the femtosecond laser was $2.4\times {10}^6\mathrm{W}∕{\mathrm{cm}}^2$ , while for the picosecond laser it was $4.1\times {10}^7\mathrm{W}∕{\mathrm{cm}}^2$ , a twenty-fold difference in average irradiance but with only a three-fold difference in fluence (Table 2).^{14, 18}

Second, the individual microtubule depolymerization rates for most of the experiments reported here were generally less than those reported in the literature, whether for laser-cutting experiments, classic nonlaser UV cutting, or noncutting experiments where control shrinkage rates were determined. For example, Colombelli ¹² reported an average shrinkage rate of $19.5\mathrm{\mu }\mathrm{m}∕\min$ for individual YFP-transfected microtubules in PtK2 cells severed with the 355-nm picosecond UV laser. In nonlaser UV cutting of the plus ends of microtubules in vitro, an average shortening rate of $21\mathrm{\mu }\mathrm{m}∕\min$ ¹⁵ was measured in one study and $22\mathrm{\mu }\mathrm{m}∕\min$ was reported in another study.¹⁹ Similarly in PtK2 and LLCPK cells, Rusan ²⁰ reported shrinkage rates for nonsevered microtubules of 14.8 and $13.1\mathrm{\mu }\mathrm{m}∕\min$ for rhodamine tubulin and eGFP transfected tubulin, respectively. Also, Shelden and Wadsworth²¹ measured shrinkage rates of $19.8\mathrm{\mu }\mathrm{m}∕\min$ for PtK1 cells and $32.2\mathrm{\mu }\mathrm{m}∕\min$ in Chinese hamster ovary (CHO) cells. In Taricha granulosa newt lung cells, average shortening rates of $17.3\mathrm{\mu }\mathrm{m}∕\min$ were measured,²² and in the South African clawed toad Xenopus laevis, average shortening rates were $12.8\mathrm{\mu }\mathrm{m}∕\min$ .²³ Slower average shrinkage rates of $7.4\mathrm{\mu }\mathrm{m}∕\min$ were measured in PtK1 cells,²⁴ and $4.3\mathrm{\mu }\mathrm{m}∕\min$ in human neonatal fibroblasts.²⁵ The shrinkage rates reported in these latter two studies more closely approximated the rates we have observed in CFP-labeled microtubules in PtK2 cells (3.72 and $5.43\mathrm{\mu }\mathrm{m}∕\min$ ).

It is clear that the variability is great between cell types, and even from different laboratories working with the same cell type, i.e., PtK1 cells with shrinkage rates of $7.4\mathrm{\mu }\mathrm{m}∕\min$ ²⁶ and $19.8\mathrm{\mu }\mathrm{m}∕\min$ .²¹ In this case, the experiments were done six years apart, and it is possible that the passage numbers for the PtK1 cells used were substantially different. The many years of selection pressure in vitro could have resulted in physiological and genetic changes in the cells themselves. However, a large variation in depolymerization rates for spontaneously depolymerizing microtubules was observed between day-to-day measurements: 5.43 to $3.72\mathrm{\mu }\mathrm{m}∕\min$ (Fig. 5). In addition, the microtubule location within the cytoplasm was shown to be a factor in the rate of depolymerization (see later discussion).

Classic UV laser-cutting experiments by Walker, Inoue, and Salmon have proposed a UV-activated mechanism to explain the rapid stabilization of the minus end.¹⁵ Observations of ablated microtubules by both the near-IR femtosecond laser and 532-nm $\mathrm{Nd}:\mathrm{YAG}$ laser are inconsistent with this theory, as they show the same stabilization as observed with the UV laser (Figs. 1 and 4). The stability of newly formed minus ends seems to be a characteristic of the microtubule itself rather than a mechanism dependent on laser wavelength.

Third, we observed a highly significant difference $(\mathrm{p}\ll 0.01)$ between average shrinkage rates of cut microtubules in the periphery of the cell (3.44 to $3.95\mathrm{\mu }\mathrm{m}∕\min$ ) compared to shrinkage rates of cut microtubules directly above or below the cell nucleus $(13.66\mathrm{\mu }\mathrm{m}∕\min )$ . This observation may be relevant to differences in plus-end shrinkage rates observed in migrating newt cells when microtubules were either parallel to the leading edge of the cell $(7.6\mathrm{\mu }\mathrm{m}∕\min )$ or perpendicular to the leading edge $(5.2\mathrm{\mu }\mathrm{m}∕\min )$ .²⁶ Our experimental results are consistent with these observations, and they may be explained by differences in the local structure and chemistry at the cell edge versus the nucleus. This is also consistent with recent studies that suggest stress fibers and the surrounding environment, specifically motor proteins and extracellular matrix components, can affect cytoskeletal dynamics.⁶ Also, it is possible that the biomechanics and fluid mechanics within the cytoplasm are different between the thin lamellae regions of the cell, where single tubes are easily distinguished, and the thicker region around the nucleus, where the biochemical and biomechanical environments may be different. Notwithstanding these possible explanations, the statistical difference between depolymerization of cut microtubules at the periphery of the cell and above/below the nucleus is highly significant.

We have demonstrated that the near-IR femtosecond laser can be used for subcellular nanosurgery of individual microtubules in living cells. However, we have found no significant advantage of this laser over the picosecond green laser, or other lasers reported in the literature, for ablating single microtubules. In addition, the studies reported here have revealed several interesting observations on the dynamics of single microtubules, especially with respect to rates of depolymerization in different regions of the cell.

4.

Materials and Methods

4.3.

Laser Ablation

The femtosecond laser ablation system used is diagrammed in Fig. 7 . A Coherent Mira 900 Ti:sapphire laser (Coherent Incorporated, Santa Clara, California) emitting 200 femtosecond pulses at $76\mathrm{MHz}$ was used for ablation at $800\mathrm{nm}$ . Laser power was controlled manually by a neutral density wheel, which allowed regulation of beam power from 0 to $700\mathrm{mW}$ . Laser power was measured by a FieldMaxII TOP power meter coupled to a PM3 probe (Coherent Incorporated, Santa Clara, California) with a 19-mm-diameter sensor. The probe was placed directly after the objective where the diameter of the laser beam was smaller than the sensor diameter. This allowed for collection of all light exiting the objective, which resulted in an estimate of the power level at the focal point of the specimen. The diameter of the laser beam at the focal point was calculated from the equation $d=1.22\lambda ∕\mathrm{NA}$ , where $\lambda$ is the wavelength and NA is a numerical aperture of the objective. Laser exposure time was controlled by a motorized Oriel (Stratford, Connecticut) electronic shutter controller, which allowed a minimum of 5-ms exposure time. Laser exposure times were set at $150\mathrm{ms}$ based on previously published work.⁵ The laser beam was directed into the epi-illumination port (optics removed) of a Zeiss Axiovert S100 2TV microscope (Carl Zeiss Incorporated, Thornwood, New York) by a dichroic filter designed to pass visible arc lamp emission and reflect the 800-nm laser light. The beam was focused by a Zeiss $63\times \mathrm{PH}3$ oil immersion apochromat objective lens (numerical aperture 1.40). Initial microtubule dynamics experiments were conducted at a warm room temperature of $23°\mathrm{C}$ , and later experiments were conducted using an air stream stage incubator (Nevtec, Burnsville, Virginia) that maintained the cell culture medium at $34°\mathrm{C}$ . A comparison of the microtubule depolymerization rates at the two temperatures using the rank-sum statistical test showed that there was no difference in the rates between the two temperatures $\mathrm{p}>0.95$ . Thus, in this study the two groups are combined.

Fig. 7

Schematic diagram of imaging and laser ablation system. The Ti:sapphire laser beam is expanded and passes through a shutter and neutral density wheel before being directed through the microscope and objective. The beam is focused at the imaging plane. A dichroic mirror in the laser path reflects the beam and allows for fluorescence excitation light to transmit into the objective and illuminate the specimen.

Table 2

Comparison of laser parameters for femtosecond and picosecond laser cutting thresholds of CFP tubulin in PtK2 cells.

	Femtosecond laser	Picosecond laser
Repetition rate	$76\mathrm{MHz}$	$76\mathrm{MHz}$
Pulse duration	$200\mathrm{fs}$	$80\mathrm{ps}$
Wavelength	$800\mathrm{nm}$	$532\mathrm{nm}$
Focused spot diameter (from 1.22 l/NA)	$0.7\mathrm{mm}$	$0.46\mathrm{mm}$
Focused spot area	$3.82\times {10}^{-9}{\mathrm{cm}}^2$	$1.66\times {10}^{-9}{\mathrm{cm}}^2$
Energy per pulse	$0.11\mathrm{nJ}$	$0.92\mathrm{nJ}$
Peak irradiance	$157\mathrm{GW}∕{\mathrm{cm}}^2$	$7\mathrm{GW}∕{\mathrm{cm}}^2$
Exposure time (average irradiance)	$150\mathrm{ms}$	$3\mathrm{ms}$
Deposited energy/area (energy density)	$3.6\times {10}^5\mathrm{J}∕{\mathrm{cm}}^2$	$1.24\times {10}^5\mathrm{J}∕{\mathrm{cm}}^2$
Average irradiance	$2.4\times {10}^6\mathrm{W}∕{\mathrm{cm}}^2$	$4.1\times {10}^7\mathrm{W}∕{\mathrm{cm}}^2$

Targeting was accomplished by moving the stage (Ludl Incorporated, Hawthorne, New York) by joystick control until the region of interest was positioned at the prealigned laser focal point. Laser ablation was accomplished by stage movement, such that the target region was exposed to the focused laser. Stage movement was controlled by a PCI-7344 motion controller and a MID-7604, 4 Axis Integrated Stepper Driver Power Unit (National Instruments), all controlled by our custom-made Robolase software.²⁸

The picosecond laser ablation system used is described in detail by Botvinick and Berns.²⁸ The laser system was comprised of a 532-nm Spectra-Physics Vanguard laser with 76-MHz repetition rate and 80-ps pulse duration. Average power was attenuated by rotation of a neutral density wheel.