2.1.

Two-Photon Fluorescence Recovery after Photobleaching for Analyzing Histone with High Mobility In Vivo

The packaging of eukaryotic DNA is performed by histones, which are mainly classified into H1, H2A, H2B, H3, and H4. The H2A, H2B, H3, and H4 histones are core histones that assemble into an octamer as a nucleosome core particle. The H1 histone is a linker histone that connects one nucleosome core particle to the next. Data regarding the mobility of GFP-tagged histone H1, H2A, H2B, H3 and H4 proteins in living human cells have been reported.^{40, 41, 42, 43} In those studies, single-photon excitation was used to obtain the mobility. However, photobleaching using single-photon absorption has a disadvantage, namely, collateral absorption outside the focal volume, as mentioned before. Therefore, it is difficult to observe the mobility of a fluorophore along the beam propagation axis ( $z$ axis) by single-photon excitation. With two-photon bleaching, the photobleached volume along the $z$ axis can be limited.^{7, 9}

We established a HeLa cell line to stably express enhanced GFP (EGFP)-histone H1.2.⁴⁴ Using this cell line, we measured the dynamics of EGFP-histone H1.2 in a selectively bleached heterochromatic region of the nucleus by single-photon and two-photon FRAP (Fig. 1 ). In the case of single-photon FRAP, the GFP fluorescence of bleached areas recovered within a maximum of $900\mathrm{s}$ , as shown by the representative recovery curve. In contrast, with two-photon FRAP using the same region of interest (ROI) size, the EGFP fluorescence recovered within $200\mathrm{s}$ . Thus, the fluorescence recovery rate $\left(t_{1∕2}\right)$ for two-photon excitation was much shorter than that for single-photon excitation. The difference in the fluorescence recovery rates was due to differences in the bleaching properties between single- and two-photon excitation. Similar to fixed cells, in living cells, EGFP molecules existing above and below the focal plane were completely bleached by single-photon excitation, whereas in two-photon excitation, the bleached area was limited to the vicinity of the focal plane.^{9, 45} Therefore, EGFP fluorescence recovered more rapidly in two-photon FRAP.

Fig. 1

FRAP analyses of EGFP-histone H1.2. (a) The heterochromatic region within an open box of living HeLa cell nuclei was excited and bleached by single-photon excitation using ${\mathrm{Ar}}^{+}$ laser light with a wavelength of $488\mathrm{nm}$ . Scale bar indicates $5\mu \mathrm{m}$ . (b) The heterochromatic region within an open box of living HeLa cell nuclei was excited and bleached by two-photon excitation using femtosecond laser light with a wavelength of $928\mathrm{nm}$ . Scale bar indicates $5\mu \mathrm{m}$ . (c) Circles and squares indicate fluorescence recovery curve obtained from single- and two-photon FRAP, respectively.

We established a tobacco BY-2 cell line stably expressing histone H1-sGFP (a GFP variant in which threonine is substituted for serine at 65) and analyzed the histone H1 mobility in plant cultured cells by two-photon FRAP (Fig. 2 ). The BY-2 cells have spherical nuclei, which are different from the flat nuclei in mammalian adhesive cultured cells. The recovery rate in two-photon FRAP using a tobacco BY-2 cell was also approximately four times faster than that in single-photon FRAP. This result indicates that two-photon FRAP is also suitable for investigating molecules in thick subcellular organelles. Furthermore, living cells are damaged to a lesser extent when two-photon excitation is used for in-vivo analyses, because a near-infrared laser is employed for the excitation. We compared the recovery rate of GFP-tagged histone H1 between single- and two-photon FRAP in tobacco BY-2 cells, and obtained similar results both in HeLa and tobacco BY-2 cells. We found that the recovery rate is not mainly dependent on the kinds of host cells, but types of target proteins.

Fig. 2

FRAP analysis of histone H1-sGFP in BY-2 cells. (a) The heterochromatic region of living BY-2 cell nuclei was excited and bleached by two-photon excitation using femtosecond laser light with a wavelength of $928\mathrm{nm}$ . Arrow shows the bleaching area. Scale bar indicates $5\mu \mathrm{m}$ . (b) Fluorescence recovery curve obtained from two-photon FRAP.

2.2.

Two-Photon Fluorescence Recovery after Photobleaching for Analyzing Histone with Low Mobility In Vivo

FRAP is an indispensable method for analyzing the molecular dynamics in living cells. Analysis of the dynamics of molecules with low mobility by conventional single-photon FRAP is time consuming, as observed for core histones whose $t_{1∕2}$ of the slow fraction is more than $510\min$ .⁴² We also established a HeLa cell line to stably express one of the core histones, namely, histone H2A (Fig. 3 ).⁹ In the case of histone H2A, the recovery rate in two-photon FRAP has been reported to be twice faster than that in single-photon FRAP.⁹ As described in the prevous section, in the case of histone H1, the recovery rate can be reduced to approximately one-fourth using two-photon FRAP. This demonstrates that two-photon FRAP is a powerful tool to analyze molecules with low mobility in vivo.

Fig. 3

FRAP analysis of EGFP-histone H2A. The heterochromatic region of living HeLa cell nuclei was excited and bleached by two-photon excitation using femtosecond laser light with a wavelength of $928\mathrm{nm}$ . Scale bar indicates $5\mu \mathrm{m}$ .

3.1.

Difference between Bleaching and Intracellular Ablation

Cell and intracellular surgery based on femtosecond laser pulses is attractive in cell biology because it allows noninvasive intracellular ablation at subdiffraction resolution to be performed within vital cells. ^{10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23} A femtosecond laser pulse at higher energies can produce localized energy absorption, and intracellular ablation occurs only in and around the focal volume, allowing site-specific dissection, removal, or disruption of organelles to be carried out.

We previously investigated fluorescence recovery after femtosecond laser irradiation to distinguish between bleaching and intracellular ablation.¹⁷ Here, we used the EGFP-tagged histone H1 for visualization of the nucleus.⁴⁴ Figure 4 shows fluorescence images of the EGFP-tagged nucleus before and after femtosecond laser irradiation with different energies (0.26 and $0.39\mathrm{nJ}$ /pulse) at a wavelength of $930\mathrm{nm}$ . A nuclear region of $1.5\times 1.5\mu {\mathrm{m}}^2$ in a living HeLa cell expressing EGFP-histone H1 was irradiated. Figure 5 shows the relative fluorescence intensity of the bleached area after femtosecond laser irradiation. We defined the relative fluorescence intensity as the fluorescence intensity after femtosecond laser irradiation divided by that before irradiation. At the energy of $0.39\mathrm{nJ}$ /pulse, fluorescence disappeared after irradiation and fluorescence recovery was not observed. At the energy of $0.26\mathrm{nJ}$ /pulse, fluorescence disappeared after irradiation; however, fluorescence recovery was observed. This result demonstrates that the fluorophore was bleached; however, subsequent recovery of fluorescence in the bleached region occurred due to the exchange of unbleached fluorophore molecules.

Fig. 4

FRAP analysis of enhanced green fluorescent protein (EGFP)-labeled nucleus at different energies. An EGFP-labeled nuclear region within an open box of $1.5\times 1.5\mu {\mathrm{m}}^2$ in a living HeLa cell was irradiated. Scale bar indicates $5\mu \mathrm{m}$ .

Fig. 5

Fluorescence intensity of the femtosecond laser irradiated area.

After femtosecond laser irradiation, we restained the cell with Hoechest 33342. The nuclei normally became uniformly stained $40\min$ after the addition of $0.1\mu \mathrm{g}∕\mathrm{mL}$ of Hoechest 33342, and therefore, we obtained fluorescence images after $45\min$ . When intracellular ablation of the nucleus was performed at $0.39\mathrm{nJ}$ /pulse, no fluorescence was observed from EGFP or Hoechest 33342 in the laser-irradiated region. When bleaching occurred at $0.26\mathrm{nJ}$ /pulse, fluorescence was observed from both EGFP and Hoechest 33342. These results confirm that fluorescence recovery is an indicator of intracellular ablation of organelles in living cells. Table 1 summarizes the differences between bleaching and intracellular ablation.

Table 1

Difference between bleaching and intracellular ablation.

3.2.

Intracellular Ablation of Mitochondria

We previously showed intracellular ablation of individual mitochondria in living HeLa cells using a femtosecond laser.¹⁷ Here, we show the viability of the cells after femtosecond laser radiation by use of a femtosecond laser oscillator at a repetition rate of $76\mathrm{MHz}$ . The cells expressed the enhanced yellow fluorescent protein (EYFP) in the mitochondria. Femtosecond laser pulses with an energy of $0.39\mathrm{nJ}$ /pulse were focused on the cells. Figures 6a and 6b show the stacked confocal images obtained before and after irradiation. The figures show that fluorescence from a single mitochondrion disappeared. That is, a mitochondrion irradiated by the femtosecond laser pulses did not exhibit fluorescence recovery. We also confirmed intracellular ablation of the mitochondrion by restaining with MitoTracker Red. Note that displacement of the mitochondria outside the focal region between the before and after images was attributed to cytoplasmic streaming, which indicated the viability of the cells. The viability of the cells after intracellular ablation was also verified by the observation of cell division. These experiments demonstrate that femtosecond laser pulses can be used to ablate a specific organelle from living cells without compromising their viability.

Fig. 6

Stacked 3-D confocal images (a) before and (b) after femtosecond laser irradiation with $0.39\mathrm{nJ}$ /pulse (exposure time: $32\mathrm{ms}$ ). Target mitochondrion is indicated by the arrow. Scale bar indicates $10\mu \mathrm{m}$ .

The viability of the cells after femtosecond laser radiation was ascertained by using PI. The membranes of living cells are impermeable to PI; it only penetrates dead or apoptotic cells with compromised membranes. PI was added to the medium just after irradiation of the femtosecond laser pulses, and fluorescence images were observed after $30\min$ . Cells whose mitochondria were ablated at the energy of $0.39\mathrm{nJ}$ /pulse were not stained by the PI. The viability of the cells after femtosecond laser irradiation was ascertained by impermeability of PI, as well as by the presence of cytoplasmic streaming. These experiments demonstrate that the cells remained viable when mitochondria were ablated by femtosecond laser pulses. In contrast, at the energy of $0.55\mathrm{nJ}$ /pulse, red fluorescence derived from PI was observed in the nuclei, because the plasma membrane was distorted or destroyed by laser irradiation, allowing PI to penetrate the cell (Fig. 7 ), even if the focal point was a mitchondrion beneath the cell membrane. At the energy of $0.55\mathrm{nJ}$ /pulse, cytoplasmic streaming in the cells was not observed. To perform intracelluar ablation of mitochondria in a living cell without compromising cell viability, it is important to select optimal laser energy.

Fig. 7

Confocal image of the cells after femtosecond laser irradiation and the addition of PI. Just after irradiation of femtosecond laser pulses, PI was added to the culture. The fluorescence images were observed $30\min$ after addition of the PI. At an energy of $0.55\mathrm{nJ}$ /pulse, red fluorescence derived from PI was observed in the nuclei. Scale bar indicates $5\mu \mathrm{m}$ .

6.1.

Optical Setup

Figure 10 shows a schematic diagram of the setup used for imaging of fluorescence-labeled organelles in living cells using continuous wave (cw) lasers and manipulation of organelles using a femtosecond laser oscillator. We used a confocal microscope to image the fluorescence. The laser scanning microscope was adapted from an Olympus FV300 scanning unit (Olympus Corp., Tokyo, Japan) combined with an Olympus IX71 inverted microscope. The cw beams from a He-Ne laser (wavelength $543\mathrm{nm}$ ) and an ${\mathrm{Ar}}^{+}$ laser (wavelength $488\mathrm{nm}$ ) were reflected by dichroic mirrors DM2 and DM3 and then focused into a BY-2 cell through an oil-immersion objective lens (OB, Olympus, $\mathrm{PlanApo}60\times \mathrm{O}2$ , NA 1.4). Fluorescence was excited by the cw laser light, and the backpropagating fluorescence was collected using the same objective lens and detected with photomultiplier tubes, after passing through a bandpass filter. 2-D confocal crosssectional images were obtained by scanning the focused laser beams in the $xy$ plane with a pair of high-speed galvanometer mirrors (GM) inside the laser-scanning microscope. Scanning in the depth direction ( $z$ direction) was achieved by moving the objective lens with a stepping motor to obtain 3-D confocal fluorescence images. To construct the stacked 3-D images, we obtained 13 confocal crosssectional images by translating the objective lens by $2.4\mu \mathrm{m}$ in the depth $\left(z\right)$ direction in steps of $0.5\mu \mathrm{m}$ . Imaging and marking by laser irradiation were performed using Fluoview software.

Fig. 10

Schematic diagram of the experimental setup. M, mirror; L, lens; DM, dichroic mirror; OB, objective lens; GM, pair of galvanometer mirrors; and PMT, photomultiplier tube.

6.2.

Tobacco BY-2 Cells

Histone H1-sGFP-expressing tobacco BY-2 cells were prepared according to the previously reported procedure.⁴⁷ Kaede-expressing tobacco BY-2 cells were prepared by a previously described method,³⁷ and the cells were maintained as previously described by Nagata, Nenoto, and Hasezawa.⁴⁸ The cells were cultured in modified Linsmaier and Skoog medium in a rotary shaker at $25°\mathrm{C}$ in the dark. Two-day-old BY-2 cells, after subculturing, were used for our analyses.

6.3.

HeLa Cells

HeLa human carcinoma cells were cultured and maintained as previously reported.⁴⁹ For visualization of the chromosomes, the HeLa cells were transfected by pEGFP-C1/histone H1.2 using LipofectAMINE plus reagent (Invitrogen Corp., Calsbad, California).⁴⁴

Fusion constructs containing a gene encoding enhanced yellow fluorescent protein EYFP and the mitochondrial targeting sequence from human cytochrome c oxidase (pEYFP-Mito, Clonetech) were transfected into HeLa cells using the calcium phosphate method. In the restaining experiments, mitochondria of the HeLa cells were stained with $2.5\text{-}\mathrm{ng}∕\mathrm{ml}$ MitoTracker Red CMH₂XRos(chloramethyl-X-rosamine, Molecular Probes), which is a mitochondria-selective dye, for $30\min$ at $37°\mathrm{C}$ .

To determine the effects on cell viability after femtosecond-laser irradiation, $1\text{-}\mu \mathrm{g}∕\mathrm{ml}$ PI (Sigma, St. Louis, Missouri) was added to the medium $30\min$ after the irradiation.

6.4.

Single-Photon and Two-Photon Fluorescence Recovery after Photobleaching

HeLa cells were transfected with the constructed vectors using Lipofectamine (Invitrogen) or FuGENE6 (Roche Diagnostics, Basel, Switzerland) following the corresponding manufacturer’s protocols. Transfectants were selected with $800\mu \mathrm{g}∕\mathrm{ml}$ of G418 (Sigma) and cloned by the limiting dilution method as described previously.⁷

HeLa cells were inoculated onto $35\text{-}\mathrm{mm}$ glass-bottomed dishes (Matsunami Glass, Inc., Kishiwada, Japan), maintained in Dulbecco’s modified Eagle’s medium (DMEM) without phenol-red (Invitrogen) with $10\text{-}\mathrm{mM}$ 2-[4-(2-hydroxyethyl)-1-piperadinyl] ethansulfonic acid (HEPES, pH 7.2) in a humidified atmosphere on a heated stage, and were imaged using an inverted fluorescence microscope (IX-71, Olympus) with a $60\times$ objective lens. Single-photon FRAP was performed using an FV300 confocal microscopic system ( $10\text{-}\mathrm{mW}$ ${\mathrm{Ar}}^{+}$ laser, wavelength $488\mathrm{nm}$ ). To carry out single-photon FRAP, the entire image was taken using illumination from the ${\mathrm{Ar}}^{+}$ laser at a mean power of $0.1\mathrm{mW}$ ( $5\mathrm{nW}$ at the sample) before the microscope, and then a nuclear region of $2.3\times 2.3\mu {\mathrm{m}}^2$ was bleached, while scanning with the galvanometer mirrors, by increasing the power of the ${\mathrm{Ar}}^{+}$ laser to $2\text{to}4\mathrm{mW}$ ( $110\text{to}220\mathrm{nW}$ at the sample). After $20\mathrm{s}$ , subsequent single-photon fluorescence images were captured with the original settings.

Two-photon FRAP with femtosecond laser pulses was performed using a mode-locked Ti:sapphire laser oscillator with a wavelength of $928\mathrm{nm}$ and a repetition rate of $76\mathrm{MHz}$ (Coherent, Mira, Santa Clara, California). The laser pulses passed through a series of SF10 prisms to compensate for the dispersion of the optical components in the light path and the microscope. The entire imaging was performed using two-photon excitation at a wavelength of $928\mathrm{nm}$ with a mean power of $20\text{to}30\mathrm{mW}$ ( $2\text{to}3\mathrm{mW}$ at the sample). A nuclear region of $2.3\times 2.3\mu {\mathrm{m}}^2$ was bleached at a power of $70\text{to}80\mathrm{mW}$ ( $7\text{to}8\mathrm{mW}$ at the sample) by scanning with the galvanometer mirrors. After bleaching, subsequent images were captured with the original settings.

6.5.

Intracellular Ablation

Intracellular ablation of mitochondria was performed using a mode-locked Ti:sapphire laser oscillator with a wavelength of $800\mathrm{nm}$ and a repetition rate of $76\mathrm{MHz}$ (Coherent, Mira). To perform ablation in the nuclear region, an EGFP-labeled nuclear region in a living HeLa cell was irradiated at a wavelength of $930\mathrm{nm}$ . To obtain fluorescence images of GFP and YFP, bandpass filters BP1 (transmission wavelength: $510\text{to}540\mathrm{nm}$ ) and BP2 (transmission wavelength: $560\text{to}600\mathrm{nm}$ ) were placed before photomultiplier tubes PMT1 and PMT2, respectively. The number of pulses supplied was selected by an electromagnetic shutter (Sigma Koki, $\Sigma$ -65L, Tokyo, Japan).

6.6.

Photoconversion of Kaede

Photoconversion of Kaede was performed using near-infrared femtosecond laser pulses from a mode-locked Ti:sapphire laser oscillator with a wavelength of $750\mathrm{nm}$ . The green fluorescence was obtained using a single-photon fluorescence microscope. ${\mathrm{Ar}}^{+}$ laser light passed through a bandpass filter BP1 (transmission wavelength: $510\text{to}540\mathrm{nm}$ ) and the green fluorescence was detected with a photomultiplier tube (PMT1). Red fluorescence after photoconversion of Kaede was excited by the He-Ne laser light (wavelength $543\mathrm{nm}$ ) and detected with a photomultiplier tube (PMT2), after passing through a bandpass filter BP2 (transmission wavelength: $560\text{to}600\mathrm{nm}$ ).