The cytoplasm is a restrictive medium for the diffusion of charged compounds and ions such as ROS because of its highly reducing environment. Thus, intracellular signaling systems utilizing ROS frequently operate via local communication between the sources and targets [53, 54]. The endoplasmic reticulum (ER) can be influenced by the ROS produced by mitochondria, due to its close proximity to mitochondria and abundance throughout the cytoplasm [55, 56]. Established ROS-dependent regulators include Ca2+ channels (ryanodine receptors; RyRs and inositol 1,4,5-triphosphate receptors; InsP3Rs), cAMP-dependent kinases (PKA), and Ca2+/calmodulin-dependent kinases (CaMK), that can associate with Ca2+ transport proteins via anchoring proteins (Fig. 2) [57-60]. All of these scenarios suggest that ER-mitochondrial coupling serve as the center stage for ROS-Ca2+ cascade.
Recent biophotonic studies have indicated that femtosecond-pulsed lasers stimulation could modulate many biological functions, regardless of cell type by controlling intracellular Ca2+ concentrations [20, 22, 25, 35]. Localized Ca2+ release from the ER through Ca2+ channels such as InsP3 or RyRs, initiates calcium-induced calcium release (CICR) . CICR, which is related to ER Ca2+ release channels, plays a role in generation of intracellular Ca2+ waves and is important in the excitation of muscle cells and neurons [62-64]. The intracellular Ca2+ wave propagates to adjacent cells though gap junctions, which are intercellular connections that allow various molecules and ions to pass freely between cells . The processes mentioned above modulate Ca2+-dependent signaling in tissues. Cells are classified into two types: excitable cells that are able to produce and respond to electrical signals, called action potentials; and nonexcitable cells that also react to electrical signals, but cannot produce action potentials. Although the excitability of these cell types differs, the intracellular Ca2+ level of both is tightly regulated due to its physiological importance [66, 67].
2.4.1. Optical modulation of cellular functions in excitable cells
Excitable cell types including neurons, muscles, and secretory cells, require electrical signals to regulate their functions. The difference in calcium ion concentration between the inside and outside of the cell induces an electrical potential, leading to changes in cellular activities. The entry of calcium ions into neurons causes action potentials and neurotransmitter release. Ca2+ is also an essential molecule for regulation of contraction of all types of muscle .
Recently, optical methods of inducing action potentials on neurons have emerged. One such method is use of photoactivatable chemical molecules. Many chemical compounds that could release bioactive molecules such as glutamate or Ca2+ have been developed, and such compounds are metaphorically termed “caged compounds” or “caged molecules” . The exposure of light alters the chemical structure of caged compounds liberating (“uncaging”) the caged bioactive molecule. Caged compounds along with focused light irradiation could directly trigger membrane depolarization and action potential in the neuron. However, the compounds should be introduced into the neuron, and they have off-target effects that limit functional specificity. Hence, caged compounds are largely limited to in vivo applications [69, 70].
Another optical method for manipulation of neuronal activity using femtosecond-pulsed lasers has been developed. Hirase et al.  showed that exposure of an 76-MHz, 13-fs laser pulses of 780–800 nm produced an action potential on a pyramidal neuron in the absence of any exogenous molecules like caged compounds. The authors found that only mode-lock pulsed laser stimulation could induce this action potential on the neuron, but CW laser irradiation could not. This result indicated that the nonlinear effect, especially multiphoton absorption, is crucial to depolarization. Stimulation at low intensity and for a longer exposure duration showed different responses compared to a higher-intensity and shorter-duration exposure. The former showed induction of sustained depolarization, which was mediated by ROS, but the latter produced rapid depolarization, which might be the result of membrane pore formation due to photomechanical effects. Liu et al.  found that femtosecond-pulsed laser stimulation triggered a calcium wave in the irradiated hippocampal neuron. Then, the laser-induced calcium wave propagated to adjacent neurons, which allowed the authors to identify neural circuits ex vivo. Because there was no need for an exogenous probe or genetic modification, the femtosecond-pulsed laser was proposed as a useful optical tool for neurophysiology studies.
Femtosecond-pulsed laser stimulation can modulate muscle contractility. There have been many reports that femtosecond laser pulses generate intracellular calcium waves in a variety of cell types [20, 24, 73]. By controlling intracellular Ca2+ concentration, femtosecond-pulsed laser stimulation could induce muscle contraction, in which Ca2+ plays a critical role. Muscle is classified into three types; skeletal, smooth, and cardiac muscle. Skeletal muscle involves a voluntary action that has a distinct series of alternating light and dark bands perpendicular to the long axis. Skeletal muscle fibers can be detected by label-free imaging techniques, using autofluorescence or second-harmonic generation . After laser stimulation, skeletal muscle shows rapid twitch contraction and returns to its basal length within several minutes (Fig. 3a). Smooth muscle, located within the walls of blood vessels, the urinary bladder, and respiratory tract, lacks the distinct banding pattern found in skeletal muscle, and nerves innervating smooth muscle are derived from the autonomic division. Thus, smooth muscle is not normally under direct voluntary control. Femtosecond-pulsed laser irradiation changes the cytosolic Ca2+ concentration, leading to smooth muscle contraction without nerve activity. This method can induce the contraction of arterial blood vessels without use of exogenous probes, such as caged molecules. Laser irradiation focused in the brain artery wall caused localized circular contraction; the artery recovered its basal lumen diameter within a few minutes (Fig. 3b) . Laser irradiation also caused bladder smooth muscle contraction (Fig. 3c) . The bladder wall has a smooth muscle layer that controls its capacity. Laser irradiation of dissected bladder smooth muscle tissue induced localized increases in calcium ion concentration, followed by whole smooth muscle tissue contraction. The bladder smooth muscle fibers recovered to their basal length within a few minutes. This optical method for modulation of muscle contractility can be used as an alternative therapeutic tool in neuromuscular diseases.
Figure 3. (online color at: www.ann-phys.org) Optical modulation of contraction of different types of muscle. (a) Laser-induced skeletal muscle contraction. After intravenous injection of 2 MDa FITC-dextran (green fluorescence), the dorsal skinfold chamber model in mice was imaged with two-photon microscopy. Red fluorescence indicates autofluorescence of skeletal muscle fibers under two-photon excitation using a 760-nm Ti:Sapphire laser. The white dashed square in the baseline image indicates the region of laser irradiation. White dashed lines indicate the baseline positions of capillaries. Yellow lines and white arrows indicate changes in capillary position caused by skeletal muscle contraction. Scale bar, 50 µm. (b) Laser-induced artery contraction in vivo. Green fluorescence indicates the lumen of blood vessels. The red dot and dashed line indicate the irradiated region and baseline vessel wall, respectively. Scale bar, 20 µm. (c) Laser-induced urinary bladder tissue contraction. Urinary bladder tissue was stained with the calcium indicator Fluo4-AM. Green fluorescence indicates the intracellular calcium level of urinary bladder smooth muscle fibers. The red dot and white dashed line indicate the irradiated region and baseline position of smooth muscle fibers, respectively. The white arrow and yellow line indicate changes in smooth muscle fiber length caused by smooth muscle contraction. Scale bar, 50 µm.
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Femtosecond laser pulses can alter the intracellular Ca2+ concentrations in cardiomyocytes, a type of muscle cell that provides contractility to the heart. Using an 82-MHz, 80-fs laser pulses of 780 nm, an 8-ms exposure induced an intracellular Ca2+ wave, and the Ca2+ signal propagated to nearby regions. Cardiomyocyte beat rate was synchronized to laser irradiation frequency [23, 24]. Jenkins et al.  showed that a pulsed infrared diode laser (λ = 1.875 µm) coupled light into a multimode fiber 400 µm in diameter and modulated pacing of the embryonic quail heart in vivo. This study used relatively long (millisecond) pulses and a long wavelength, without sufficient photon energy to overcome the bandgap energy. Thus, the laser intensity was insufficient to generate low-density plasma directly. Although the mechanisms remain unclear, both studies were remarkable in that optical stimulation was shown to have the potential to act as a pacemaker.
2.4.2. Optical modulation of cellular functions in nonexcitable cells
The role of Ca2+ in nonexcitable cell types including epithelial cells, endothelial cells, and astrocytes, is different from that in excitable cell types. Ca2+ signaling in nonexcitable cells is closely involved in cell death, migration, and cell differentiation. Thus, nonexcitable cells maintain low intracellular Ca2+ concentrations by tightly regulating the flux of Ca2+ between cellular compartments . Despite their nonexcitability, femtosecond-pulsed lasers can control the Ca2+ signaling and subsequent cellular functions of nonexcitable cells.
Femtosecond-pulsed laser stimulation can modulate blood–brain barrier (BBB) permeability in vivo . Brain microvascular endothelial cells are linked by tight junctions that interconnect adjacent endothelial cells, forming a physiological barrier, called the BBB. Most endogenous and exogenous macromolecules do not cross the blood vessel wall due to BBB. Thus, exogenous delivery of molecular probes or drugs is widely used for in vivo brain research and brain-disease therapy. Recent studies showed that unamplified 80-MHz, 120-fs pulsed laser stimulation could modulate BBB permeability in vivo. Brief laser exposure of the brain vein wall caused a transient break in tight junctions and extravasation of plasma into the brain parenchyma (Fig. 4a). We have observed that the irradiated blood vessel wall and BBB were recovered within several minutes after stimulation . By combining this method with systemic injection, laser-induced extravasation can be used for local delivery of functional molecular probes, such as the astrocyte staining dye SR101 (Fig. 4b), nuclear staining probe Hoechst 33342 (Fig. 4c), nanoparticles, and adenovirus, into the brain. This optical method has the advantages of noninvasive introduction of macromolecules into the brain without opening the skull.
Figure 4. (online color at: www.ann-phys.org) Optical modulation of BBB permeability. (a) Time-series two-photon laser scanning microscopic images of a cortical vein in the brain. After intravenous injection of 2 MDa FITC–dextran, the thinned-skull window was imaged with two-photon microscopy. The red dot indicates the region of laser irradiation. Scale bar, 50 µm. (b) Staining of astrocytes in the brain using laser-induced extravasation. Red fluorescence indicates astrocytes stained with the astrocyte specific dye SR101. Scale bar, 50 µm. (c) Local nuclear staining in the brain cortex with Hoechst 33342. Image was taken 30 min after induction of extravasation. Scale bar, 20 µm.
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Astrocytes are the most abundant cell type in the central nervous system (CNS). Unamplified 80-MHz femtosecond-pulsed laser irradiation focused on a single astrocyte induced intracellular calcium wave generation in the irradiated astrocyte in vitro and in vivo [21, 35]. In response to elevation of intracellular calcium, astrocytes release neuromodulatory signaling molecules that modulate vasomotion of the brain arteries. After a short femtosecond-pulsed laser irradiation exposure of an astrocyte wrapped around an artery, the astrocyte showed rapid increases in levels of intracellular calcium ions, followed by artery dilation in vivo (Fig. 5). This optical method has drawbacks compared to other techniques, such as caged molecules or optogenetics, because it does not target specific molecular events. However, it has the advantages of being label-free, noninvasive, and does not show deleterious effects, such as microglial activation.
Figure 5. (online color at: www.ann-phys.org) Vasodilation of the cerebral artery by optical activation of surrounding astrocytes. Temporal dynamics of astrocyte-mediated vasodilation. The dotted lines demarcate the arterial lumen at the baseline and the outer yellow line demarcates the arterial lumen at 30 s. The white dot indicates the irradiated region. Scale bar, 10 µm.
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