Calcium release domains in mammalian skeletal muscle studied with two-photon imaging and spot detection techniques

The spatiotemporal characteristics of the Ca(2+) release process in mouse skeletal muscle were investigated in enzymatically dissociated fibers from flexor digitorum brevis (FDB) muscles, using a custom-made two-photon microscope with laser scanning imaging (TPLSM) and spot detection capabilities. A two-microelectrode configuration was used to electrically stimulate the muscle fibers, to record action potentials (APs), and to control their myoplasmic composition. We used 125 muM of the low-affinity Ca(2+) indicator Oregon green 488 BAPTA-5N (OGB-5N), and 5 or 10 mM of the Ca(2+) chelator EGTA (pCa 7) in order to arrest fiber contraction and to constrain changes in the [Ca(2+)] close to the release sites. Image and spot data showed that the resting distribution of OGB-5N fluorescence was homogeneous along the fiber, except for narrow peaks ( approximately 23% above the bulk fluorescence) centered at the Z-lines, as evidenced by their nonoverlapping localization with respect to di-8-ANEPPS staining of the transverse tubules (T-tubules). Using spot detection, localized Ca(2+) transients evoked by AP stimulation were recorded from adjacent longitudinal positions 100 nm apart. The largest and fastest DeltaF/F transients were detected at sites flanking the Z-lines and colocalized with T-tubules; the smallest and slowest were detected at the M-line, whereas transients at the Z-line showed intermediate features. Three-dimensional reconstructions demonstrate the creation of two AP-evoked Ca(2+) release domains per sarcomere, which flank the Z-line and colocalize with T-tubules. In the presence of 10 mM intracellular EGTA, these domains are formed in approximately 1.4 ms and dissipate within approximately 4 ms, after the peak of the AP. Their full-width at half-maximum (FWHM), measured at the time that Ca(2+) transients peaked at T-tubule locations, was 0.62 mum, similar to the 0.61 mum measured for di-8-ANEPPS profiles. Both these values exceed the limit of resolution of the optical system, but their similarity suggests that at high [EGTA] the Ca(2+) domains in adult mammalian muscle fibers are confined to Ca(2+) release sites located at the junctional sarcoplasmic reticulum (SR).     

Direct in vivo monitoring of sarcoplasmic reticulum Ca2+ and cytosolic cAMP dynamics in mouse skeletal muscle

Skeletal muscle contraction depends on the release of Ca(2+) from the sarcoplasmic reticulum (SR), but the dynamics of the SR free Ca(2+) concentration ([Ca(2+)](SR)), its modulation by physiological stimuli such as catecholamines, and the concomitant changes in cAMP handling have never been directly determined. We used two-photon microscopy imaging of GFP-based probes expressed in mouse skeletal muscles to monitor, for the first time in a live animal, the dynamics of [Ca(2+)](SR) and cAMP. Our data, which were obtained in highly physiological conditions, suggest that free [Ca(2+)](SR) decreases by approximately 50 microM during single twitches elicited through nerve stimulation. We also demonstrate that cAMP levels rise upon beta-adrenergic stimulation, leading to an increased efficacy of the Ca(2+) release/reuptake cycle during motor nerve stimulation.     

Multicolor imaging of Ca(2+) and protein kinase C signals using novel epifluorescence microscopy.

Dynamic changes in intracellular free Ca(2+) concentrations ([Ca(2+)](i)s) control many important cellular events, including binding of Ca(2+)-calmodulin (Ca(2+)-CaM) and phosphorylation by protein kinase C (PKC). The two signals compete for the same domains in certain substrates, such as myristoylated alanine-rich PKC-substrate (MARCKS). To observe the convergence and relative time of arrival of CaM and PKC signals at their shared domain of MARCKS, we need to image cells that are loaded with more than two fluorescent dyes at a reasonable speed. We have developed a simple and powerful multicolor imaging system using conventional fluorescence microscopy. The epifluorescence configuration uses a glass reflector and rotating filter wheels for excitation and emission paths. As it is free of dichroic (multichroic) mirrors, multiple fluorescence images can be acquired rapidly regardless of the colors of fluorophores. We visualized Ca(2+)-CaM and PKC together with the dynamics of their common target, MARCKS, in single live cells. Receptor-activation resulted in translocation of MARCKS from the plasma membrane to cytosol through its phosphorylation by PKC. By observing fluorescence resonance energy transfer, we also obtained direct evidence that Ca(2+)-CaM binds MARCKS to drag it away from the membrane in circumstances when Ca(2+)-mobilization predominates over PKC activation.     

Ca2+ fluorescence imaging with pico- and femtosecond two-photon excitation: signal and photodamage.

The signal and limitations of calcium florescence imaging using nonresonant multiphoton absorption of near-infrared femto- and picosecond laser pulses were examined. The fluorescence changes of various Ca(2+)-indicators induced by transient increases of the intradendritic calcium concentration were evaluated by evoking physiological activity in neocortical neurons in rat brain slices. Photodamage was noticeable as irreversible changes in the parameters describing the calcium fluorescence transients. At higher two-photon excitation rates, a great variety of irregular functional and structural alterations occurred. Thus, signal and observation time were limited by phototoxic effects. At lower excitation rates, photodamage accumulated linearly with exposure time. Femtosecond and picosecond laser pulses were directly compared with respect to this cumulative photodamage. The variation of the pulse length at a constant two-photon excitation rate indicated that a two-photon excitation mechanism is mainly responsible for the cumulative photodamage within the investigated window of 75 fs to 3.2 ps. As a direct consequence, at low excitation rates, the same image quality is achieved irrespective of whether two-photon Ca(2+)-imaging is carried out with femto- or picosecond laser pulses.     

Improved calcium imaging in transgenic mice expressing a troponin C-based biosensor

Fluorescent Ca(2+) indicator proteins (FCIPs) are attractive tools for studying Ca(2+) dynamics in live cells. Here we describe transgenic mouse lines expressing a troponin C (TnC)-based biosensor. The biosensor is widely expressed in neurons and has improved Ca(2+) sensitivity both in vitro and in vivo. This allows FCIP-based two-photon Ca(2+) imaging of distinct neurons and their dendrites in vivo, and opens a new avenue for structure-function analysis of intact neuronal circuits.     

Direct monitoring of vesicular release and uptake in brain slices by multiphoton excitation of the styryl FM 1-43

Fluorescence imaging using FM 1-43 and related styryl dyes has provided invaluable insights into presynaptic function of synapses in culture preparations, but has been limited in use for studying central synapses in vivo or in brain slices, because of excessive fluorescence background due to nonspecific membrane binding of dye. We demonstrate here that focal excitation of FM dyes using two-photon laser-scanning microscopy (TPLSM) provides high resolution of FM 1-43-labeled nerve terminals in brain slices by suppressing out-of-focus background and that a readily releasable pool of vesicles can be selectively and stably labeled by hypertonic shock despite slice diffusion barriers. We find direct TPLSM of FM 1-43-labeled nerve terminals to be superior to treatment of slices with either the fluorescent quencher sulforhodamine 101 or dye scavenger ADVASEP-7 in resolving nerve terminal against background fluorescence, enabling continuous monitoring of vesicular uptake, and release of styryl dyes from individual nerve terminals in brain slices.     

Calcium microdomains at presynaptic active zones of vertebrate hair cells unmasked by stochastic deconvolution

Signal transduction by auditory and vestibular hair cells involves an impressive ensemble of finely tuned control mechanisms, strictly dependent on the local intracellular Ca(2+) concentration ([Ca(2+)](i)). The study of Ca(2+) dynamics in hair cells typically combines Ca(2+)-sensitive fluorescent indicators (dyes), patch clamp and optical microscopy to produce images of the patterns of fluorescence of a Ca(2+) indicator following various stimulation protocols. Here we describe a novel method that combines electrophysiological recordings, fluorescence imaging and numerical simulations to effectively deconvolve Ca(2+) signals within cytoplasmic microdomains that would otherwise remain inaccessible to direct observation. The method relies on the comparison of experimental data with virtual signals derived from a Monte Carlo reaction-diffusion model based on a realistic reconstruction of the relevant cell boundaries in three dimensions. The model comprises Ca(2+) entry at individual presynaptic active zones followed by diffusion, buffering, extrusion and release of Ca(2+). Our results indicate that changes of the hair cell [Ca(2+)](i) during synaptic transmission are primarily controlled by the Ca(2+) endogenous buffers both at short (<1mu) and at long (tens of microns) distances from the active zones. We provide quantitative estimates of concentration and kinetics of the hair cell endogenous Ca(2+) buffers and Ca(2+)-ATPases. We finally show that experimental fluorescence data collected during Ca(2+) influx are not interpreted correctly if the [Ca(2+)](i) is estimated by assuming that Ca(2+) equilibrates instantly with its reactants. In our opinion, this approach is of potentially general interest as it can be easily adapted to the study of Ca(2+) dynamics in diverse biological systems.     

Single-molecule imaging of l-type Ca(2+) channels in live cells

L-type Ca(2+) channels are an important means by which a cell regulates the Ca(2+) influx into the cytosol on electrical stimulation. Their structure and dynamics in the plasma membrane, including their molecular mobility and aggregation, is of key interest for the in-depth understanding of their function. Construction of a fluorescent variant by fusion of the yellow-fluorescent protein to the ion channel and expression in a human cell line allowed us to address its dynamic embedding in the membrane at the level of individual channels in vivo. We report on the observation of individual fluorescence-labeled human cardiac L-type Ca(2+) channels using wide-field fluorescence microscopy in living cells. Our fluorescence and electrophysiological data indicate that L-type Ca(2+) channels tend to form larger aggregates which are mobile in the plasma membrane.     

中枢神经系统去甲肾上腺素分泌的实时检测

为了探讨与中枢神经系统单胺类递质分泌失调有关疾病的中枢机制,人们对单胺类递质分泌动力学的研究越来越有兴趣。去甲肾上腺素是中枢神经系统重要的递质和调质,本文介绍了我们实验室最近发展的实时检测中枢神经系统去甲肾上腺素分泌的一些技术方法,并比较了电化学微碳纤电极（carbon fiber electrode,CFE）测量与电生理、荧光显微测量技术优缺点,阐述了CFE技术在神经科学研究中的一个基本应用。     

In vivo monitoring of Ca(2+) uptake into mitochondria of mouse skeletal muscle during contraction

Although the importance of mitochondria in patho-physiology has become increasingly evident, it remains unclear whether these organelles play a role in Ca(2+) handling by skeletal muscle. This undefined situation is mainly due to technical limitations in measuring Ca(2+) transients reliably during the contraction-relaxation cycle. Using two-photon microscopy and genetically expressed "cameleon" Ca(2+) sensors, we developed a robust system that enables the measurement of both cytoplasmic and mitochondrial Ca(2+) transients in vivo. We show here for the first time that, in vivo and under highly physiological conditions, mitochondria in mammalian skeletal muscle take up Ca(2+) during contraction induced by motor nerve stimulation and rapidly release it during relaxation. The mitochondrial Ca(2+) increase is delayed by a few milliseconds compared with the cytosolic Ca(2+) rise and occurs both during a single twitch and upon tetanic contraction.     

Long-term two-photon fluorescence imaging of mammalian embryos without compromising viability.

A major challenge for fluorescence imaging of living mammalian cells is maintaining viability following prolonged exposure to excitation illumination. We have monitored the dynamics of mitochondrial distribution in hamster embryos at frequent intervals over 24 h using two-photon microscopy (1,047 nm) while maintaining blastocyst, and even fetal, developmental competence. In contrast, confocal imaging for only 8 h inhibits development, even without fluorophore excitation. Photo-induced production of H2O2 may account, in part, for this inhibition. Thus, two-photon microscopy, but not confocal microscopy, has permitted long-term fluorescence observations of the dynamics of three-dimensional cytoarchitecture in highly photosensitive specimens such as mammalian embryos.     

Multimodal Optical Microscopy Methods Reveal Polyp Tissue Morphology and Structure in Caribbean Reef Building Corals

An integrated suite of imaging techniques has been applied to determine the three-dimensional (3D) morphology and cellular structure of polyp tissues comprising the Caribbean reef building corals Montastraeaannularis and M. faveolata. These approaches include fluorescence microscopy (FM), serial block face imaging (SBFI), and two-photon confocal laser scanning microscopy (TPLSM). SBFI provides deep tissue imaging after physical sectioning; it details the tissue surface texture and 3D visualization to tissue depths of more than 2 mm. Complementary FM and TPLSM yield ultra-high resolution images of tissue cellular structure. Results have: (1) identified previously unreported lobate tissue morphologies on the outer wall of individual coral polyps and (2) created the first surface maps of the 3D distribution and tissue density of chromatophores and algae-like dinoflagellate zooxanthellae endosymbionts. Spectral absorption peaks of 500 nm and 675 nm, respectively, suggest that M. annularis and M. faveolata contain similar types of chlorophyll and chromatophores. However, M. annularis and M. faveolata exhibit significant differences in the tissue density and 3D distribution of these key cellular components. This study focusing on imaging methods indicates that SBFI is extremely useful for analysis of large mm-scale samples of decalcified coral tissues. Complimentary FM and TPLSM reveal subtle submillimeter scale changes in cellular distribution and density in nondecalcified coral tissue samples. The TPLSM technique affords: (1) minimally invasive sample preparation, (2) superior optical sectioning ability, and (3) minimal light absorption and scattering, while still permitting deep tissue imaging.     

Ca2+]i signaling between mitochondria and endoplasmic reticulum in neurons is regulated by microtubules. From mitochondrial permeability transition pore to Ca2+-induced Ca2+ release

The positioning and dynamics of organelles depend on membrane-cytoskeleton interactions. Mitochondria relocate along microtubules (MT), but it is not clear whether MT have direct effects on mitochondrial function. Using two-photon microscopy and the mitochondrial fluorescent dyes rhodamine 123 and Rhod-2, we showed that Taxol and nocodazole, which correspondingly stabilize and disrupt MT, decreased potential and Ca(2+) in the mitochondria of brain stem pre-Botzinger complex neurons. Without changing basal cytoplasmic Ca(2+) ([Ca(2+)](i)), Taxol promoted the generation of [Ca(2+)](i) spikes in dendrites. These spikes were abolished after blockade of Ca(2+) influx and after depletion of internal Ca(2+) stores, indicating the involvement of Ca(2+)-induced Ca(2+) release. Nocodazole decreased mitochondrial potential and [Ca(2+)](m) and produced a long lasting increase in [Ca(2+)](i). MT-acting drugs depolarized single immobilized mitochondria and released previously stored Ca(2+). All of these effects were inhibited by pretreatment with blockers of mitochondrial permeability transition pore (mPTP), cyclosporin A, and 2-aminoethoxydiphenyl borate. Induction of mPTP by Taxol and nocodazole was confirmed by using a calcein/Co(2+) imaging technique. Electron and optical microscopy revealed tubulin bound to mitochondria. Mitochondria, MT, and endoplasmic reticulum (ER) showed strong co-localization, the degree of which decreased after MT were disrupted. We propose that changes in the structure of MT by Taxol and nocodazole promote the induction of mPTP. Subsequent Ca(2+) efflux stimulates the Ca(2+) release from the ER that drives spontaneous [Ca(2+)](i) transients. Thus, close positioning of mitochondria to the ER as determined by MT can be essential for the local [Ca](i) signaling in neurons.     

Observations of calcium dynamics in cortical secretory vesicles

Calcium (Ca(2+)) dynamics were evaluated in fluorescently labeled sea urchin secretory vesicles using confocal microscopy. 71% of the vesicles examined exhibited one or more transient increases in the fluorescence signal that was damped in time. The detection of transient increases in signal was dependent upon the affinity of the fluorescence indicator; the free Ca(2+) concentration in the secretory vesicles was estimated to be in the range of ～10 to 100μM. Non-linear stochastic analysis revealed the presence of extra variance in the Ca(2+) dependent fluorescence signal. This noise process increased linearly with the amplitude of the Ca(2+) signal. Both the magnitude and spatial properties of this noise process were dependent upon the activity of vesicle p-type (Ca(v)2.1) Ca(2+) channels. Blocking the p-type Ca(2+) channels with ω-agatoxin decreased signal variance, and altered the spatial noise pattern within the vesicle. These fluorescence signal properties are consistent with vesicle Ca(2+) dynamics and not simply due to obvious physical properties such as gross movement artifacts or pH driven changes in Ca(2+) indicator fluorescence. The results suggest that the free Ca(2+) content of cortical secretory vesicles is dynamic; this property may modulate the exocytotic fusion process.     

Real-Time dynamics of Ca2+, caspase-3/7, and morphological changes in retinal ganglion cell apoptosis under elevated pressure

Quantitative information on the dynamics of multiple molecular processes in individual live cells under controlled stress is central to the understanding of the cell behavior of interest and the establishment of reliable models. Here, the dynamics of the apoptosis regulator intracellular Ca(2+), apoptosis effector caspase-3/7, and morphological changes, as well as temporal correlation between them at the single cell level, are examined in retinal gangling cell line (differentiated RGC-5 cells) undergoing apoptosis at elevated hydrostatic pressure using a custom-designed imaging platform that allows long-term real-time simultaneous imaging of morphological and molecular-level physiological changes in large numbers of live cells (beyond the field-of-view of typical microscopy) under controlled hydrostatic pressure. This examination revealed intracellular Ca(2+) elevation with transient single or multiple peaks of less than 0.5 hour duration appearing at the early stages (typically less than 5 hours after the onset of 100 mmHg pressure) followed by gradual caspase-3/7 activation at late stages (typically later than 5 hours). The data reveal a strong temporal correlation between the Ca(2+) peak occurrence and morphological changes of neurite retraction and cell body shrinkage. This suggests that Ca(2+) elevation, through its impact on ion channel activity and water efflux, is likely responsible for the onset of apoptotic morphological changes. Moreover, the data show a significant cell-to-cell variation in the onset of caspase-3/7 activation, an inevitable consequence of the stochastic nature of the underlying biochemical reactions not captured by conventional assays based on population-averaged cellular responses. This real-time imaging study provides, for the first time, statistically significant data on simultaneous multiple molecular level changes to enable refinements and testing of models of the dynamics of mitochondria-mediated apoptosis. Further, the platform developed and the approach has direct significance to the study of a variety of signaling pathway phenomena.     

Construction of a two-photon microscope for video-rate Ca imaging

We describe the construction of a video-rate two-photon laser scanning microscope, compare its performance to a similar confocal microscope, and illustrate its use for imaging local Ca(2+) transients from cortical neurons in brain slices. Key features include the use of a Ti-sapphire femtosecond laser allowing continuous tuning over a wide (700-1000 nm) wavelength range, a resonant scanning mirror to permit frame acquisition at 30 Hz, and efficient wide-field fluorescence detection. Two-photon imaging provides compelling advantages over confocal microscopy in terms of improved imaging depth and reduced phototoxicity and photobleaching, but the high cost of commercial instruments has limited their widespread adoption. By constructing one's own system the expense is greatly reduced without sacrifice of performance, and the microscope can be more readily tailored to specific applications.     

Smooth muscle cells and interstitial cells of blood vessels

A rise in intracellular ionised calcium concentration ([Ca(2+)](i)) at sites adjacent to the contractile proteins is a primary signal for contraction in all types of muscles. Recent progress in the development of imaging techniques with special accent on the fluorescence confocal microscopy and new achievements in the synthesis of organelle- and ion-specific fluorochromes provide an experimental basis for study of the relationship between the structural organisation of the living smooth muscle myocyte and the features of calcium signalling at subcellular level. Applying fluorescent confocal microscopy and tight-seal recording of transmembrane ion currents to freshly isolated vascular myocytes we have demonstrated that: (1) Ca(2+) sparks originate from clustered opening of ryanodine receptors (RyRs) and build up a cell-wide increase in [Ca(2+)](i) upon myocyte excitation; (2) spontaneous Ca(2+) sparks occurred at the highest rate at certain preferred locations, frequent discharge sites (FDS), which are associated with a prominent portion of the sarcoplasmic reticulum (SR) located close to the cell membrane; (3) Ca(2+)-dependent K(+) and Cl(-) channels sense the local changes in [Ca(2+)](i) during a calcium spark and thereby couple changes in [Ca(2+)](i) within a microdomain to changes in the membrane potential, thus affecting excitability of the cell; (4) an intercommunication between RyRs and inositol trisphosphate receptors (IP(3)Rs) is one of the important determinants of intracellular calcium dynamics that, in turn, can modulate the cell membrane potential through differential targeting of calcium dependent membrane ion channels. Furthermore, using immunohystochemical approaches in combination with confocal imaging we identified non-contractile cells closely resembling interstitial cells (ICs) of Cajal (which are considered to be pacemaker cells in the gut) in the wall of portal vein and mesenteric artery. Using electron microscopy, tight-seal recording and fluorescence confocal imaging we obtained information on the morphology of ICs and their possible coupling to smooth muscle cells (SMCs), calcium signalling in ICs and their electrophysiological properties. The functions of these cells are not yet fully understood; in portal vein they may act as pacemakers driving the spontaneous activity of the muscle; in artery they may have other a yet unsuspected functions.     

Visualization and quantification of endoplasmic reticulum Ca2+ in renal cells using confocal microscopy and Fluo5F

Sarcoplasmic/endoplasmic reticulum (ER) Ca(2+) is the most abundant store of intracellular Ca(2+), and its release is an important trigger of physiological and cell death pathways. Previous work in our laboratory revealed the importance of ER Ca(2+) in toxicant-induced renal proximal tubular cell (RPTC) death. The purpose of this study was to evaluate the use of confocal microscopy and Fluo5F, a low affinity Ca(2+) indicator, to directly monitor changes in RPTC ER Ca(2+). Fluo5F staining reflected ER Ca(2+), resolved ER structure, and showed no colocalization with tetramethyl rhodamine methyl ester (TMRM), a marker of mitochondrial membrane potential. Thapsigargin, an ER Ca(2+) pump inhibitor, decreased ER fluorescence by 30% and 55% at 5 and 15 min, respectively, whereas A23187, a Ca(2+) ionophore caused more rapid ER Ca(2+) release (55% and 75% decrease in fluorescence at 5 and 15 min). Carbonylcyanide-p-trifluoromethoxyphenylhydrazone (FCCP), a mitochondrial uncoupler, added at the end of the experiment, further decreased ER fluorescence after thapsigargin treatment, revealing that thapsigargin did not release all ER Ca(2+). In contrast, FCCP did not decrease ER fluorescence after A23187 treatment, suggesting complete ER Ca(2+) release. ER Ca(2+) release in response to A23187 or thapsigargin resulted in a modest but significant decrease in mitochondrial membrane potential. These data provide evidence that confocal microscopy and Fluo5F are useful and effective tools for directly monitoring ER Ca(2+) in live cells.     

Role of Junctin protein interactions in cellular dynamics of calsequestrin polymer upon calcium perturbation

Calsequestrin (CSQ), the major intrasarcoplasmic reticulum calcium storage protein, undergoes dynamic polymerization and depolymerization in a Ca(2+)-dependent manner. However, no direct evidence of CSQ depolymerization in vivo with physiological relevance has been obtained. In the present study, live cell imaging analysis facilitated characterization of the in vivo dynamics of the macromolecular CSQ structure. CSQ2 appeared as speckles in the presence of normal sarcoplasmic reticulum (SR) Ca(2+) that were decondensed upon Ca(2+) depletion. Moreover, CSQ2 decondensation occurred only in the stoichiometric presence of junctin (JNT). When expressed alone, CSQ2 speckles remained unchanged, even after Ca(2+) depletion. FRET analysis revealed constant interactions between CSQ2 and JNT, regardless of the SR Ca(2+) concentration, implying that JNT is an essential component of the CSQ scaffold. In vitro solubility assay, electron microscopy, and atomic force microscopy studies using purified recombinant proteins confirmed Ca(2+) and JNT-dependent disassembly of the CSQ2 polymer. Accordingly, we conclude that reversible polymerization and depolymerization of CSQ are critical in SR Ca(2+) homeostasis.     

Adaptive movement compensation for in vivo imaging of fast cellular dynamics within a moving tissue

In vivo non-linear optical microscopy has been essential to advance our knowledge of how intact biological systems work. It has been particularly enabling to decipher fast spatiotemporal cellular dynamics in neural networks. The power of the technique stems from its optical sectioning capability that in turn also limits its application to essentially immobile tissue. Only tissue not affected by movement or in which movement can be physically constrained can be imaged fast enough to conduct functional studies at high temporal resolution. Here, we show dynamic two-photon Ca(2+) imaging in the spinal cord of a living rat at millisecond time scale, free of motion artifacts using an optical stabilization system. We describe a fast, non-contact adaptive movement compensation approach, applicable to rough and weakly reflective surfaces, allowing real-time functional imaging from intrinsically moving tissue in live animals. The strategy involves enslaving the position of the microscope objective to that of the tissue surface in real-time through optical monitoring and a closed feedback loop. The performance of the system allows for efficient image locking even in conditions of random or irregular movements.