Ca²⁺, an intracellular second messenger, is crucial for cell proliferation, differentiation, metabolism, and programmed cell death by participating in gene transcription, protein modification, adenosine triphosphate (ATP) generation, and the initiation of numerous signaling pathway cascades. Intracellular Ca²⁺ is first arrested by the plasma membrane, the first barrier that maintains an approximately 20000-fold Ca²⁺ concentration gradient from the extracellular environment to the cytosol, by controlling Ca²⁺ influx and efflux through a series of Ca²⁺ channels. Cytoplasmic Ca²⁺ concentration is maintained at approximately 100 nmol/L in most non-excitable cells. Excess cytosolic Ca²⁺ can be chelated by Ca²⁺-binding proteins, compartmentalized into intracellular Ca²⁺ stores, or extruded into the extracellular environment. Organelles typically play the role of subcellular Ca²⁺ stores (or temporary Ca²⁺ buffers) and further synergize with each other to form a Ca²⁺ network. As nodes on the network, they coordinate with the plasma membrane (PM) to regulate intracellular Ca²⁺ signaling. The endoplasmic reticulum (ER) is the primary Ca²⁺ store, with a resting Ca²⁺ concentration of approximately 200 μmol/L, releasing free Ca²⁺ into the cytoplasm. Mitochondria are temporary calcium buffers that transiently accommodate excess Ca²⁺ in the cytosol. Lysosomes and Golgi are small, but important in Ca²⁺ signaling. Among these subcellular structures, the nucleus appears to be a mist for cell calcium research. However, little is known about the existence of Ca²⁺ stores in the nucleus, how nuclear Ca²⁺ is regulated, or how Ca²⁺ is transported through the nuclear membrane. There are lots of membrane contact sites (MCSs) between organelles, such as ER-mitochondria, ER-PM, and ER-nucleus contacts. Local Ca²⁺ interactions at the MCSs also perform vital cellular functions.

A ubiquitous broad group of Ca²⁺ sensors, channels, pumps, exchangers, and binding proteins (receptors) in subcellular structures regulate intracellular Ca²⁺ for dynamic hemostasis. Ca²⁺ signaling modulation techniques with high spatial and temporal resolutions can facilitate the study of subcellular Ca²⁺ modulation mechanisms. To the best of our knowledge, owing to the difficulty of isolating Ca²⁺ solely in the submicron domain, some parts of the intracellular Ca²⁺ network, for example, Ca²⁺ regulation in some MCSs and nuclei, remain unclear. Thus, the conductive method of precisely regulating Ca²⁺ in subcellular organelles may provide further insights into the mechanisms of Ca²⁺ hemostasis and Ca²⁺-involved cell processes. Pharmacological reagents have been extensively used to control intracellular Ca²⁺ signaling in Ca²⁺ research. Ideally, subcellular Ca²⁺ stores can be precisely modulated by these reagents if they activate the target calcium channels/receptors, or organelles with high specificity. However, in practice, owing to poor specificity, most reagents simultaneously bind with a broad spectrum of molecules and perturb the entire cell, finally leading to a significant calcium increase or Ca²⁺ oscillations that smear all interactions between organelles under physiological conditions. In another dimension, the targeting of pharmacological drugs to organelles deteriorates significantly owing to the global distribution of Ca²⁺ channels/receptors in the entire cell. To improve spatial resolution, Ca²⁺ modulation technologies based on lasers have been developed, which can theoretically work in the submicron region by utilizing lasers and microscopic systems. Progress has been made, but their applications vary and are limited because of their different methodological properties. Therefore, it is important and necessary to discuss the methodological advances and limitations of laser-based techniques to provide a systematic reference for researchers in bio-photonics and related biological fields.

In this review, three laser-based modulation methods of Ca²⁺ signaling are summarized. It is sensible to take advantage of lasers to precisely control cellular Ca²⁺ at a submicron resolution if the photon energy can be transformed to modulate biochemical processes for Ca²⁺ release. This concept is realized by uncaging (Fig. 1) and optogenetics (Fig. 3), which utilize lasers to excite Ca²⁺-caged compounds or optogenetic proteins that have been introduced into cells and are sensitive to lasers for Ca²⁺ modulation. Photo-uncaging and optogenetics are effective and powerful tools for studying neuroscience. Theoretically, lasers can achieve diffraction-limited spatial resolution, but the specificity and efficiency of these two methods are limited by the intracellular distribution of Ca²⁺ caging compounds or the expression of optogenetic genes. Recently, all-optical technology using a laser to control cellular Ca²⁺ with high resolution and specificity has been reported (Fig. 4). Specifically, we provide a deep insight into low-power near-infrared femtosecond laser modulation technologies for precise control of intracellular calcium and derive possible mechanisms for subcellular calcium regulation.

Laser-based Ca²⁺ modulation techniques are powerful tools for cellular calcium research. In particular, the multiphoton excitation of cells with a near-infrared femtosecond laser contributes to high spatial resolution and facilitates the study of subcellular Ca²⁺ modulation mechanisms in organelle contacts and the nucleus. We propose that lasers are capable of bringing breakthroughs in the calcium theory. The interaction mechanism of lasers with cells requires in-depth and detailed exploration to promote the development of subcellular Ca²⁺ signaling.