Zinc (Zn2+) is an important component of various proteins and plays important roles in many biological processes. It is buffered and regulated by three major protein families: zinc exporters (ZnTs), zinc importers (ZIPs), and metallothioneins (MTs) [1]. The membrane-localized ZIP and ZnT transporters control uptake and export pathways [2,3] while MTs may act as intracellular storage sites. The Zn2+ levels in different cells may vary significantly to cater for certain physiological functions or survival [2,4, 5, 6, 7]. For instance, high concentrations of Zn2+ can be found in the intracellular vesicles of pancreatic islet β-cells, in which Zn2+ forms a complex with insulin [8]••. In contrast, labile Zn2+ exhibits an inhibitory effect on aerobic respiration and proliferation of cells, such that cells with high metabolic activity or rapid proliferation, such as cancer cells, tend to maintain lower concentrations of Zn2+ in mitochondria and/or the cytosol compared to their counterparts [9,10]. Depending on the specific function, various tissues and organs contain different amounts of zinc, as indicated by the mass fractions shown in Figure 1 [9,11,12]. Given the potential role of Zn2+ as a second messenger [13], organs that are involved in neuronal signal transduction contain higher levels of zinc. Among all human organs, the prostate contains the highest amount of zinc, which is used to prevent abnormal growth and inhibit aconitase from accumulating sufficient citrate, which is released into the prostate fluid to ensure fertility [14]. Therefore, biological Zn2+ is maintained at distinct levels that match the specific physiological functions. Deviations from the normal concentration range often result in malfunction and can also be found during carcinogenesis. Therefore, rapid spatial-temporal monitoring of Zn2+ level in organelles, cells, and live organisms is essential for studying zinc-related physiological processes.

Various imaging techniques have been developed to visualize Zn2+, and among them, fluorescence microscopy stands out owing to its high sensitivity and simple operation [15, 16, 17]. However, due to the absorption and scattering of light in tissues, conventional fluorescence microscopy suffers from a shallow penetration depth and low resolution, unsuitable for deep-tissue imaging. In contrast, two-photon (TP) microscopy uses a long wavelength (700–1000 nm) femtosecond-pulsed infrared laser as the excitation source, resulting in much-improved penetration depth in biological tissues, thus enabling the identification of individual cells and determining the uptake of traces of drug carriers in tissues with a penetration depth of greater than 500 μm [18].

As a complementary imaging tool, photoacoustic (PA) imaging technology relies on the detection of ultrasonic waves generated by the thermoelastic expansion of biological tissue upon laser excitation to generate an image based on the optical absorption contrast of the tissue. By relying on ultrasonic transducers rather than optical detectors employed in fluorescence imaging, PA imaging circumvents the limited penetration depth caused by optical scattering [19]. Capable of deep-tissue imaging up to 5 cm depth, PA imaging greatly exceeds the soft limit of traditional optical imaging around 1 mm, and yields high-resolution images across multiple lengths scales from cells to tissues and even whole organs [20]. While these in vivo imaging methods are particularly useful for visualizing deep structures within a sample, it is not possible to capture detailed structures at the 100 nm length-scale.

Super-resolution imaging has broken the boundaries imposed by the diffraction limit and effectively promoted rapid progress in the life sciences. Based on a range of different physical principles, the major super-resolution imaging technology include stochastic optical reconstruction microscopy (STORM), stimulated emission depletion (STED), photoactivated localization microscopy (PALM), and structured illumination microscopy (SIM) [21]. Among them, SIM shows a typical spatial resolution of 80–100 nm, and the fast-imaging speed and moderate photostability requirement make it suitable for observing organelle interactions and real-time in-situ tracking of small molecules [22,23].

Considering the significance of real-time in situ tracking of Zn2+ levels both in subcellular structures and in live organisms, this review focuses on the most recent developments and applications of Zn2+ fluorescent probes and advances in emerging imaging techniques for tracing biological Zn2+ (Table 1). Our goal is to inspire the development of new Zn2+ imaging probes, which will be of great importance for exploring the physiological and pathological significance of Zn2+ and for developing new diagnostic and treatment approaches for major diseases, especially in neurobiology.