Studying the ultra-fine structure and functions of mitochondria at a nanoscale level has garnered tremendous attention from biologists. Mitochondria perform many more functions than merely generating adenosine triphosphate (ATP), and their functions can vary in different eukaryotic cells. In place of diffractionlimited conventional imaging techniques, advanced nanoscopic technologies have been devised in the past decades to explore the unknown aspects of mitochondrial dynamics and complex structures with a sub-diffraction resolution. The success of these super-resolution microscopy and nanoscopy techniques is complemented by the advancements in designing smart fluorescent probes that target mitochondria. Therefore, this review includes the comprehensive aspects of the recent progress in developing fluorogenic systems for nanoscopic imaging of mitochondria. The review also critically assesses the associated benefits and limitations of such fluorophores when they are employed in practical experiments. Future scope and challenges in developing suitable fluorophores for several nanoscopic techniques are also judiciously evaluated.

A rationally designed simple organic molecule exhibits white-light emission in methanol/water and acetonitrile/water mixtures.

Two cyanine-based fluorescent probes, ( E)-2-(4-(diethylamino)-2-hydroxystyryl)-3-ethyl-1,1-dimethyl-1 H-benzo[ e]indol-3-ium iodide (L) and ( E)-3-ethyl-1,1-dimethyl-2-(4-nitrostyryl)-1 H-benzo[ e]indol-3-ium iodide (L), have been designed and synthesized. Of these two probes, the twisted-intramolecular-charge-transfer (TICT)-based probe, L, can preferentially self-assemble to form nanoaggregates. L displayed a selective turn-on fluorescence response toward human and bovine serum albumin (HSA and BSA) in ∼100% aqueous PBS medium, which is noticeable with the naked eye, whereas L failed to sense these albumin proteins. The selective turn-on fluorescence response of L toward HSA and BSA can be attributed to the selective binding of probe L with HSA and BSA without its interfering with known drug-binding sites. The specific binding of L with HSA led to the disassembly of the self-assembled nanoaggregates of L, which was corroborated by dynamic-light-scattering (DLS) and transmission-electron-microscopy (TEM) analysis. Probe L has a limit of detection as low as ∼6.5 nM. The sensing aptitude of probe L to detect HSA in body fluid and an artificial-urine sample has been demonstrated.

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A versatile twisted-intramolecular-charge-transfer (TICT)-based near-infrared (NIR) fluorescent probe (L) has been judiciously designed and synthesized that could be utilized for potential cancer diagnosis and to track lymph node(s) in mice through distinct emission signals. Essentially, the probe rendered the capability to preferentially recognize the cancer cells over the noncancer cells by polarity-guided lipid droplet specific differential bioimaging (in green emission channel) studies. The probe also exhibited selective turn-on fluorescence response toward HSA/BSA in physiological media (aqueous PBS buffer; pH 7.4) at far-red/NIR regions, because of the 1:1 chelation between the probe and HSA/BSA. Therefore, the fluorescent probe was then maneuvered to track the draining lymphatic system and sentinel lymph node in tumor mice model by fluorescence imaging (NIR/deep-red channel), wherein the accumulated albumin protein in the draining tumor lymphatic system facilitated the in situ formation of the fluorescent albumin–L complex.

An aggregation-induced emission (AIE) active probe (L) displayed TURN-ON fluorescence response toward Al(3+) under physiological conditions and in HeLa cells. The L-Al(3+) ensemble could subsequently facilitate tracking of interaction with DNA in solution.

Various strategies for TSQ-induced fluorophore stabilization and their application in sm-FRET as well as in super-resolution imaging microscopy are thoroughly reviewed.

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