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Spectral imaging is a fluorescence microscopy technique with several applications, including imaging of environment-sensitive probes, spectral unmixing and identification of fluorescent species. In confocal microscopes not equipped with a spectral detection unit, spectral images can be obtained using the lambda scan mode of the microscope, namely the sequential acquisition of images using a tunable emission filter or other dispersive optical elements. Unfortunately, the lambda scan mode has poor temporal resolution, is a photon-wasting technique, and is not ideal for the spectral imaging of live samples. Here, we describe a spectral imaging method that can be implemented on commercial confocal microscopes not equipped with a spectral detector. The method is based on simultaneous image acquisition in 4 contiguous spectral channels and spectral phasor analysis. We demonstrate that this method can be easily implemented on a Leica confocal laser scanning microscope, with better photon efficiency and temporal resolution than the lambda scan mode. We perform a 4-channel (4 C) spectral phasor analysis of live cells stained with the environment-sensitive ACDAN and Nile Red dyes. We can distinguish changes in spectral emission in the order of 5 nm between different subcellular compartments. We show that 4 C-spectral phasor can be used to decompose the Nile Red signal into 2 components and perform 3-color imaging in combination with a DNA dye in live organoids. Finally, we show that the 4 C-spectral phasor can be also used to unmix the signal of fluorescent proteins with overlapping emission spectra such as mEmerald and EYFP.

A new type of fluorogenic and fluorochromic probe based on the reduction of weakly fluorescent 4-azido-6-(4-cyanophenyl)cinnoline to the corresponding fluorescent cinnoline-4-amine was developed. We found that the fluorescence of 6-(4-cyanophenyl)cinnoline-4-amine is strongly affected by the nature of the solvent. The fluorogenic effect for the amine was detected in polar solvents with the strongest fluorescence increase in water. The environment-sensitive fluorogenic properties of cinnoline-4-amine in water were explained as a combination of two types of fluorescence mechanisms: aggregation-induced emission (AIE) and excited state intermolecular proton transfer (ESPT). The suitability of an azide–amine pair as a fluorogenic probe was tested using a HepG2 hepatic cancer cell line with detection by fluorescent microscopy, flow cytometry, and HPLC analysis of cells lysates. The results obtained confirm the possibility of the transformation of the azide to amine in cells and the potential applicability of the discovered fluorogenic and fluorochromic probe for different analytical and biological applications in aqueous medium.

Amyloid formation plays a major role in a number of neurodegenerative diseases, including Alzheimer’s disease. Amyloid-β peptides (Aβ) are one of the primary markers associated with this pathology. Aβ aggregates exhibit a diverse range of morphologies with distinct pathological activities. Recognition of the Aβ aggregates by using small molecule-based probes and sensors should not only enhance understanding of the underlying mechanisms of amyloid formation, but also facilitate the development of therapeutic strategies to interfere with amyloid neurotoxicity. BODIPY (boron dipyrrin) dyes are among the most versatile small molecule fluorophores. BODIPY scaffolds could be functionalized to tune their photophysical properties to the desired ranges as well as to adapt these dyes to various types of conditions and environments. Thus, BODIPY dyes could be viewed as unique platforms for the design of probes and sensors that are capable of detecting and tracking structural changes of various Aβ aggregates. This review summarizes currently available examples of BODIPY dyes that have been used to investigate conformational changes of Aβ peptides, self-assembly processes of Aβ, as well as Aβ interactions with various molecules.

Hydrophobic fluorescence： Tan and his colleagues recently introduced a brand new chemotype of environment-sensitive fluorescent turn-on probes to detect the hydrophobic ligand-binding domain by using SBD fluorophore. The design strategy described in this report generalized the environment sensitivity turn-on mechanism to recognize a specific protein, which provides a robust breakthrough for interchanging fluorescence in conventional small-molecule fluorescent imaging.

Because of their deep penetration capability in tissue, red or near infrared (NIR) fluorophores attract much attention in bio-optical imaging. Among these fluorophores, the ones that respond to the immediate microenvironment (i.e., temperature, polarity, pH, viscosity, hypoxia, etc.) are highly desirable. We studied the response of six NIR aza-BODIPY-based and structurally similar fluorophores to polarity and viscosity for incorporation inside Pluronic nanoparticles as switchable fluorescent probes (SFPs). Based on our results, all of these fluorophores were moderately to strongly sensitive to the polarity of the microenvironment. We concluded that attaching amine groups to the fluorophore is not necessary for having strong polarity sensitive probes. We further studied the response of the fluorophores when embedded inside Pluronic nanoparticles and found that four of them qualified as SFPs. We also found that the switching ratio of the fluorophore-encapsulated Pluronic nanoparticles (ION-to-IOFF) is related to the length of the hydrophobic chain of the Pluronic tri-block copolymers. As such, the highest switching ratio pertained to F-68 with the lowest hydrophobic block poly (propylene oxide) (PPO chain of only 30 units).

Highly environment-sensitive fluorophores have been desired for many biomedical applications. Because of the noninvasive operation, high sensitivity, and high specificity to the microenvironment change, they can be used as excellent probes for fluorescence sensing/imaging, cell tracking/imaging, molecular imaging for cancer, and so on (i.e., polarity, viscosity, temperature, or pH measurement). In this work, investigations of the switching mechanism of a recently reported near-infrared environment-sensitive fluorophore, ADP(CA)2, were conducted. Besides, multiple potential biomedical applications of this switchable fluorescent probe have been demonstrated, including wash-free live-cell fluorescence imaging, in vivo tissue fluorescence imaging, temperature sensing, and ultrasound-switchable fluorescence (USF) imaging. The fluorescence of the ADP(CA)2 is extremely sensitive to the microenvironment, especially polarity and viscosity. Our investigations showed that the fluorescence of ADP(CA)2 can be switched on by low polarity, high viscosity, or the presence of protein and surfactants. In wash-free live-cell imaging, the fluorescence of ADP(CA)2 inside cells was found much brighter than the dye-containing medium and was retained for at least two days. In all of the fluorescence imaging applications conducted in this study, high target-to-noise (>5-fold) was achieved. In addition, a high temperature sensitivity (73-fold per Celsius degree) of ADP(CA)2-based temperature probes was found in temperature sensing.

The embrittlement of austempered ductile iron has been studied as a function of aqueous solution composition and of applied potentials. The extent of the embrittlement does not show marked dependence upon potential or the species present in water, and only the latter appears to be the critical substance for embrittlement. It is tentatively suggested that the water is effective in lowering the surface energy for fracture, which is accompanied by some small amounts of plastic deformation.