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Although fluorescence offers ultrasensitivity, real-world applications of fluorescence techniques encounter many practical problems. As a noninvasive means to investigate biomolecular mechanisms, pathways, and regulations in living cells, the intrinsic heterogeneity and inherent complexity of biological samples always generates optical interferences such as autofluorescence. Therefore, innovative fluorescence technologies are needed to enhance measurement reliability while not compromising sensitivity. In this review, we present current strategies that use photoswitchable nanoparticles to address these real-world challenges. The unique feature in these photoswitchable nanoparticles is that fundamental molecular photoswitches are playing the critical role of fluorescence modulation rather than traditional methods like modulating the light source. As a result, new innovative technologies that have recently emerged include super-resolution imaging, frequency-domain imaging, antiphase dual-color correlation, etc. Some of these methods improve imaging resolution down to the nanometer level, while others boost the detection sensitivity by orders of magnitude and confirm the nanoparticle probes unambiguously. These enhancements, which are not possible with non-photoswitching molecular probes, are the central topics of this review.

Our understanding of molecules has stagnated at a single quantum system, with atoms as Newtonian particles and electrons as quantum particles. Here, however, we reveal that both atoms and electrons in a molecule are quantum particles, and their quantum–quantum interactions create a previously unknown, newfangled molecular property—supracence. Molecular supracence is a phenomenon in which the molecule transfers its potential energy from quantum atoms to photo-excited electrons so that the emitted photon has more energy than that of the absorbed one. Importantly, experiments reveal such quantum energy exchanges are independent of temperature. When quantum fluctuation results in absorbing low-energy photons, yet still emitting high-energy photons, supracence occurs. This report, therefore, reveals novel principles governing molecular supracence via experiments that were rationalized by full quantum (FQ) theory. This advancement in understanding predicts the super-spectral resolution of supracence, and molecular imaging confirms such innovative forecasts using closely emitting rhodamine 123 and rhodamine B in living cell imaging of mitochondria and endosomes. [ABSTRACT FROM AUTHOR]

Monitoring multiple molecular probes simultaneously establishes their correlations and reveals the holistic mechanism. Current fluorescence imaging, however, is limited to about four colors because of typically circa 100‐nm spectral width. Herein, we show that molecular supracence imparts superior spectral resolution, resolving eight colors in 300‐nm width, about 37.5‐nm per color. A recently discovered light‐molecule interacting phenomenon, supracence only measures molecular emission above its excitation energy due to entanglement between atomic quantum system and electronic quantum system. As such, supracence takes advantage of sharp spectral edge of a single pathway and excitation specificity to produce narrow bands, whereas fluorescence has to deal with multiple pathways spilling out low‐energy long tail, that causes poor resolution. Thus, supracence enables myriad innovative molecular spectroscopy and microscopic imaging with profound impact broadly. [ABSTRACT FROM AUTHOR]

The currently understood principles about light–molecule interactions are limited, and thus scientific scope beyond current theories is rarely harvested. Herein we demonstrate supracence phenomena, in which the emitted photons have more energy than the absorbed photons. The extra energy comes from couplings of the absorbed and emitted photon to molecular phonons, whose potentials are constantly exchanging with molecular quantum energy and the environment. Thus, supracence is a linear optical process rather than a nonlinear optical process, such as second harmonic generation. Because supracence results in cooled molecular phonons and thus cooled molecules, behavior opposite to that of hot fluorescing emitters is expected. This report reveals certain supracence principles while contrasting fluorescence with supracence in high‐resolution imaging. [ABSTRACT FROM AUTHOR]

Folding–unfolding imparts fluorescence dual color switching, thus a novel concept to switch fluorescence between two distinct colors while avoiding traditional bond rupturing and bond forming in photoswitching. Because folding and unfolding minimize the wear and tear on molecular structures, the new systems have excellent reversibility and fatigue resistance. [ABSTRACT FROM AUTHOR]

The fluorescence brightness of a molecular probe determines whether it can be effectively measured and its water solubility dictates if it can be applied in real-world biological systems. However, molecules brighter than the most efficient fluorescent dyes or particles brighter than quantum dots are hard to come by, especially when they must also be soluble in water. In this report, chromophoric phosphoramidites are used in a solid-state synthesis to construct functional dendrimers. When highly twisted chromophores are chosen and the proper spacers and dendrons are introduced, the resultant dendrimers emit exceptionally bright fluorescence. Chromophores, spacers, and dendrons are stitched together by efficient phosphoramidite reagents, which afford high-yield water-soluble phosphodiester linkages after deprotection. The resulting water-soluble dendrimers are exceptionally bright. [ABSTRACT FROM AUTHOR]

Core-shell polymeric nanoparticles (NPs) containing photoswitchable spiropyrans (SP) and spirooxazines (SO) within the cores were fabricated. Fluorescence photoswitching studies revealed that the substituent groups on the photochromic SP/SO structures exerted significant influence on the fluorescence photoswitching behaviors of the as-prepared polymeric NPs, such as the reversibly interchangeable fluorescence color and the photoswitching kinetics. The electron-releasing and electron-withdrawing abilities of the substituent groups are believed to play important roles in controlling the electron configuration of the photochromic component and therefore be responsible for the tunable dual-color photoswitching performance. [ABSTRACT FROM AUTHOR]

Because of its ultrasensitivity, fluorescence offers a noninvasive means to investigate biomolecular mechanisms, pathways, and regulations in living cells, tissues, and animals. However, real-world applications of fluorescence technologies encounter many practical challenges. For example, the intrinsic heterogeneity of biological samples always generates optical interferences. High background such as autofluorescence can often obscure the desired signals. Finally, the wave properties of light limit the spatial resolution of optical microscopy. [ABSTRACT FROM AUTHOR]

Using photo-actuated unimolecular logical switching attained reconstruction (PULSAR) nanoscopy, the structures of photoswitchable polymeric nanoparticles self-assembled on the surfaces of CaCl2crystals at the nanoscale were revealed; the photoswitching events and the locations of the photoswitchable fluorescent dyes inside the hydrophobic cores of the core–shell type polymeric nanoparticles were determined. [ABSTRACT FROM AUTHOR]

Polymeric fluorescent nanoparticles with covalently embedded perylene fluorophores were developed by facile synthesis strategy and their advanced features of extremely high fluorescence intensity, non-photoblinking and excellent photostability were experimentally confirmed at the single nanoparticle level. [ABSTRACT FROM AUTHOR]

A pair of reversible photochemical reactions correlates their reactant and product specifically, and such a correlation uniquely distinguishes their correlated signal from others that are not linked by this reversible reaction. Here a nanoparticle-shielded fluorophore is photodriven to undergo structural dynamics, alternating between a green-fluorescence state and a red-fluorescence state. As time elapses, the fluorophore can be in either state but not both at the same time. Thus, the red fluorescence is maximized while the green fluorescence is minimized and vice versa. Such an antiphase dual-color (AD) corelationship between the red and green fluorescence maxima as well as between their minima can be exploited to greatly improve the signal-to-noise ratio, thus enhancing the ultimate detection limit. Potential benefits of this correlation include elimination of all interferences originating from single-color dyes and signal amplification of AD photoswitching molecules by orders of magnitude. [ABSTRACT FROM AUTHOR]

We report that two molecular building blocks differ only by two protons, yet they form totally different nanostructures. The protonated one self-organized into hollow nanospheres (200 nm), whereas the one without the protons self-assembled into rectangular plates. Consequently, the geometrically defined nanoassemblies exhibit radically different properties. As self-assembly directing units, protons impart ion-pairing and hydrogen-bonding probabilities. The plate-forming nanosystem fluoresces weakly, probably due to energy transfer among chromophores (Φ < 0.2), but the nanospheres emit strong yellow fluorescence (Φ ≈ 0.58-0.85). [ABSTRACT FROM AUTHOR]

The article demonstrates that single-molecule spiropyran-to-merocyanine photoswitching imparts strong red fluorescence and that the resulting merocyanine offers good photophysical characteristics. The authors report that such fluorescence photoswitching enables nanometer spatial resolution PULSAR microscopy.

The article presents information on a hard-and-soft hybrid nanosystem whose fluorescence intensity can be reversibly modulated using specific bandwidths of light. There is considerable current interest within the rapidly expanding field of nanotechnology in integrating hard and soft structures into complex functional nanosystems that have stimuli responsive properties. The study specifically attaches a spiropyran via a thiol-metal linkage to a core-shell semiconductive CdSe/ZnS nanocrystal. Preparation of both the spiropyran and the core-shell nanocrystals is a straightforward procedures.