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Precisely identifying biological targets and accurately extracting their relatively weak signals from complicated physiological environments represent daunting challenges in biological detection and biomedical diagnosis. Fluorescence techniques have become the method of choice and offer minimally invasive and ultrasensitive detections, thus, providing a wealth of information regarding the biological mechanisms in living systems. Despite fluorescence analysis has advanced remarkably, conventional detections still encounter considerable limitations. This stems from the fact that the fluorescence intensity signal (I) is sensitive and liable to numerous external factors including temperature, light source, medium characteristics, and dye concentration. The interferences exasperatingly undermine the precision of measurements, and frequently render the signal undetectable. For example, fluorescence from single-molecule emitters can be measured on glass substrates under optimum conditions, but single-molecule events in complicated physiological environments such as live cells can hardly be detected because of autofluorescence interference and other factors. Furthermore, traditional intensity (I) and wavelength (λ) measurements do not reveal the interactive nature between the donor and the acceptor. Thus, innovative detection strategies to circumvent these aforementioned limitations of the conventional techniques are critically needed. With the use of photoswitching-induced donor-acceptor-fluorescence double modulations, we present a novel strategy that introduces three additional physical parameters: modulation amplitude (A), phase shift (ΔΦ), and lock-in frequency (ω), and demonstrate that such a strategy can circumvent the limitation of the conventional fluorescence detection techniques. Together, these five physical quantities (I, λ, A, ΔΦ, ω) reveal insightful information regarding molecular interactive strength between the probe and the analyte and enable extracting weak-fluorescence spectra from large interfering noises in complex environments.

This minireview highlights recent advances of research dedicated to photoswitchable fluorescent nanoparticles and their applications. Recently, several strategies have been developed to synthesize nanoparticles with optically switchable emission properties: either fluorescence on/off or dual-alternating-color fluorescence photoswitching. The underlying mechanisms of fluorescence photoswitching enable many different types of photoswitchable fluorescent nanoparticles to change fluorescence colors, thus validating the basis of the initial photoswitching design. Among all possible applications, the usage of photoswitchable fluorescent nanoparticles to empower super-resolution fluorescence imaging and to label biological targets was subsequently reviewed. Finally, we summarize the important areas regarding future research and development on photoswitchable fluorescent nanoparticles.

Although H5N1 avian influenza has not yet acquired the capacity to readily infect humans, should it do so, this viral pathogen would present an increasing threat to the immunologically naïve human population. Subunit vaccines based on the viral glycoprotein hemagglutinin (HA) can provide protective immunity against influenza. Polyanhydride nanoparticles have been shown to enhance efficacy of subunit vaccines, providing the dual advantages of adjuvanticity and sustained delivery resulting in enhanced protein stability and immunogenicity. In this work, a recombinant trimer of H5 (H53 ) was encapsulated and released from polyanhydride nanoparticles. Release kinetics of the encapsulated H53 were found to be dependent on polymer chemistry (i.e., hydrophobicity and molecular weight). Polyanhydride nanoparticles composed of sebacic anhydride and 1,6-bis(p-carboxyphenoxy)hexane (CPH; that degrade into more acidic monomers) released structurally stable HA H53 , while H53 released from formulations composed of CPH and 1,8-bis(p-carboxyphenoxy)-3,6-dioxaoctane (CPTEG) (that are amphiphilic and whose degradation products are less acidic) displayed unfolding of tertiary structure. However, the antigenicity of the H53 based on binding of a H5-specific monoclonal antibody was preserved upon release from all the formulations studied, demonstrating the value of polyanhydride nanoparticles as a viable platform for HA-based influenza vaccines.

We have developed a class of spiropyran dyes and their fluorescence colors can be reversibly photoswitched from red color to green, blue, or nearly dark, thus alternating between two colors. Such individual dyes emit either one color or the other, but not both simultaneously. These photoswitchable dyes-enabled nanoparticles, however, emit either one pure color or a combination of both colors because the nanoparticle fluorescence originates from multiple dyes therein. As a result, the nanoparticle shines >30 times brighter than the state-of-the-art organic dyes such as fluorescein. Interestingly, these copolymer nanoparticles exhibit tunable non-specific interactions with live cells and nanoparticles containing properly balanced butyl acrylate and acrylamide monomers render essentially very little non-specific binding to live cells. Decorated with HMGA1 protein, these optically switchable dual-color nanoparticles undergo endocytosis and unambiguously identify themselves from fluorescence interference including autofluorescence, thus enabling a new tool for live cell imaging.

Beyond-diffraction-limit optical imaging of cells will reveal biological mechanisms, cellular structures, and physiological processes in nanometer scale. Harnessing the photoswitching properties of spiropyran fluorophores, we achieved nanoresolution fluorescence imaging using photoactuated unimolecular logical switching attained reconstruction (PULSAR) microscopy. The PULSAR microscope successfully resolved nanostructures and subcellular organelles when the photoswitchable nanoparticles containing spiropyran dyes were used as fluorescent probes.

Optically switchable dual-color fluorescent nanoparticles that incorporate two classes of dyes into the polymeric chains have been synthesized using an emulsion polymerization method. The nanoparticles consist of an organic photoisomerizable dye, spiropyran, as an optically responsive component and another fluorescent dye, perylene diimide, as a high-energy emitter. Under UV irradiation, the colorless spiropyran undergoes photoisomerization to yield merocyanine, which absorbs at 588 nm and fluoresces strongly at 670 nm. The absorption band of the merocyanine matches well the fluorescence bands of perylene diimide (at 535 and 575 nm), and therefore fluorescence resonance energy transfer (FRET) converts the high-energy green emission of the perylene into low-energy red emission when the merocyanine form is present. Upon exposure to alternating UV ( 420 nm) light, the nanoparticles cycle between red and green fluorescence as the spiro and mero forms of the optically responsive component...

Kathleen A Ross,1 Hyelee Loyd,2 Wuwei Wu,2 Lucas Huntimer,3 Shaheen Ahmed,4 Anthony Sambol,5 Scott Broderick,6 Zachary Flickinger,2 Krishna Rajan,6 Tatiana Bronich,4 Surya Mallapragada,1 Michael J Wannemuehler,3 Susan Carpenter,2 Balaji Narasimhan1 1Chemical and Biological Engineering, Iowa State University, Ames, IA, USA; 2Animal Science, Iowa State University, Ames, IA, USA; 3Veterinary Microbiology and Preventive Medicine, Iowa State University, Ames, IA, USA; 4Pharmaceutical Sciences, University of Nebraska Medical Center, Omaha, NE, USA; 5Pathology and Microbiology, University of Nebraska Medical Center, Omaha, NE, USA; 6Materials Science and Engineering, Iowa State University, Ames, IA, USA Abstract: H5N1 avian influenza is a significant global concern with the potential to become the next pandemic threat. Recombinant subunit vaccines are an attractive alternative for pandemic vaccines compared to traditional vaccine technologies. In particular, polyanhydride nanoparticles encapsulating subunit proteins have been shown to enhance humoral and cell-mediated immunity and provide protection upon lethal challenge. In this work, a recombinant H5 hemagglutinin trimer (H53) was produced and encapsulated into polyanhydride nanoparticles. The studies performed indicated that the recombinant H53 antigen was a robust immunogen. Immunizing mice with H53 encapsulated into polyanhydride nanoparticles induced high neutralizing antibody titers and enhanced CD4+ T cell recall responses in mice. Finally, the H53-based polyanhydride nanovaccine induced protective immunity against a low-pathogenic H5N1 viral challenge. Informatics analyses indicated that mice receiving the nanovaccine formulations and subsequently challenged with virus were similar to naïve mice that were not challenged. The current studies provide a basis to further exploit the advantages of polyanhydride nanovaccines in pandemic scenarios. Keywords: polymer, nanoparticle, vaccine, subunit, neutralizing antibody