Liyi Zhou

Molecular Engineering of a TBET-Based Two-Photon Fluorescent Probe for Ratiometric Imaging of Living Cells and Tissues

In contrast to one-photon microscopy, two-photon probe-based fluorescent imaging can provide improved three-dimensional spatial localization and increased imaging depth. Consequently, it has become one of the most attractive techniques for studying biological events in living cells and tissues. However, the quantitation of these probes is primarily based on single-emission intensity change, which tends to be affected by a variety of environmental factors. Ratiometric probes, on the other hand, can eliminate these interferences by the built-in correction of the dual emission bands, resulting in a more favorable system for imaging living cells and tissues. Herein, for the first time, we adopted a through-bond energy transfer (TBET) strategy to design and synthesize a small molecular ratiometric two-photon fluorescent probe for imaging living cells and tissues in real time. Specifically, a two-photon fluorophore (D-π-A-structured naphthalene derivative) and a rhodamine B fluorophore are directly connected by electronically conjugated bond to form a TBET probe, or Np-Rh, which shows a target-modulated ratiometric two-photon fluorescence response with highly efficient energy transfer (93.7%) and two well-resolved emission peaks separated by 100 nm. This novel probe was then applied for two-photon imaging of living cells and tissues and showed high ratiometric imaging resolution and deep-tissue imaging depth of 180 μm, thus demonstrating its practical application in biological systems.

Alkyne-functionalized superstable graphitic silver nanoparticles for Raman imaging.

Noble metals, especially gold, have been widely used in plasmon resonance applications. Although silver has a larger optical cross section and lower cost than gold, it has attracted much less attention because of its easy corrosion, thereby degrading plasmonic signals and limiting its applications. To circumvent this problem, we report the facile synthesis of superstable AgCu@graphene (ACG) nanoparticles (NPs). The growth of several layers of graphene onto the surface of AgCu alloy NPs effectively protects the Ag surface from contamination, even in the presence of hydrogen peroxide, hydrogen sulfide, and nitric acid. The ACG NPs have been utilized to enhance the unique Raman signals from the graphitic shell, making ACG an ideal candidate for cell labeling, rapid Raman imaging, and SERS detection. ACG is further functionalized with alkyne-polyethylene glycol, which has strong Raman vibrations in the Raman-silent region of the cell, leading to more accurate colocalization inside cells. In sum, this work provides a simple approach to fabricate corrosion-resistant, water-soluble, and graphene-protected AgCu NPs having a strong surface plasmon resonance effect suitable for sensing and imaging.

Through-Bond Energy Transfer-Based Ratiometric Two-Photon Probe for Fluorescent Imaging of Pd2+ Ions in Living Cells and Tissues

Palladium can cause severe skin and eye irritation once it enters the human body. Ratiometric two-photon fluorescent probes can both eliminate interference from environmental factors and realize deep-tissue imaging with improved spatial localization. To quantitatively track Pd(2+) in biosystems, we report here a colorimetric and two-photon ratiometric fluorescent probe, termed Np-Rh-Pd, which consists of a two-photon fluorophore (naphthalene derivative with a D-π-A structure) and a rhodamine B dye. The two fluorophores are directly linked to form a two-photon ratiometric fluorescent probe for Pd(2+) based on a through-bond energy transfer (TBET) strategy. It exhibits highly efficient energy transfer (90%) with two well-resolved emission peaks (wavelength difference of 100 nm), which could efficiently diminish the cross talk between channels and is especially favorable for ratiometric bioimaging applications. A signal-to-background ratio of 31.2 was observed for the probe, which affords a high sensitivity for Pd(2+) with a detection limit of 2.3 × 10(-7) M. It was also found that acidity does not affect the fluorescent response of the probe to Pd(2+), which is favorable for its applications in practical samples. The probe was further used for fluorescence imaging of Pd(2+) ions in live cells and tissue slices under two-photon excitation, which showed significant tissue-imaging depths (90-270 μm) and a high resolution for ratiometric imaging.

Rhodamine-based fluorescent probe for direct bio-imaging of lysosomal pH changes

Intracellular pH plays a pivotal role in various biological processes. In eukaryotic cells, lysosomes contain numerous enzymes and proteins exhibiting a variety of activities and functions at acidic pH (4.5-5.5), and abnormal variation in the lysosomal pH causes defects in lysosomal function. Thus, it is important to investigate lysosomal pH in living cells to understand its physiological and pathological processes. In this work, we designed a one-step synthesized rhodamine derivative (RM) with morpholine as a lysosomes tracker, to detect lysosomal pH changes with high sensitivity, high selectivity, high photostability and low cytotoxicity. The probe RM shows a 140-fold fluorescence enhancement over a pH range from 7.4 to 4.5 with a pKa value of 5.23. Importantly, RM can detect the chloroquine-induced lysosomal pH increase and monitor the dexamethasone-induced lysosomal pH changes during apoptosis in live cells. All these features demonstrate its value of practical application in biological systems.

A highly sensitive and reductant-resistant fluorescent probe for nitroxyl in aqueous solution and serum

A novel coumarin-based fluorescent probe, P-CM, for quantitative detection of nitroxyl (HNO) was developed. P-CM exhibits a selective response to HNO over other biological reductants and was also applied for quantitative detection of HNO in bovine serum with satisfactory results.

Bispyrene–Fluorescein Hybrid Based FRET Cassette: A Convenient Platform toward Ratiometric Time-Resolved Probe for Bioanalytical Applications

Pyrene excimer possesses a large Stokes shift and long fluorescence lifetime and has been widely applied in developing time-resolved biosensing systems to solve the autofluorescence interference problems in biological samples. However, only a few of pyrene excimer-based small molecular probes have been reported so far. Ratiometric probes, on the other hand, can eliminate interferences from environmental factors such as instrumental efficiency and environmental conditions by a built-in correction of the dual emission bands but are ineffective for endogenous autofluorescence in biosystems. In this work, by combining the advantages of time-resolved fluorescence technique with ratiometric probe, we reported a bispyrene-fluorescein hybrid FRET cassette (PF) as a novel ratiometric time-resolved sensing platform for bioanalytical applications, with pH chosen as a biorelated target. The probe PF showed a fast, highly selective, and reversible ratiometric fluorescence response to pH in a wide range from 3.0 to 10.0 in buffered solution. By employing time-resolved fluorescence technique, the pH-induced fluorescence signal of probe PF can be well-discriminated from biological autofluorescence background, which enables us to detect pH in a range of 4.0-8.0 in cell media within a few seconds. It has also been preliminarily applied for ratiometric quantitative monitoring of pH changes in living cells with satisfying results. Since many fluorescein-based fluorescence probes have been developed, our strategy might find wide applications in design ratiometric time-resolved probes for detection of various biorelated targets.

Localizable and Photoactivatable Fluorophore for Spatiotemporal Two-Photon Bioimaging

Photoactivatable probe-based fluorescent imaging has become an efficient and attractive technique for spatiotemporal microscopic studies of biological events. However, almost all previously reported photoactivatable organic probes have been based on hydrosoluble precursors, which have produced water-soluble active fluorophores able to readily diffuse away from the photocleavage site, thereby dramatically reducing spatial resolution. Hydroxyphenylquinazolinone (HPQ), a small organic dye known for its classic luminescence mechanism through excited-state intramolecular proton transfer (ESIPT), shows strong light emission in the solid state, but no emission in solution. In this work, HPQ was employed as a precursor to develop a localizable, photoactivatable two-photon probe (PHPQ) for spatiotemporal bioimaging applications. After photocleavage, PHPQ releases a precipitating HPQ fluorophore which shows both one-photon and two-photon excited yellow-green fluorescence, thereby producing a localizable fluorescence signal that affords high spatial resolution for bioimaging, with more than 200-fold one-photon and 150-fold two-photon fluorescence enhancement.

A novel two-photon fluorescent probe for the selective detection of hydrogen peroxide based on a naphthalene derivative

In this study, we report a novel two-photon fluorescent probe for monitoring hydrogen peroxide. Probe 1 consists of a naphthalene backbone and a boric acid ester which was used as a H2O2 reporter. The reaction of probe 1 with H2O2 triggers the cleavage of a boronate-based protecting group, and as a result, restores the fluorescence of compound 2. The probe can be applied to the quantification of hydrogen peroxide with a linear range from 1.0 × 10−6 to 2.5 × 10−4 mol L−1. The detection limit of probe 1 toward H2O2 was estimated to be 0.7 μM. Furthermore, probe 1 was found to have a much higher selectivity for H2O2 than other reactive oxygen species and successfully applied to cell imaging of hydrogen peroxide using two-photon microscopy in living cells. The superior properties of the probe made it highly promising for use in chemical and biological applications.