Temperature is one of the most important of the physiological parameters that determine the biological status of living organisms. However, intracellular temperature was not imaged at the single-cell level until recently because of the lack of a molecular thermometer that can be applied to living cells. We have recently developed a method for imaging intracellular temperature using a cationic linear fluorescent polymeric thermometer (FPT) and fluorescence lifetime imaging microscopy (FLIM). The cationic linear FPT exhibits cell permeability in various mammalian cell lines and yeast cells, entering live cells within 10 min of incubation. Intracellular thermometry using the cationic linear FPT and FLIM can be used to image temperature with high temperature resolution (0.3-1.29 °C within a temperature range of 25-35 °C). The diffuse intracellular localization of the cationic linear FPT allows a high spatial resolution (i.e., the light microscope's diffraction limit, 200 nm), enabling the detection of temperature distributions at the subcellular level. This protocol, including the construction of a calibration curve and intracellular temperature imaging, requires ~14 h. Experience in handling cultured mammalian cells and use of a confocal laser-scanning microscope (CLSM) is required.

Temperature imaging using a cationic linear fluorescent polymeric thermometer and fluorescence lifetime imaging microscopy

The development of lanthanide-doped non-contact luminescent nanothermometers with accuracy, efficiency and fast diagnostic tools attributed to their versatility, stability and narrow emission band profiles has spurred the replacement of conventional contact thermal probes. The application of lanthanide-doped materials as temperature nanosensors, excited by ultraviolet, visible or near infrared light, and the generation of emissions lying in the biological window regions, I-BW (650 nm-950 nm), II-BW (1000 nm-1350 nm), III-BW (1400 nm-2000 nm) and IV-BW (centered at 2200 nm), are notably growing due to the advantages they present, including reduced phototoxicity and photobleaching, better image contrast and deeper penetration depths into biological tissues. Here, the different mechanisms used in lanthanide ion-doped nanomaterials to sense temperature in these biological windows for biomedical and other applications are summarized, focusing on factors that affect their thermal sensitivity, and consequently their temperature resolution. Comparing the thermometric performance of these nanomaterials in each biological window, we identified the strategies that allow boosting of their sensing properties.

Lanthanide doped luminescence nanothermometers in the biological windows: strategies and applications.

The upsurge interest in the development of efficient methodologies for the construction of nitrogen-containing frameworks via the use of expedient reagents have been creating a renaissance in contemporary organic chemistry. In this perspective, tertbutyl nitrite (TBN) is an emerging building block. Due to its unique structural features, it shows differential reactivity under different reaction conditions. These diverse reactivities have resulted in the construction of a diverse array of complex N-containing molecules. The primary objective of the present review is to bring the latest findings of TBN in terms of its applications in reactions (oxidation, diazotization, nitrosation, nitration, oximation, N-synthon, and miscellaneous reactions) into the limelight. For simplicity and brevity, reactions in each section are explained with the mechanism of formation and selected examples are given.

tert-Butyl Nitrite (TBN), a Multitasking Reagent in Organic Synthesis.

Sensing temperature at the subcellular level is of great importance for the understanding of miscellaneous biological processes. However, the development of sensitive and reliable organic fluorescent nanothermometers remains challenging. In this study, we report the fabrication of a novel organic fluorescent nanothermometer and study its application in temperature sensing. First of all, we synthesize a dual-responsive organic luminogen that can respond to the molecular state of aggregation and environmental polarity. Next, natural saturated fatty acids with sharp melting points as well as reversible and rapid phase transition are employed as the encapsulation matrix to correlate external heat information with the fluorescence properties of the luminogen. To apply the composite materials for biological application, we formulate them into colloidally dispersed nanoparticles by a technique that combines in situ surface polymerization and nanoprecipitation. As anticipated, the resultant zwitterionic nanothermometer exhibits sensitive, reversible, reliable, and multiparametric responses to temperature variation within a narrow range around the physiological temperature (i.e., 37 °C). Taking spectral position, fluorescence intensity, and fluorescence lifetime as the correlation parameters, the maximum relative thermal sensitivities are determined to be 2.15% °C-1, 17.06% °C-1, and 17.72% °C-1, respectively, which are much higher than most fluorescent nanothermometers. Furthermore, we achieve the multimodal temperature sensing of bacterial biofilms using these three complementary fluorescence parameters. Besides, we also fabricate a cationic form of the nanothermometer to facilitate efficient cellular uptake, holding great promise for studying thermal behaviors in biological systems.

A Sensitive and Reliable Organic Fluorescent Nanothermometer for Noninvasive Temperature Sensing.

Fluorescent thermometers with near-infrared (NIR) emission play an important role in visualizing the intracellular temperature with high resolution and investigating the cellular functions and biochemical activities. Herein, we designed and synthesized a donor-Π-acceptor luminogen, 2-([1,1'-biphenyl]-4-yl)-3-(4-((E)-4-(diphenylamino)styryl) phenyl) fumaronitrile (TBB) by Suzuki coupling reaction. TBB exhibited twisted intramolecular charge transfer-based NIR emission, aggregation-induced emission, and temperature-sensitive emission features. A ratiometric fluorescent thermometer was constructed by encapsulating thermosensitive NIR fluorophore TBB and Rhodamine 110 dye into an amphiphilic polymer matrix F127 to form TBB&R110@F127 nanoparticles (TRF NPs). TRF NPs showed a good temperature sensitivity of 2.37%·°C-1, wide temperature response ranges from 25 to 65 °C, and excellent temperature-sensitive emission reversibility. Intracellular thermometry experiments indicated that TRF NPs could monitor the cellular temperature change from 25 to 53 °C for Hep-G2 cells under the photothermal therapy agent heating process, indicating the considerable potential applications of TRF NPs in the biological thermometry field.

TICT-Based Near-Infrared Ratiometric Organic Fluorescent Thermometer for Intracellular Temperature Sensing.

A cationic fluorescent nanogel thermometer based on thermo-responsive N-isopropylacrylamide and environment-sensitive benzothiadiazole was developed with a new azo compound bearing imidazolium rings as the first cationic radical initiator. This cationic fluorescent nanogel thermometer showed an excellent ability to enter live mammalian cells in a short incubation period (10 min), a high sensitivity to temperature variations in live cells (temperature resolution of 0.02-0.84 °C in the range 20-40 °C), and remarkable non-cytotoxicity, which permitted ordinary cell proliferation and even differentiation of primary cultured cells.

A Cell‐Targeted Non‐Cytotoxic Fluorescent Nanogel Thermometer Created with an Imidazolium‐Containing Cationic Radical Initiator

The method is effective for the synthesis of xanthone, thioxanthone, acridone, and anthrone oximes, which are difficult to obtain by conventional dehydrative condensation of corresponding ketones.

Formation of xanthone oxime and related compounds using a combination of tert-butyl nitrite and potassium hexamethyldisilazide

A formal synthesis of a hepatitis C virus (HCV) inhibitor is described. The synthesis features a benzyne-mediated one-pot indoline formation–methylation sequence and an In(OTf) 3 -mediated mild oxa-Pictet–Spengler reaction for the synthesis of substituted electron-rich pyranoindole.

Formal Synthesis of Hepatitis C Virus NS5B Polymerase Inhibitor

Synthetic studies on discorhabdin V: Construction of the A–F hexacyclic framework

FORMATION OF XANTHONE OXIME AND RELATED COMPOUNDS USING A COMBINATION OF tert-BUTYL NITRITE AND POTASSIUM HEXAMETHYLDISILAZIDE (Dedicated to Professor Victor Snieckus on the occasion of his 77th birthday)

Takahiro Noro

Papers

A Cell‐Targeted Non‐Cytotoxic Fluorescent Nanogel Thermometer Created with an Imidazolium‐Containing Cationic Radical Initiator

Formation of xanthone oxime and related compounds using a combination of tert-butyl nitrite and potassium hexamethyldisilazide

Formal Synthesis of Hepatitis C Virus NS5B Polymerase Inhibitor

Synthetic studies on discorhabdin V: Construction of the A–F hexacyclic framework

FORMATION OF XANTHONE OXIME AND RELATED COMPOUNDS USING A COMBINATION OF tert-BUTYL NITRITE AND POTASSIUM HEXAMETHYLDISILAZIDE (Dedicated to Professor Victor Snieckus on the occasion of his 77th birthday)