Delayed Increase in Near-Infrared Fluorescence in Cultured Murine Cancer Cells Labeled with Oxygen-Doped Single-Walled Carbon Nanotubes.

The labeling technique for cells with over-thousand-nanometer near-infrared (OTN-NIR) fluorescent probes has attracted much attention for in vivo deep imaging for cell tracking and cancer metastasis, because of low scattering and absorption of OTN-NIR light by biological tissues. However, the intracellular behavior following the uptake of the single-walled carbon nanotubes (SWCNTs), an OTN-NIR fluorophore, remains unknown. The aim of this study is to investigate the time-dependent change in OTN-NIR fluorescence images of cultured murine cancer cells (Colon-26) following treatment with a recently developed OTN-NIR fluorescent probe, epoxide-type oxygen-doped SWCNTs (o-SWCNTs). The o-SWCNTs were synthesized by oxygenation of SWCNTs by ozone under ultraviolet irradiation and were dispersed in an aqueous solution of N-(carbonyl-methoxypolyethyleneglycol 2000)-1,2-distearoyl- sn-glycero-3-phosphoethanolamine to prepare biocompatible o-SWCNTs (o-SWCNT-PEG). OTN-NIR fluorescent o-SWCNT-PEG showed an abnormal behavior following cellular uptake. OTN-NIR fluorescence was not observed in the cells after 24 h incubation with the o-SWCNT-PEG, but clearly increased with longer incubation time from three days after the treatment. This result was further confirmed by Raman microscopy, suggesting that OTN-NIR fluorescence intensity was associated with the cellular uptake of the o-SWCNT-PEG. These results suggest that the Colon-26 cells were successfully labeled by the o-SWCNT-PEG that emit OTN-NIR fluorescence. The o-SWCNT-PEG may aggregate in the cells over time, which could favor their internalization. This delayed concentration followed by a long retention of the o-SWCNT-PEG in cells will facilitate further biotechnological applications of the o-SWCNTs to in vivo deep OTN-NIR fluorescent imaging.

Heat Treatment Effects for Controlling Dye Molecular States in the Hydrophobic Core of Over-1000 nm Near-Infrared (NIR-II) Fluorescent Micellar Nanoparticles.

Organic molecules that emit near-infrared (NIR) fluorescence at wavelengths above 1000 nm, also known as the second NIR (NIR-II) biological window, are expected to be applied to optical in vivo imaging of deep tissues. The study of molecular states of NIR-II dye and its optical properties are important to yield well-controlled fluorescent probes; however, no such study has been conducted yet. Among the two major absorption peaks of the NIR-II dye, IR-1061, the ratio of the shorter wavelength (900 nm) to the longer one (1060 nm) increased with an increase in the dye concentration in tetrahydrofuran, suggesting that the 900 nm peak is due to the dimer formation of IR-1061. Both absorption peaks are also observed when IR-1061 is encapsulated in the hydrophobic (stearyl) core of micellar nanoparticles (MNPs) of a phospholipid-poly(ethylene glycol). The dimers in the MNP cores decreased via dimer dissociation by enhancing the mobility of the hydrophobic stearyl chains by heat treatment of the dye-encapsulating MNPs at 50-70 °C. The MNPs maintained the dissociated IR-1061 monomers in the core after recooling to 25 °C and showed a higher NIR-II fluorescence intensity than those before heat treatment. This concept will provide better protocols for the preparation of NIR-II fluorescent probes with well-controlled fluorescence properties.

Rapid increase in transparency of biological organs by matching refractive index of medium to cell membrane using phosphoric acid.

Tissue clearing is a fundamental challenge in biology and medicine to achieve high-resolution optical imaging of tissues deep inside intact organs. The clearing methods reported up to now require long incubation times or physical/electrical pressure to achieve tissue clearing, which is done by matching the refractive indices of the whole sample and medium to that of the lipid layer. Here we show that phosphoric acid increases the refractive index of the medium and can increase the transparency of formalin-fixed tissue samples rapidly. While phosphoric acid (8.5-14.2 M) suppresses bright signals on the boundary of cells in their phase-contrast images, it does not damage the morphology of the phospholipid cell membrane. Immersion of fixed tissues of mice in phosphoric acid solutions (8.5-14.2 M) increased their transparency within 60 min in the case of 3 mm-thick fixed tissue specimens. Although further investigations are needed to apply this protocol to three-dimensional fluorescence imaging or immunohistochemistry, the protocol presented herein may contribute to developing better and faster soaking methods for tissue clearing than previously reported protocols.

Biological Deep Temperature Imaging with Fluorescence Lifetime of Rare-Earth-Doped Ceramics Particles in the Second NIR Biological Window.

Contactless thermal imaging generally relies on mid-infrared cameras and fluorescence imaging with temperature-sensitive phosphors. Fluorescent thermometry in the near-infrared (NIR) region is an emerging technique for analysing deep biological tissues but still requires observation depth calibration. We present an NIR fluorescence time-gated imaging (TGI) thermometry technology based on fluorescence lifetime, an intrinsic fluorophore time constant unrelated to observation depth. Fluorophore used is NaYF4 co-doped with Nd3+ and Yb3+ that emits fluorescence at 1000 nm. An agarose gel-based phantom with the fluorophore embedded at a 5-mm depth was covered by sheets of meat to vary the observation depth. The temperature was determined independently from depth by sequences of NIR fluorescence decay images, and the rate of change in the fluorescence lifetime per temperature was almost constant (-0.0092 ~ -0.010 °C-1) at depths ranging from 0 to 1.4 mm of meat, providing non-contact and absolute measurements of temperature in deep biological tissues.

Temperature Sensing of Deep Abdominal Region in Mice by Using Over-1000 nm Near-Infrared Luminescence of Rare-Earth-Doped NaYF4 Nanothermometer.

Luminescence nanothermometry has attracted much attention as a non-contact thermal sensing technique. However, it is not widely explored for in vivo applications owing to the low transparency of tissues for the light to be used. In this study, we performed biological temperature sensing in deep tissues using ß-NaYF4 nanoparticles co-doped with Yb3+, Ho3+, and Er3+ (NaYF4: Yb3+, Ho3+, Er3+ NPs), which displayed two emission peaks at 1150 nm (Ho3+) and 1550 nm (Er3+) in the >1000 nm near-infrared wavelength region, where the scattering and absorption of light by biological tissues are at the minimum. The change in the luminescence intensity ratio of the emission peaks of Ho3+ and Er3+ (IHo/IEr) in the NaYF4: Yb3+, Ho3+, Er3+ nanothermometer differs corresponding to the thickness of the tissue. Therefore, the relationship between IHo/IEr ratio and temperature needs to be calibrated by the depth of the nanothermometer. The temperature-dependent change in the IHo/IEr was evident at the peritoneal cavity level, which is deeper than the subcutaneous tissue level. The designed experimental system for temperature imaging will open the window to novel luminescent nanothermometers for in vivo deep tissue temperature sensing.