X‑ray-Activated Near-Infrared Persistent Luminescent Probe for Deep-Tissue and Renewable in Vivo Bioimaging

Near-infrared (NIR) persistent luminescence nanoparticles (PLNPs) are considered as new alternative optical probes due to being free of autofluorescence, benefited from the self-sustained emission after excitation and high signal-to-noise ratio. However, the NIR-emitted PLNPs always present a short decay time and require excitation by ultraviolet or visible light with a short penetrable depth, remarkably hindering their applications for in vivo long-term tracking and imaging. Therefore, it is important to develop NIR-emitted PLNPs with in vivo activation nature by new excitation sources with deeper penetrating depths. Here, we propose a new type of X-ray-activated ZnGa₂O₄:Cr PLNPs (X-PLNPs) with efficient NIR persistent emission and rechargeable activation features, in which both the excitation and emission possess a high penetrable nature in vivo. These X-PLNPs exhibit long-lasting, up to 6 h, NIR emission at 700 nm after the stoppage of the X-ray excitation source. More importantly, the designed X-PLNPs can be readily reactivated by a soft X-ray excitation source with low excitation power (45 kVp, 0.5 mA) to restore in vivo bioimaging signals even at 20 mm depth. Renewable in vivo whole-body bioimaging was also successfully achieved via intravenous injection/oral administration of X-PLNPs after in situ X-ray activation. This is the first time that NIR-emitted PLNPs have been demonstrated to be recharged by X-ray light for deep-tissue in vivo bioimaging, which paves the way for in vivo renewable bioimaging using PLNPs and makes the PLNPs more competitive in bioimaging area

<i>In vitro</i> propagation of <i>Paphiopedilum</i> orchids

<p><i>Paphiopedilum</i> is one of the most popular and rare orchid genera. Members of the genus are sold and exhibited as pot plants and cut flowers. Wild populations of <i>Paphiopedilum</i> are under the threat of extinction due to over-collection and loss of suitable habitats. A reduction in their commercial value through large-scale propagation <i>in vitro</i> is an option to reduce pressure from illegal collection, to attempt to meet commercial needs and to re-establish threatened species back into the wild. Although they are commercially propagated <i>via</i> asymbiotic seed germination, <i>Paphiopedilum</i> are considered to be difficult to propagate <i>in vitro</i>, especially by plant regeneration from tissue culture. This review aims to cover the most important aspects and to provide an up-to-date research progress on <i>in vitro</i> propagation of <i>Paphiopedilum</i> and to emphasize the importance of further improving tissue culture protocols for <i>ex vitro</i>-derived explants.</p

Effect of organic amendments on seed germination of <i>Renanthera imschootiana</i> at 150 DAP on 1/4 MS containing 0.5 mg l⁻¹ NAA, 10 g l⁻¹ sucrose and 1.0 g l⁻¹ AC for 75 days in culture.

<p>For each treatment, approximately 300 seeds (in three replicates) were cultured in a 500-ml culture flask containing 90 ml of medium. All experiments consisted of three independent replicates with 10 culture flasks per replicate. Values followed by different letters within a column are significantly different at <i>P</i><0.05 (DMRT). Percentage data were arcsin transformed before being subjected to ANOVA. Each mean is based on microscopic observations. BH, banana homogenate; CW, coconut water; MS, Murashige and Skoog medium; NAA, α-naphthaleneacetic acid; PH, potato homogenate.</p><p>Effect of organic amendments on seed germination of <i>Renanthera imschootiana</i> at 150 DAP on 1/4 MS containing 0.5 mg l⁻¹ NAA, 10 g l⁻¹ sucrose and 1.0 g l⁻¹ AC for 75 days in culture.</p

Effect of NAA and banana homogenate (BH) concentration on <i>Renanthera imschootiana</i> plantlet growth on Hyponex N016 medium with 1.0 g l⁻¹ peptone, 20 g l⁻¹ sucrose, 10% CW and 1.0 g l⁻¹ AC after 60 days in culture.

<p>* +, ++, +++ represents poor, normal, and good growth, respectively (see text for detailed explanation). All experiments consisted of three independent replicates with 10 culture flasks per replicate and 20 PLBs per flask. Values followed by different letters within a column are significantly different at <i>P</i><0.05 (DMRT).</p><p>Effect of NAA and banana homogenate (BH) concentration on <i>Renanthera imschootiana</i> plantlet growth on Hyponex N016 medium with 1.0 g l⁻¹ peptone, 20 g l⁻¹ sucrose, 10% CW and 1.0 g l⁻¹ AC after 60 days in culture.</p

Effect of sucrose concentration on <i>in vitro</i> germination of <i>Renanthera imschootiana</i> on quarter-strength MS medium supplemented with 0.5 mg l⁻¹ NAA, 20% CW and 1.0 g l⁻¹ AC 75 days after culture.

<p>Different letters indicate significant differences between days at <i>P</i><0.05 (DMRT).</p

<i>In vitro</i> seed culture, seedling development and reintroduction of <i>Renanthera imschootiana</i> Rolfe.

<p>(A) Stage 0, seed under scanning electron microscopy, ungerminated. (B) Stage 1, testa ruptured. (C) Stage 2, appearance of rhizoids. (D) Stage 3, emergence and elongation of first leaf. (E) Stage 4, one leaf and root present. (F) Stage 5, presence of two or more leaves. (G) Proliferation of PLBs on quarter-strength MS (1/4 MS) medium supplemented with 1.0 mg l⁻¹ BA, 1.0 mg l⁻¹ NAA, 1.0 g l⁻¹ peptone and 20% CW. (H) Differentiation of PLBs on 1/4 MS medium supplemented with 1.0 mg l⁻¹ NAA, 1.0 g l⁻¹ peptone, 100 g l⁻¹ BH, and 1.0 g l⁻¹ AC. (I) Development of seedlings on Hyponex N016 medium supplemented with 0.5 mg l⁻¹ NAA, 1.0 g l⁻¹ peptone, 150 g l⁻¹ BH, and 1.0 g l⁻¹ AC. (J) Transplanted plantlets 6 months after acclimatization in the greenhouse. (K) Reintroduced flowering plantlets from <i>in vitro</i>-derived seedlings on the trunk of <i>Pinus massoniana</i> on Huolu Mountain, Guangzhou. (L) Reintroduced plantlets from <i>in vitro</i>-derived seedlings at the Orchids Garden, South China Botanical Garden. Bars: (A) 50 µm, (B) 100 µm, (C) 0.05 mm, (D) 0.35 mm, (E) 0.40 mm, (F) 0.60 mm, (G, H) 3.0 cm, (I) 4.0 cm, (J, K) 5.0 cm, (L) 3.0 cm.</p

Seed germination and seedling developmental growth stages of <i>Renanthera imschootiana</i> (modified from Zeng et al., 2012).

<p>Seed germination and seedling developmental growth stages of <i>Renanthera imschootiana</i> (modified from Zeng et al., 2012).</p

Survival of <i>Renanthera imschootiana</i> plantlets grown on different substrates after transplanting for 60 days.

<p>Means ± SE of 300 replicates with the different letters within columns are significantly different at <i>P</i><0.05 (DMRT).</p><p>* Substrate mixture 1: commercial sand for orchids+shattered fir bark (2∶1; v/v).</p><p>** Substrate mixture 2: commercial sand for orchids+sieved peat+shattered fir bark (2∶1∶1; v/v).</p><p>Survival of <i>Renanthera imschootiana</i> plantlets grown on different substrates after transplanting for 60 days.</p

Effect of basal medium supplemented with 0.5 mg l⁻¹ NAA, 10 g l⁻¹ sucrose and 1.0 g l⁻¹ AC on germination and development of <i>Renanthera imschootiana</i> 150 DAP seed after 75 days in culture.

<p>For each treatment, approximately 300 seeds were cultured in a 500-ml culture flask containing 90 ml of medium. All experiments consisted of three independent replicates with 10 culture flasks per replicate. Values followed by different letters within a column are significantly different at <i>P</i><0.05 (DMRT). Percentage data were arcsin transformed before being subjected to ANOVA. Each mean is based on microscopic observations. DAP, days after pollination; KC, Knudson's C medium; MS, Murashige and Skoog medium; NAA, α-naphthaleneacetic acid; RE, Robert Ernst medium; VW, Vacin and Went medium.</p><p>*Lin et al. (2008).</p><p>Effect of basal medium supplemented with 0.5 mg l⁻¹ NAA, 10 g l⁻¹ sucrose and 1.0 g l⁻¹ AC on germination and development of <i>Renanthera imschootiana</i> 150 DAP seed after 75 days in culture.</p

Survival rates of different ages of transplanted <i>Renanthera imschootiana</i> seedlings after 360- or 720-day reintroduction.

<p>Means ± SE of 300 replicates with the different letters are significantly different at <i>P</i><0.05 (DMRT).</p><p>Survival rates of different ages of transplanted <i>Renanthera imschootiana</i> seedlings after 360- or 720-day reintroduction.</p