Research in novel materials has been extremely active over the past few decades, wherein a major area of interest has been nanoparticles with special optical properties. These structures can overcome some of the intrinsic limitations of contrast agents routinely used in medical practice, while offering additional functionalities. Materials that absorb or scatter near infrared light, to which biological tissues are partially transparent, have attracted significant attention and demonstrated their potential in preclinical research. In this review, we provide an at-a-glance overview of the most recent developments in near infrared nanoparticles that could have far-reaching applications in the life sciences. We focus on materials that offer additional functionalities besides diagnosis based on optical contrast: multiple imaging modalities , sensing of physical and chemical cues (multivariate diagnosis), or therapeutic activity (theranostics). Besides presenting relevant case studies for each class of optically active materials, we discuss their design and safety considerations, detailing the potential hurdles that may complicate their clinical translation. While multifunctional nanomaterials have shown promise in preclinical research, the field is still in its infancy; there is plenty of room to maximize its impact in preclinical studies as well as to deliver it to the clinics. This review highlights the very recent examples of near infrared contrast agents employed for multivariate diagnostics, multimodal imaging, and theranostic. Considerations on how to further advance these probes towards real-life use are also given. The strive for even faster, more cost-effective, and less invasive ways to address healthcare problems nonetheless continues, and is being developed at an unprecedented speed when it comes to optical tools.Current clinical diagnostic methods take advantage of several mature imaging modalities – magnetic resonance imaging (MRI), X-ray computer tomography (CT), positron emission tomography (PET), ultrasonography, 1–4 While intrinsic tissue fluorescence in the NIR can provide diagnostic information in some specific scenarios, analogous to MRI or PET techniques, the potential of light-based imaging is maximized when paired with contrast agents, i.e., markers that can absorb (or scatter) NIR wavelengths. The use of contrast agents enables NIR photoluminescence (PL),5 photoacoustic (PA),6 or optical coherence tomography (OCT) imaging.7 Nanotechnology has further allowed to design NIR contrast agents that accommodate multiple imaging modalities at once,8,9 enable multivariate diagnostics – combining image acquisition with qualitative or quantitative measurement of chemical or physical cues,10–12 and provide concurrent therapeutic possibilities (the principle of theranostics) , can be generated with inexpensive, compact, easy to operate sources (lasers or lamps), and can provide real-time imaging at the microscale. The success of optical diagnostics, however, largely depends on an appropriate selection of the irradiation wavelengths. NIR light (“light” here is used as a shorthand for radiation) interacts less with biological tissues than light of shorter wavelengths, and thus can propagate deeper within them. NIR wavelengths which experience reduced absorption and scattering by endogenous components of tissues cover two spectral regions – NIR-I (750–950 nm) and NIR-II (1000–1700 nm).nostics) .13–1516 Despite the potential of multifunctional NPs to revolutionize clinical diagnosis and therapy, turning that into reality requires the development of the most suitable materials and fully ascertaining their effectiveness and safety for use in personalized medicine.NPs that act as multimodal contrast agents for two or more complementary imaging modalities remove the need for multiple contrast agents and allow achieving a more complete diagnostics picture. NPs capable of multivariate diagnostic could similarly reduce the number of diagnostic tests the patients would be subjected to. These NPs would provide clinicians with ample information  that would facilitate accurate diagnosis. Beyond diagnostics, multifunctional theranostic NPs combine disease diagnosis and therapy. However, particular caution has to be taken with theranostic NPs to minimize damage to healthy tissues by timely activating the therapeutic function and limiting it to the diseased tissue.In this review, we discuss the most recent developments in multimodal imaging, multivariate diagnostics, and theranostics using optical NPs, providing the reader with an at-a-glance overview of the most promising materials studied for these applications .We focus on the four classes of materials that have attracted major research efforts over the past few years – Pb- and Cd-based semiconductor nanocrystals (SNCs), Cu- and In-based SNCs, Ag-based SNCs, and rare-earth doped nanoparticles (RENPs), with a final section focused on novel and hybrid systems. For each material, we present recent relevant case studies that we believe have significant implications for the future development of these tools targeted for personalized medicine. We also discuss the potential shortcomings that could limit the applicability of these NPs and outline the attempts that are being made to improve the existing materials, create novel ones, or form hybrid-structures that combine the strengths of their individual components. Finally, we propose a set of practices to be considered when designing, testing, and applying NIR-activated multifunctional NPs, highlighting the need for two-way communication between the professionals of physical and medical sciences.2.17,18 Lead chalcogenide SNCs are particularly relevant for NIR imaging, as their PL emission peak can be easily tuned to span wavelengths from 800 to 1800 nm coinciding with NIR-I or NIR-II spectral ranges.19SNCs based on lead and cadmium are found in element groups II-VI  and IV-VI . This type of SNCs fulfil multiple requirements imposed over luminescent markers to be used for bioimaging, including large extinction coefficients spanning broad spectral ranges, narrow emission lines with high degree of spectral tunability, stability against photobleaching, and even near-unity PL quantum yields (PLQY).2+, Cd2+) in their composition, which confers their unique spectral characteristics, while intrinsic toxicity raises well-warranted concerns about the safety and potential clinical translation of these NPs.20 Nonetheless, this is less of a concern for in vitro and some preclinical studies, where Pb- and Cd-based SCNs can excel. Research into deep-tissue PL imaging,21 targeted NP delivery and pharmacokinetics,22 and real-time monitoring of physiological events,23 have relied on these SNCs. The performance of other types of NPs is also often benchmarked against that of heavy-metal SNCs. Besides, colloidal synthesis methods employed in the preparation of Pb- and Cd-based SCNs have evolved to accommodate SNCs of various compositions, shapes, and sizes, down to a monolayer precision.24 In turn, this facilitates both the versatility and the reproducibility in SNCs manufacturing, which guarantees more predictable behavior in model biological organisms.The distinctive qualifier of these SNCs is the presence of heavy metal ions  experiments showed that the Ag2Se shell over the PbS core of SNCs prevented Pb2+ leaching into the environment, poisoning the solution and decomposing the SNCs, as was the case for core-only PbS SNCs. The Ag2Se shell also shifted the PL of SNCs from ∼1586 nm for core-only PbS to around 1616 nm for PbS/Ag2Se. To selectively image ischemic stroke regions, these SNCs were functionalized so that their emission was turned on in the presence of peroxynitrite (ONOO−), a marker of early onset of an ischemic stroke. The PbS/Ag2Se SNCs were coated with phospholipids functionalized with VHPKQHR peptide and Cy7.5 organic dye  illuminated the entire brain, with no apparent delineation between healthy and injured tissue  with PbS/CdS SNCs .31 Although the latter avoids the need for radioactive agents, it still suffers from photobleaching, shallow imaging depth, and poor imaging contrast. Thus, there is an incentive for better suited lymphoscintigraphy PL contrast agents capable to detect metastasis in sentinel LNs during their intraoperative excision. In their study, the authors imaged primary metastatic tumor and sentinel LNs simultaneously with the help of a custom-synthesized donor–acceptor–donor (D–A–D) infrared fluorescent dye (IR-FD) and PbS/CdS SNCs, respectively  IR-FD dye endowed it with high PLQY (∼6%) and long circulation time in the bloodstream, allowing it to be accumulated and observed in U87MG tumor-bearing mice. The dual imaging of tumors and LNs was tested in mice bearing U87MG and 4T1 cancer models. The IR-FD dye would be administered first to coarsely visualize all tumor sites, primary and metastatic, while subsequently injected PbS/CdS SNCs clearly demarcated the sentinel LNs , as glioma imaging agents (emission peak around 800 nm) , Cd-based SNCs emitting in NIR-I can be used for subcutaneous imaging. Liang  800 nm) .32 Nude y period . Prolongvia non-invasive PL imaging in the NIR.Small size and bright PL in NIR-I and NIR-II of Cd- and Pb-based SNCs make them potent candidates to study NP/drug pharmacokinetics in cerebrospinal fluid of the brain, brain lesion formation, and detection of its early onsite markers, as well as observation of blood perfusion in cerebral vessels 3.i.e., cadmium and lead) in their composition represents a roadblock towards their application. While this aspect is of limited concern for in vitro and preclinical applications, it is unlikely that heavy-metal-containing SNCs will ever be injected in humans.35 Moreover, processes involved in the preparation and disposal of such SNCs might have an impact on the environment and the wildlife. To allay these concerns, heavy-metal-free SNCs are sought-after materials as possible contrast agents for different optical imaging modalities.As discussed in Section 2, Pb- and Cd-based SNCs are the staple as far as the brightness is concerned. However, the presence of intrinsically toxic elements x, InP, and InAs being arguably among the most relevant examples in this context. While In3+ is considered a non-toxic metal ion, Cu+/2+ is to some extent cytotoxic.36 Therefore, it is debatable whether copper-based SNCs could be considered intrinsically biocompatible.37In particular, copper- and indium-based SNCs have been the subject of intense investigation. SNCs made of CuInGiven the different optical properties featured by the above-mentioned SNCs, it is useful to split the discussion into two separate blocks: one dealing with plasmonic SNCs and the other discussing luminescent SNCs.3.1x2−E, where E = S, Se, Te and x = 1–2) SNCs have emerged as an alternative to noble-metal-based plasmonic materials. In Cux2−E SNCs, the localized plasmon resonance (LSPR) arises from the collective motion of vacancies rather than electrons.38 These vacancies are localized at the top of the valence band, and they originate from the defective stoichiometry displayed by some of the compositions. For example, Cu2S  is a fully stoichiometric material and does not generally display LSPR, but rather NIR-II PL.39 On the contrary, CuS  showcases strong NIR-centered LSPR. Although the absorption cross section of these materials is smaller than the one displayed by metallic plasmonic NPs based on, e.g., gold or silver (10−14 to 10−13 cm2vs. 10−11 to 10−9 cm2), they have the unique advantage of featuring LSPR peaks across NIR-I and NIR-II even at sizes as small as few nanometers. This characteristic makes them attractive contrast agents for NIR-based imaging techniques, as well as light-to-heat converters for photothermal therapy (PTT).40Copper chalcogenide  coating , the SNCs featured emission centered at 700 nm and were preferentially taken up by cancer cells, wherein H2O2 is present at higher concentration than in healthy cells. The authors went on to prove that Cu2S:Zn2+ SNCs produce OH radicals in the presence of H2O2, justifying the cytotoxic effect preferentially induced in tumor cells. This chemodynamic therapeutic effect was later employed in vivo to suppress the tumor growth in Balb/c mice with a 56% inhibition rate compared to a control group treated with saline.As mentioned earlier, Cu2. Their synthesis has been developed relatively recently, hence there is ample room for improvement in terms of control of their morphology, core/shell architecture,45 and size dispersion. The latter is an issue when aiming for biological applications due to the different fate experienced in the body by NPs of different sizes.46 Controlling the synthesis of ternary CuIn2 SNCs requires great care, since the reactivity of Cu+ and In3+ is different, and thus fine-tuning of the reaction mixture composition is necessary. The emission of CuIn2 SNCs is within the visible or NIR spectral range (750–1300 nm),47–50 and arises from carrier recombination events that involve copper-related interbandgap states and strong electron-phonon coupling.51 The non-excitonic nature of the emission translates to a large Stokes-shift and PL lifetimes up to several hundreds of nanoseconds – 1–2 orders of magnitude longer than the excitonic emission typical of PbS and CdSe SNCs. This extended lifetime was harnessed by Pons et al. to perform in vivo imaging of single cells in the bloodstream. The authors prepared alloyed Cu–Zn–In–Se SNCs shelled with ZnS and modified their surface with poly(imidazole-b-sulfobetaine) block polymer.47 The SNCs boasted an 800 nm emission under 660 nm excitation, PLQY as high as 30% in water, and lifetime on the order of 150–300 ns. After labelling rat red blood cells (RBCs) with SNCs via pinocytosis and electroporation, the cells were re-injected into the animal. The authors microscopically imaged single RBCs in small vessels (few micrometers in diameter) with slow blood flow. A20 lymphoma cells – a type of circulating tumor cells (CTCs) – were also labelled and imaged in larger blood vessels after injection into the animal, enabling the estimation of their concentration in the bloodstream as well as the local blood flow. A similar 800 nm emission was featured by the CuInS2/ZnS SNCs prepared by Huang et al., who pushed the emission of these SNCs towards the NIR by using InI3 as the indium source instead of the more commonly employed In-acetate.52 Upon growing the ZnS shell, partial etching of the CuInS2 core was observed , resulting in an overall blue-shift of the emission in hexane (from 933 to 813 nm) and PLQY of 94.8% of 94.8% . These Sof 94.8% . Upon ilof 94.8% . Very re2-based SNCs is the difficulty to push their emission beyond 1300 nm.48 On the contrary, this could be accomplished with In-based binary SNCs containing pnicogens , whose emission can span the whole 500–1600 nm range.55–58 The chemistry of these SNCs is yet less explored than the one of CuIn2 SNCs and their synthesis usually involves long reaction times and toxic, oxygen-sensitive precursors such as pyrophoric tris(trimethylsilyl/germanyl) arsine or metal hydrides.56 The Bawendi group proposed an alternative and safer, redox-based process where In+ precursors are used along with As-amines. Unfortunately, the reported PLQY of so-produced InAs SNCs is below 1%, overcoming 12% only after the growth of a CdSe shell. Clearly, there is plenty of room for improvement in the synthesis processes of In-based SNCs.Despite these results, an important limitation of CuIn59 similarly to what has been reported for CuIn2 SNCs. Strategies to increase the brightness of these SNCs include the growth of several shells of controlled composition,57,60,61 and treatment with HF to passivate In3+-related surface trap states.62,63 Doping with Cu+ was also proposed to tune the emission of InP SNCs towards the NIR, harnessing the energy transfer from the semiconductor matrix to the transition metal.64,65The emission mechanism in III–V In–SNCs remains a matter of investigation. Electron-phonon coupling seems to play a relevant role in determining the broadband emission spectrum, along with carrier recombination events involving trap states,et al. performed multiplexed in vivo imaging in NIR-I using ZnSe/InP/ZnS SNCs  SNCs have recently came to light in bio-oriented nanotechnology research as a direct response to Cd- and Pb-based SNCs, meeting many of the pre-requisites of deep-tissue PL imaging in the NIR.66 Despite being Cd2+ and Pb2+ free, Ag+ can be cytotoxic, yet low solubility of Ag2S ensures negligible release of free Ag+ into the biological milieu and reduces concerns related to the silver poisoning . In its bulk form, Ag2S has a direct band gap of 1 eV, thus having large extinction coefficient for wavelengths greater than 850 nm, relevant for NIR excitation.67 Furthermore, Ag2S emission usually peaks around 1200 nm, and due to the small Bohr exciton radius (∼2 nm) of the material, Ag2S SNCs larger than 4.5 nm have their PL band centered in the NIR-II. Despite certain difficulties associated with the synthesis of high-purity and high-PLQY Ag2S SNCs,68,69 the PL band is practically insensitive to the dimensions of these SNCs and guarantees NIR-II emission for nanocrystals >4 nm in diameter. However, the synthetic route employed in the preparation of Ag2S SNCs seems to have a large effect on their spectral tunability.68 Similarly to Ag2S, Ag2Se SNCs can be made to exhibit PL in the 1000–1600 nm range, and are also potent NIR imaging contrast agents.Silver chalcogenide  with emission around 1350 nm were coated with PEGylated phospholipids, heparin, and stromal-cell-derived factor-1α (SDF-1α), and loaded with chemotherapeutic drug doxorubicin (DOX). Meanwhile, TAT – cell-penetrating peptide – grafted Ag2S SNCs  were preloaded into natural killer cells (NK-92) destined for adaptive cellular immunotherapy. The premise of this therapeutic approach relied on the initial accumulation of S&D@Ag2Se in the tumor, where under low pH the DOX could be released; at the same time, SDF-1α would act as a beacon71 – selectively guiding the NK-92 cells towards the tumor site to induce cancer cell death , DOX, and a small molecular NIR absorber A1094.73 Er3+ bait-ions were introduced to cross-link histidine imidazole groups in Fmoc-His and carboxyl groups in the MPA-Ag2S SNCs, while DOX and A1094 facilitated the multi-component self-assembly by a number of intramolecular interactions. As prepared, this theranostic system is non-photoluminescent, due to Ag2S PL quenching by A1094, but at low pH conditions the system disassembles prompting rapid PL response from Ag2S SNCs and DOX release. The importance of site-specific PL turn ON systems lies in their ability to only image the area of interest, increasing the signal-to-background ratio and accurately delineating the diseased tissue. In the work by Li et al. response to ONOO− was used to bleach the A1094 conjugated on the surface of Ag2S SNCs together with VCAM1 binding peptide.74 In the presence of ONOO−, found at sites of traumatic brain injury, the PL quenching of Ag2S by A1094 would cease, allowing high contrast NIR-II PL imaging of the injured area.In a similar vein, Ling 2Se SNCs coupled with gadopentetic acid (Gd-DTPA), a clinically approved MRI contrast agent, could be used for PL as well as MR imaging,75 whereas Cu+-doped Ag2S SNCs can achieve NIR PL and PA imaging, as well as PTT.76 The presence of Cu+ in Agx2−CuxS SNCs induced a bathochromic shift of the emission peak from 765 to around 820 nm and granted the SNCs a light-to-heat conversion efficiency up to 44%, successfully utilized by both PA imaging and PTT.Furthermore, Ag-based SNCs serve as NIR PL contrast agents in multimodal imaging systems. For instance, Ag2S SNCs can be used to measure local temperature non-invasively and conduct multivariate diagnostics. Santos et al. showed that NIR-II PL of Ag2S SNCs can be used not only for tumor visualization, but also to more accurately discriminate between healthy and malignant tissues by means of PL thermometry  as dopants . Fourth, their emissions present narrow bandwidths, reducing the possibility of channel crosstalk during spectral multiplexing. Finally, their luminescent properties stand out among other PL materials due to their high resistance to photoblinking and photobleaching. Besides emission in the NIR, rationally designed RENPs allow for upconversion PL – emission of photons of higher energy than initially absorbed ones. Nevertheless, this comes at a cost in the form of low radiative transition probabilities, yielding poor Ln3+ molar extinction coefficients on the order of 0.1–10 M−1 cm−1.85 Although different strategies aimed to improve the absorption (that translates to brightness) of RENPs have been developed, it still represents an important bottleneck for these nanomaterials.85,86RENPs bring together highly desired optical properties for NIR bioimaging and multivariate diagnostics. First, they feature very long-lived emissions, in the range of μs to ms (in contrast to the ns- or μs-long PL associated with the best performing SNCs), which easily allow for time-gated imaging and lifetime barcoding.3+ within the crystalline structure of a proper dielectric host material which has three main fundamental roles: (i) crystal structure asymmetry and crystal field can be used to increase the probability of the 4f–4f transitions; (ii) low cutoff phonon energies reduce non-radiative quenching of the excited states, and (iii) acting as a platform where different sensitizers and activators can be combined to achieve specific luminescent features.87–89Generally, RENPs are the result of embedding Ln3+ precursors at high temperatures. This synthetic strategy is closely followed by the thermal co-precipitation, which is associated with relatively non-toxic precursors and milder conditions, although it involves a larger number of synthetic steps. These methods offer high synthetic control, allowing tunability of size, shape and nano-architecture, size monodispersity, and high crystallinity of the resulting RENPs.90,91RENPs can be produced by multiple approaches, which can be generally split into syntheses performed in organic solvents or aqueous media. Thermal decomposition is a common method to produce RENPs due to its relative simplicity, consisting of the decomposition of organo-metallic Lnin vivo bioimaging, but in the number of those that explore their PL properties for novel applications in the fields of drug delivery, multivariate diagnostics, and theranostics.92–95 Zhong et al. combined RENPs and PbS SNCs in a multiplexed diagnostic strategy, that also offered therapeutic properties, due to their functionalization with immunomodulating antibodies , where autofluorescence is negligible and the light penetration depth into tissues is maximized. Alpha-phase NaYbF4: 2 mol% Er3+, 2 mol% Ce3+, 10 mol% Zn2+/NaYF4 RENPs showcased an absolute PLQY of ∼5% and an ∼11-fold increase in their emission at 1550 nm over previous beta-phase compositions.97 The subsequent surface functionalization of the RENPs with hydrophilic and cross-linked coating layers provided water dispersibility and biocompatibility, and resulted in a biliary excretion of up to 90% of the injected RENPs in a two week period. Further surface modification allowed to attach anti-PD-L1 mAb (atezolizumab) to the RENPs for the specific targeting of CT-26 colon cancer cells, which over-express PD-L1 ligand, responsible for the evasion of the immune system. The remarkable long PL lifetime exhibited by these RENPs (4.3 ms) allowed for easy in vivo multiplexed imaging when combined with PbS SNCs (emission ∼1600 nm). PbS SNCs exhibited a lifetime of 46 μs upon excitation at 980 nm  system working in NIR-II that combined Nd3+-doped core/shell RENPs as FRET donors and a cyanine dye (MY-1057) grafted to their surface as an energy acceptor  radiative transition. Yet, the ONOO− dependent degradation of MY-1057 resulted in diminished FRET and consequently in an increase in the lifetime of the 1060 nm emission  or are result-products of it, such as peroxynitrite, hypochlorous acid, or hydrogen peroxide.93,98–101 As a consequence, their oxidative properties are used to chemically modify the FRET acceptor, usually a cyanine dye that acts by quenching one of the RENPs emission bands. Upon oxidation, the optical properties  of the FRET acceptor change and the otherwise quenched emission band from RENPs is restored. This strategy evidences that the oxidation of the FRET acceptor implies the non-reversibility of the sensing process. In this regard, the design of reversible probes for in vivo multivariate diagnosis and an expansive library of potentially detectable analytes represent exciting research opportunities.Similar 103 Temperature-dependent alteration of non-radiative relaxations within specific Ln3+ excited states results in a change of the RENPs PL intensity and the excited state lifetime. This lifetime temperature dependence enables measuring temperature more reliably at different tissue depths and obtaining 2D thermal images, as shown by the work of Tan et al.  after the excitation is ceased, so they could be optically excited before being injected into an animal, eliminating side effects associated with the continuous irradiation of the subject and interference of the excitation light and autofluorescence in PL images. The most studied persistent PL perovskites for in vivo applications to date are Cr3+-activated phosphors emitting in the NIR-I. Additional or alternative doping with Sr3+ or Ln3+ ions could enhance the persistence of the luminescence and shift it to reach NIR-II.119,120 Pellerin et al., showed that co-doping of LaAlO3:Cr3+ nanoperovskites with Sm3+ can improve their persistent radiance 35-fold after ceasing ultraviolet charging.121 After PEGylation, these NPs presented increased circulation time in blood vessels and could be used for NIR-I bioimaging of live mice. Although promising, persistent PL bioimaging presents some drawbacks, such as the impossibility of recharging the probes since that are excited with ultraviolet and visible light. As a first step to overcome this limitation, Hang et al. developed persistent Cr3+-activated Na0.5Gd0.5TiO3 perovskite-like NPs. These NPs could be excited at the lower bound of the NIR-I for in situ recharging with low-power incoherent light, solving the problem of fast decay that hampers the use of persistent NPs as continuous PL contrast agents.122 In a similar direction, a recent study demonstrated the use of RENPs as persistent luminescence NIR contrast agents upon X-ray charging.123Other major example of alternative NIR agents are perovskites. They are known for their successful application in photovoltaics and recently have attracted attention in other research fields, such as 3+ 124–126 could lead to the development of NPs with combined functionalities to overcome the limitations of the individual materials. For example, despite the outstanding properties of RENPs (see Section 5), they present low absorption. This drawback can be minimized combining them with more absorbing NPs, such as Ag2S SNCs, which can act as sensitizers to enhance the emission of the RENPs through an energy transfer process. This type of system has been used to detect the existence of tumors in vivo when the two types of NPs are crosslinked via glutathione, which is highly abundant in tumor microenvironments.127 Other examples of energy transfer-based sensors use RENP as donors to create nanocomposites in combination with SNCs for tumor detection. Chan et al. detected the disassembly of the RENPs-SNCs hybrid structure  at the tumor site in a mouse model, produced by the digestion of the link between the two PL agents by the matrix metalloproteinases (MMP2), highly expressed at the tumoral site . This change in the emission signal was used to detect the presence of glutathione in human serum samples.Energy-transfer-based sensors can suffer from low energy transfer efficiency, low photostability, complex preparation methods and quenching of the donor emission due to the action of the acceptor, all of which should be considered when choosing the building blocks of the hybrid NPs. This latter drawback, nonetheless, can be converted into an asset for the development of a sensing platform. Shu in situ growth methods present some advantages compared to other methods used to combine different NPs, such as simplicity and mild reaction conditions, and the possibility of obtaining more uniform and stable nanoplatforms. As an example, Ag2S SNCs can be grown in situ on the surface of chitosan-coated NaYF4:Yb3+, Er3+/NaLuF4:Nd3+, Yb3+/NaLuF4 RENPs .132 After PEGylation and modification with c(RGDfC) peptide, the dual imaging capabilities  of the nanocomposite was demonstrated both in vitro and in vivo. Specific recognition of HeLa and MCF-7 tumor cells was observed via both CT and PL imaging with the peptide-modified nanocomposite. Similarly, after injection of the nanocomposite in a nude mouse bearing a HeLa transplanted tumor, the malignant tissue started to become visible via both imaging approaches 6 days after injection, indicating selective nanocomposite accumulation. Another relevant example of in situ growth of hybrid nanoprobes is provided by H. Ning et al., who developed KMnF3:Yb3+, Er3+/ZnS NPs that combine upconversion emission of RENPs with downshifting luminescence of SNCs.133 At the same time, the ZnS shell provided shielding for Ln3+ from external quenchers, thus boosting upconversion emission intensity.Regarding the synthetic process of hybrid systems, F4 RENPs .130 In ttruction . As anotet al., created a hybrid nanostructure formed by DNA-functionalized Ag2S linked to Fe3O4 magnetic NPs that were wrapped in a tumor and white blood cell hybrid membrane.134 These membranes increased the affinity for the cancerous cells and reduced the interaction with the white blood cells to minimize interference. High-specificity binding of the hybrid NPs to CTCs in peripheral blood samples from cancer patients was observed. The magnetic properties of the Fe3O4 NPs were used to efficiently isolate the cells using a magnetic field and the NIR emission of the Ag2S was used for high-sensitivity detection and quantification of CTCs. In addition, Ag2S SNCs and Fe3O4 magnetic NPs have also been combined by polymeric encapsulation for PL thermometry controlled magnetic hyperthermia.135 In this case, the temperature increase produced by the Fe3O4 NPs when subjected to an alternating magnetic field was monitored using the temperature-dependent PL of the Ag2S NPs. Thanks to the combined properties of their building blocks, the hybrid NPs were also exemplary multimodal contrast agents for MRI, PA and NIR-II PL imaging, as well as OCT and CT.Other strategies of hybrid NP preparation make use of cell membranes to wrap the individual entities together, which endows hybrid structures with additional functionalities and biocompatibility. Ding 29,136 Alternative silica nanocapsule strategies enable combination of theranostics and multimodal imaging approaches in a straightforward manner,137 while classic mesoporous silica NPs can be loaded with anticancer drugs to deliver them into the tumor region. Song et al. designed Ag2S SNCs coated with mesoporous silica and grafted with graphene oxide on its surface. The mesoporous silica was also loaded with DOX, and additionally coated with FA for tumor targeting.138 The heat generated by Ag2S NPs after 808 nm irradiation was used to perform the PTT and to trigger the release of the anticancer drug by the detachment of the graphene oxide coating due to the temperature increase. The combination of both chemotherapy and PTT increased the efficacy of the treatment and only mice treated with the hybrid NPs showed no tumor recurrence.Finally, hybrid NPs allow the combination of multiple therapies using a single agent for a greater treatment success, such as chemotherapy and PTT.In summary, judicious selection and combination of already established materials and novel ones in a single platform enables multimodal imaging and theranostics that could overcome the existing limitations of their individual components.7.This review evidences that the research on novel materials capable of multimodal imaging, multivariate diagnostics, and theranostics is a highly active and promising area. This is possible thanks to the improved communication between researchers in the materials and life sciences. Yet, to translate these nanomaterials from preclinical research to the clinics will necessitate further, extensive collaboration between multiple disciplines. At this stage, the translation of these cutting-edge materials to the clinics remains out of reach and more work needs to be done to demonstrate their viability in the clinical settings.e.g. the lack of ionizing radiation) are insufficient. This is exacerbated by the availability of true and tested traditional technologies (such as CT or MRI). Unlike these traditional technologies, however, new nanomaterials provide qualitative as well as quantitative information about physiological parameters of relevance, such as temperature or presence of chemical species in the environment that indicate disease. Further, nanomaterials can provide a therapeutic stimulus concurrent to diagnosis, resulting in targeted disease therapy with reduced side effects. In this review, we show there is substantial evidence for the potential and effectiveness of multivariate diagnostics (imaging and sensing) and theranostics (imaging and therapy) by NIR-active NPs and nanostructures. It is time to share these results with the clinicians, a significant number of whom will be undoubtedly surprised by the copious research on nanomaterials and will be interested in their use for imaging and sensing. Notwithstanding, the bio and medical communities are increasingly studying the interaction of the NPs with cells, tissues, and biological models; specifically, how the type, state, or physiology of the living organism can influence the performance of the NPs.139Usual arguments to interest clinicians in these optical materials  that would be considered as golden rules to elucidate how the organism reacts to the administration of NIR-emitting NPs. Despite recent attempts to standardize reporting in bio-nano experimental literature, there has been little uptake of these practices by the materials science community.140,141Nonetheless, even for the most interested clinicians, two looming questions prevail: “are these nanomaterials toxic?”; and “how are they cleared from the body following administration and operation?”. Here, our answers are not so convincing. Results included in this review reveal that materials science researchers are also worried about these questions, but are more focused on the improvement of nanomaterials synthesis procedures and on the design of in vivo? An ethical argument should also be made to reduce animal testing for the sake of routine proof-of-concept demonstrations, which often bring little scientific value if any at all. It is our believe and hope that more humane technologies, such as 3D cell cultures, lab grown tissues, and organs-on-a-chip, will become staple approaches to investigate nanomaterials at primary and pre-clinical stages of their development.Furthermore, materials are continuously being improved and pushed forward in consecutive steps, which demonstrates an amazing body of innovation and interest in this research area. Nonetheless, must every iteration of each nanomaterial be tested 2S SNCs are not yet considered robust clinical probes because of the lack of reproducibility in their optical properties. These depend strongly on both the synthesis and the storage conditions, resulting in a large inter- and intra-batch variability. This is also the case for some of the RENPs discussed in this review. Again, the key point here is the standardization, together with the publication of works with meticulous description of synthesis procedures. This is, without a doubt, a required step towards commercialization of NIR-emitting NPs for biomedical applications. Notwithstanding, the development of cost-effective and green synthesis procedures is highly desirable.Standardization is also lacking regarding the optical properties of most of the nanomaterials discussed in this review. Development and improvement of nanomaterials is an ever-on-going process and synthesis technologies that ensure good batch-to-batch reproducibility and high-quality NPs are not always straightforward to create. As a representative example, Agi.e. similar responses to changes in different environmental parameters) is hampering their development. To address this problem, new systems with sensitivity to a single parameter exclusively are in demand. This requires a complete knowledge of the mechanisms governing the light–matter interactions in these nanostructures so that the response to different external stimuli can be decoupled. Here, standardization is once again crucial. Further development of the field of NP-based biosensing requires the widespread acceptance by the scientific community of standards for the characterization of sensing units. Such standards will focus on the elucidation of possible bias and, in addition, will describe in detail the measuring conditions for a characterization to be accepted by the scientific community.It has been observed along this review that adding the possibility of sensing to imaging makes NPs especially suitable for advanced diagnostics. However, this is an emerging field and sensors' susceptibility to cross-talk (Despite the required work in standardizing synthesis methods, characterization techniques, and toxicity and biocompatibility assays, the works highlighted in this review showcase the fast development of NPs intended for biomedical use. This is the best evidence we have for the interest of a broad scientific community in the development of multifunctional NPs for multivariate diagnostics. This review also evidences the great potential of NIR-emitting NPs for early diagnosis and efficient treatment of different diseases, and highlights the high degree of multidisciplinary efforts necessary to spark the interest of a broader scientific community towards a unified goal. It is time to think even harder from the materials science standpoint about how to develop the required standards while designing nanomaterials with carefully tailored properties to meet a specific purpose.The authors declare that there are no conflicts of interest.


This study aimed to (1) explore the configuration of vocational identity status among emerging adults with and without hearing impairment using latent profile analysis, and (2) investigate the relationships between vocational identity status and self-esteem and subjective well-being. In total, 408 students without disabilities and 432 with hearing impairments from two Chinese higher institutions participated in the study. The Vocational Identity Status Assessment, Rosenberg Self-Esteem scale, Satisfaction with Life Scale, and Positive and Negative Affect were used to assess the major variables. The results derived five latent profiles  of vocational identity in the present sample. The students were over-represented in undifferentiated profiles and under-represented in achieved and foreclosed ones. Hearing impairment significantly affected vocational identity status profile membership. The results showed that emerging adults with achievement and foreclosure statuses displayed healthy psychological outcomes, having the highest self-esteem, life satisfaction, and positive affect, and the lowest negative affect. In contrast, the diffused group showed the most disturbing pattern with the lowest self-esteem, life satisfaction, and positive affect, and the highest negative affect. The research findings reveal some notable issues in vocational identity status for emerging Chinese adults, raising concerns about the influence of hearing impairment on vocational identity formation, and provide implications for Chinese society to facilitate college students’ career development process to promote their vocational identity status and enhance their self-esteem and subjective well-being. Vocational identity formation plays important roles in adolescents’ and young adults’ academic ,2, psychVocational identity has gained tremendous popularity in the last few decades, with most works examining the theoretical models, configurations, and associations of vocational identity. Existing studies across countries show discrepant results on the types and distributions of vocational identity statuses among adolescents and college students ,14,15. ABased on the above background, the present study explores the configuration of vocational identity status among Chinese emerging adults with and without hearing impairment, using latent profile analysis. Then, it investigates the influence of hearing impairment on vocational identity status and the effects of vocational identity status on life outcomes, including self-esteem and subjective well-being. The results enrich the knowledge base on vocational identity status and deepen understanding of the contributions of vocational identity to adjustment in emerging adults in China.The theoretical model for vocational identity is rooted in general ego identity models. Marcia’s paradigm, a traditional identity model, suggests that identity has two dimensions—exploration and commitment —that groBuilding on established conceptual identity status models, including Marcia’s paradigm , Luyckx’Before Porfeli’s conceptual framework of vocational identity, Holland’s My Vocational Status was the most widely used assessment for assessing vocational identity as a unitary construct, emphasising career choice clarity and feelings instead of a stable awareness of vocational goals and interests, and overlooking the exploration process of identity formation ,28,29,30Studies using VISA identified six vocational identity statuses—achievement, moratorium, foreclosure, searching moratorium, diffusion, and undifferentiation—among US high school and university students ,26 and KSome inconsistencies might be due to social, economic, and cultural differences; for example, research has shown that nationality is a significant discriminator of identity statuses ,33. TherThe influence of disability on identity development has long been discussed ,35. It iForty years ago, it was reported that high school students, college students, and workers with disabilities had lower levels of vocational identity than their counterparts without disabilities . A searcThe lack of investigation of individuals with hearing impairment’s vocational identity leads to an incomplete understanding of the association between hearing impairment and vocational identity status during emerging adulthood. Considering the significance of vocational identity in youth career development, discussed in the next section, this study endeavours to unravel the vocational identity status of emerging adults with hearing impairment by comparing them to their hearing peers, using the updated vocational identity conceptual framework. Over the past two decades, numerous studies have explored outcomes associated with vocational identity, with associations involving career-related outcomes ,44,45,46The present study further examines vocational identity statuses’ association with life outcomes, including self-esteem and subjective well-being, given the research gaps at the domestic and international levels. At the domestic level, existing studies mainly focus on the relationships between career-related variables with vocational identity among Chinese secondary, technical college, and elite university students ,8,16,18.Self-esteem and subjective well-being are among the psychosocial associations of vocational identity that have attracted great attention outside China. However, the worldwide research on the associations between self-esteem and subjective well-being with vocational identity is inadequate. Self-esteem refers to individuals’ subjective evaluations of the degree to which they hold attitudes of acceptance or rejection towards themselves . ProlifeAccording to Diener and colleagues, subjective well-being consists of both cognitive  and affective  components . ExistinThis study’s objectives are threefold. The first is to identify the vocational identity statuses of emerging adults with and without hearing impairment under Porfeli’s framework, using latent profile analysis. The second objective is to examine the influence of hearing impairment on the vocational identity process and status membership. As prior studies have revealed that gender, age, socioeconomic background, and institution type may affect identity formation ,33,64,65A purposive sampling approach was applied in the current study, with 840 participating students recruited from two institutions in China—a technical college and a university—representing the two types of colleges admitting students with hearing impairment in the country under the Separate Examination Separate Admission system . SESA is a college enrollment policy approved by the Ministry of Education in China, under which students with disabilities  are admitted based on separate examinations organised by individual colleges and universities instead of the National College Entrance Examination. Since its introduction in 1987, SESA has become the primary means for students with hearing or visual impairment to enter college. Several specific majors are available for these students to apply through SESA. The main majors available for SESA students with hearing impairment include art and design, computer science, and special education. Although students with disabilities could also take the National College Entrance Examination, it is considered too competitive; SESA has a high admission rate and offers more support for students once they enter college. In 2017, 28 colleges and universities admitted students with disabilities via SESA  and studThe sample in the present study consisted of 408 students without disabilities and 432 students with hearing impairment. The participants’ demographic information is shown in 2(382) = 1058.945, p < 0.001, CFI = 0.925, TLI = 0.915, RMSEA = 0.046 , SRMR = 0.052.The VISA–Chinese Version  was used2(4) = 14.772, p < 0.01, CFI = 0.993, TLI = 0.983, RMSEA = 0.057 , SRMR = 0.018. The PANAS’s two-factor model was also supported: x2(166) = 561.295, p < 0.001, CFI = 0.932, TLI = 0.922, RMSEA = 0.053 , SRMR = 0.046.The Satisfaction with Life Scale (SWLS)  and the 2(29) = 182.609, p < 0.001, CFI = 0.957, TLI = 0.933, RMSEA = 0.079 , SRMR = 0.058.Global self-esteem was measured using the Rosenberg Self-Esteem Scale . This scData were analysed using SPSS 21.0 and Mplus 8.0. Preliminary data cleaning, descriptive analysis, and reliability analysis were performed using SPSS 23. The following measurement invariance test, latent profile analysis (LPA), and regression mixture modelling (RMM) analysis were conducted in Mplus V.8.VISA measurement invariance was tested using a multi-group confirmatory factor analysis (MGCFA) and alignment method, in order to make a trustworthy comparison between students with and without hearing impairment regarding their vocational identity status profiles. As mentioned above, existing person-centred studies on vocational identity have widely used the cluster analytic approach; however, the approach has been criticised for lacking statistically objective criteria in selecting cluster solutions . The LPAThe BCH method was used in mixture models to examine how covariates predicted latent profiles and how latent profile membership affected the distal outcome . CovariaMeans, standard deviations, and correlations of the research variables are shown in MGCFA was conducted at first to test the measurement invariance of VISA across the two groups. The configural measurement invariance model showed an acceptable model fit ; the metric invariance model was also supported , with ΔCFI, ΔTLI, ΔRMSEA, and ΔSRMR less than 0.01 compared to the configural model. However, the scalar invariance model was rejected . An alignment analysis was further conducted to determine the non-invariant parameters. The estimates were achieved by the FIXED alignment model, taking the hearing group as the reference. The alignment analysis confirmed that all 30 items’ factor loadings were invariant in the two groups, indicating that metric invariance was satisfied. Partial scalar invariance was established, with three items’ intercepts being non-invariant  of sample participants; as such, it was too small to be included [p-value of the LMR test indicated that the model fit did not improve significantly from the four-profile model to the five-profile model. However, the five-profile model had lower AIC, CAIC, BIC and SABIC, and significant BLRT values. Its smallest profile proportion was about 5%, and the five profiles were all theoretically justifiable and conceptually meaningful. Therefore, the five-profile model was retained as the optimal solution in the present study. The fit statistics of the six models are presented in included , and theThe conceptual fit of the latent profile models was examined by plotting the mean z-standardised scores of the six vocational identity dimensions for each latent profile. As can be inferred from The distribution of the five vocational identity statuses in students with and without hearing impairment is presented in p < 0.01) and searching moratorium (OR = exp(0.32) = 1.38, p < 0.05) than in the undifferentiated one, meaning students whose mother held a higher educational degree were more likely to be in the searching moratorium and achievement profiles than in the undifferentiated profile. In addition, hearing impairment had a significant positive effect on the odds of being in the diffusion (OR = exp(0.58) = 1.79, p < 0.001), undifferentiated (OR = exp(0.56) =1.75, p < 0.001), and searching moratorium (OR = exp(0.66) = 1.93, p < 0.001) profiles than in the achievement profile. No other significant effects were found.The effects of covariates—including institution, age, gender, mother’s educational attainment, and hearing impairment—on latent profile membership were examined using the BCH method. The results showed that only the mother’s educational attainment and hearing impairment significantly affected vocational identity profile membership. Specifically, as shown in The effects of vocational identity profile membership on distal outcomes, including self-esteem, positive affect, negative affect, and life satisfaction, were examined using the BCH method. The results see  showed tThe present study examined (1) vocational identity profile among a sample of Chinese emerging-adult college students with and without hearing impairment, (2) the influence of hearing impairment on vocational identity status, and (3) the effects of vocational identity status profiles on self-esteem and subjective well-being. The results derived five latent profiles  of vocational identity in the present sample. The students were over-represented in undifferentiated profiles and under-represented in the achieved and foreclosed ones. Hearing impairment significantly affected vocational identity status profile membership. The results showed that emerging adults with achievement and foreclosure statuses displayed healthy psychological outcomes, having the highest self-esteem, life satisfaction, and positive affect, and the lowest negative affect. In contrast, the diffused group showed the most disturbing pattern, with the lowest self-esteem, life satisfaction, and positive affect, and the highest negative affect. Using the six vocational identity factors, the present study derived five vocational identity profiles in emerging adults with and without hearing impairment. While the profiles were generally similar to the clusters resolved in previous studies conducted among North American ,26, EuroThe low percentage of foreclosed students in the present sample contrasts the viewpoint that foreclosure status is common in a collectivist society ,78, as iThis generation, called the Buddhist-style generation, features low levels of desire . RecentlThe results highlight the significant negative influence associated with hearing impairment in light of vocational identity. Specifically, the results indicate that students with hearing impairment were more likely to fall into diffusion, undifferentiated, and searching moratorium statuses instead of achievement, echoing the view that adolescents with hearing impairment are less mature in career development than their hearing counterparts ,84,85. SThe five identity statuses were externally validated by examining their associations with self-esteem and subjective well-being. The results showed that emerging adults in the achievement and foreclosure statuses displayed positive psychological outcomes, having the highest self-esteem, life satisfaction, and positive affect, and the lowest negative affect. In contrast, the diffused group demonstrated the most disturbing pattern, with the lowest self-esteem, life satisfaction, and positive affect, and the highest negative affect. This agrees with prior studies’ findings that adolescents in achievement and foreclosure identity statuses reported equally few psychosocial problems, while high well-being and diffusion status showed the least favourable psychosocial and well-being outcomes ,22,26,86This study significantly contributes to the existing research in the following four aspects. First, it initiates efforts to explore the vocational identity of Chinese college students using a person-centred approach, deriving a five-profile solution for vocational identity statuses. This is the first study to examine identity statuses based on VISA using LPA, as extant studies classifying college students’ vocational identity adopted cluster analysis. By using LPA, this study provides more robust empirical evidence for classifying vocational identity statuses. The disparities between the present study and prior studies in other countries raise concerns about the influence of data analysis techniques on the results and cultural differences in emerging adults’ vocational identity statuses. Second, the study examined the influence of hearing impairment on vocational identity, adding new knowledge for understanding the difference in vocational identity formation between students with hearing impairment and hearing students in the Chinese context. Third, as the first study to validate VISA among students with hearing impairment, its measurement invariance tests showed evidence of VISA being interpreted similarly across students with and without hearing impairment in China. Fourth, the results regarding vocational identity status’ associations with self-esteem and subjective well-being confirmed that youths with achievement and foreclosure profiles are at the advanced statuses, those with undifferentiated profiles are in the moderate zone, those with diffused profiles are in undesirable conditions, and those with searching moratorium profiles are in a mixed state.The current study reveals some notable issues in vocational identity status for Chinese emerging adults. However, limitations must be acknowledged. Firstly, this is a cross-sectional study, and the findings are only exploratory. Vocational identity status’ associations with hearing impairment, self-esteem, and subjective well-being are relational rather than causal. Moreover, the potential sample bias inherent in purposive sampling may limit the generalisability of the results. For balance, the present study recruited participants without hearing impairment who were attending the same colleges as students with hearing impairment. Colleges admitting students with hearing impairment in China through the SESA policy are relatively low-ranking in China, and their students are often more disadvantaged in future job markets than those from elite universities. Thus, they may not represent all Chinese higher education students, especially those in more prestigious universities. Despite its limitations, this study has important implications for future practice and research. Specifically, the Chinese government, colleges, and individuals with hearing impairment are encouraged to make changes to address the challenges that college students usually encounter in advancing vocational identity, self-esteem and subjective well-being. For example, more favourable policies should be developed to facilitate college students’ career development, and thus, promote their vocational identity status and enhance their self-esteem and subjective well-being. In addition, college career-counselling services are urged to develop effective programs to improve students’ vocational identity by providing more career exploration workshops and opportunities to facilitate students with hearing impairment’s vocational identity construction. These services should take young people’s changing values into account. It is even more urgent that special attention be paid to college students with hearing impairment’s vocational identity development. Moreover, students with hearing impairment are encouraged to take active initiatives to broaden their career development paths and obtain more autonomy in choosing majors, which are of crucial importance for their career development and psychological health. Future research, such as longitudinal experimental studies, are recommended to explore the factors influencing students with hearing impairment’s vocational identity development and identify effective interventions.


Octopus minor, Uroteuthis edulis, Sepia esculenta and Sthenoteuthis oualaniensis are important economic cephalopod species in coastal waters of China. They are very important role in marine ecosystems as relevant prey for large marine fish, and marine mammals are a critical component of the food chain. As the main feeding organ of cephalopods, the beak has a stable structure and is resistant to corrosion. However, the sexual dimorphism on the beak of the O. minor, U. edulis and S. esculenta and Sthenoteuthis oualaniensis at different ontogenetic stages is unknown, neither has it been determined whether variation of beak shape relates to changes in habitat environment and feeding preference at different ontogenetic stages. Using a geometric morphometrics approach, we found that the beaks of O. minor, U. edulis, S. esculenta, and Sthenoteuthis oualaniensis showed a pattern of variation, displaying sexual dimorphism and allometry between ontogenetic stages. Habitat may drive variation in beak shape. This study has furthered our understanding of the beak shape ontogenetic variation among the four species. We discuss the potential factors underlying the beak shape variation and provide a basis for the understanding of cephalopod phenotypic plasticity and its ecological significance.Octopus minor, Uroteuthis edulis, Sepia esculenta and Sthenoteuthis oualaniensis of the China’s coastal waters, the differences between immature and mature stages and the sex-linked differences in the beak shape and size were analyzed with geometric morphometrics methods in this study. The results of Procrustes analysis of variance, principal component analysis and multivariate regression showed that the shapes of the upper beaks of O. minor, U. edulis and S. esculenta differed significantly among various ontogenetic stages (p < 0.05). The shapes of the lower beaks of U. edulis, S. esculenta and Sthenoteuthis oualaniensis were also significantly different among various ontogenetic stages (p < 0.05). The results of thin-plate spline deformation grids showed that the beaks of the four cephalopod species presented different variation patterns. This study gives us basic beak geometry morphology information for Octopus minor, Uroteuthis edulis, Sepia esculenta and Sthenoteuthis oualaniensis present in China’s coastal waters. The ontogenetic differences in beak shape might be related to extrinsic factors (diet difference and intra and interspecific competition) in habitat.Investigating the ontogenetic variation of biological individuals helps us to fully understand the characteristics of evolution. In order to explore the ontogenetic variation and sexual dimorphism of the beak shape in  Biological individuals in nature often face different habitat environments which can drive phenological variation  and the Diplodus puntazzo [Munida rugosa [Lates niloticus [Hysterocarpus traskii [Two methods are generally used to explore ontogenetic and sexual dimorphism. First, based on traditional morphometrics where the linear distance difference among individuals is analyzed by multivariate statistical methods. However, the shape variation of biological individuals involves geometric information, which traditional morphometrics tend to ignore . Secondlpuntazzo , Munida a rugosa , Lates niloticus  and Hyst traskii . Assessi traskii ,19,20.Octopus tehuelchus in response to the environment. Fang et al. [Ommastrephes bartramii in different aspects. Therefore, exploring the life cycle of different cephalopods is prerequisite for sustainable exploitation and utilization of this resource.Cephalopoda contains around 850 extant species , which ag et al.  discusseIllex argentinus did not vary between cohorts and sexes nor between different ontogenetic phases. Jones et al. [Doryteuthis gahi) populations had complex structures and high intra-species variations in body shape. Sexual dimorphism of the beak is a common phenomenon in some cephalopods [As the main feeding organ of cephalopods, the beaks are located in the buccal mass and divided into the upper beak and the lower beak ,28,29,30s et al.  confirmehalopods ,34,35. HO. minor, U. edulis, S. esculenta, and Sthenoteuthis oualaniensis, representatives of Octopodiformes and Decapodiformes in the subclass Coleoidea, are important economic cephalopod species in coastal waters of China [Cephalopods are diverse in China seas ,36,37,38of China ,40,41. TO. minor, 205 U. edulis, 198 S. esculenta, and 218 Sthenoteuthis oualaniensis  of each specimen was measured by measuring tape and the sex and gonadal maturity of each species were then determined ,43,44. Iculenta: ,44; U. eWe focused on lateral profiles of the upper and lower beaks , which wThe images of beaks were each named according to species, sex, ontogenetic stage, and the specimen number. We added semilandmarks to better represent beak shape . AccordiO. minor, U. edulis, S. esculenta, and Sthenoteuthis oualaniensis were analyzed with GM methods.The ontogenetic variation and sexual dimorphism of beak shape of All morphological measurements and statistical analyses were performed in the R 4.0.5 “geomorph” package . Firstlyp < 0.05; S. esculenta had the largest beaks, followed by U. edulis and Sthenoteuthis oualaniensis. The beaks of O. minor were the smallest. The upper beaks are slightly larger than the lower beaks in all four species , hood , wing (Landmarks 4 and 20) and lateral wall   . Althoug thicker a,c, but  thinner b. The hond wider a,b, whermpressed c. The lampressed a–d and tmpressed a–d.p < 0.05; U. edulis, S. esculenta, and Sthenoteuthis oualaniensis were significantly different between immature and mature stages , hood (Landmark 17), wing  and lateral wall   . In matural wall b and simaniensis c,d. In pe curved b,c, wherlattened d. Moreovlattened b–d. Compwer beak d and thee stages c, right.O. minor, U. edulis and Sthenoteuthis oualaniensis were significantly different between female and male . S. lt stage ,65. Due on stage c. After ing prey ,68. As aectively . These mectively . In addiectively ,69,70. TO. minor are benthic cephalopod species [U. edulis and S. esculenta inhabit the demersal, and Sthenoteuthis oualaniensis inhabits the pelagic [O. minor and S. esculenta are likely more useful for breaking shells   also showed significant difference between females and males [Gender selection, ecological differences, etc. may cause the differences in size and shape between female and male individuals and the shape differences of biological individuals between female and male individuals are called sexual dimorphism ,18. Some < 0.05) . Female c stages . The uppof males a,d. In tof males d. Similand males . The femO. minor, U. edulis, and S. esculenta show faster variation rates of shape in their immature stages  may drive ontogenetic variation in beak shape. This study has furthered our understanding of the beak development pattern among four cephalopods species. The detection of additional sexual differences and their expression during ontogeny in other cephalopod species is also highly interesting and necessary, considering the strongly specialized life history in cephalopods, and different habitats.Through geometric morphometrics analysis, we found that the beaks of


Newborn hearing screening (NHS) has been covered by national health insurance since October 2018 in Korea. However, the results of the NHS are not reported due to the absence of a follow-up tracking system. This study analyzed the status and the predicted referral rates of NHS after the Korean national health insurance coverage by analyzing the National Health Insurance Service database in 2019 and 2020. The NHS coverage was 91.7% of total birth in 2019 and 92.1% in 2020. The predicted referral rate of NHS calculated by the duplicated NHS cases was 1.05% in 2019 and 0.99% in 2020. However, another predicted referral rate calculated by the number of diagnostic auditory brainstem responses (ABRs) performed was 1.44% in 2019 and 1.43% in 2020. The first NHS was performed within one day of birth for 96.5% of the babies and within three days of birth for 97%. However, diagnostic ABR was adequately performed within three months of birth for only 4.3%, while 82.3% performed the test after six months which delays appropriate intervention for hearing loss. National support such as national coordinators, follow-up tracking, and data management systems are needed for early hearing detection and intervention of newborns and infants in Korea. The incidence of permanent newborn hearing loss is between 1 and 3 for every 1000 births ,3,4,5. BWhen the screening tests are not financially supported by national health insurance, some babies, especially in low-income families, may skip the test and be exposed to undetected hearing loss. However, the national health insurance fully supported the cost of NHS for all the children in Korea in October 2018, with the expectation of an increase in the performance of the NHS and, consequently, a decrease in the incidence of babies with hearing loss. It is recommended, in the international and Korean NHS guidelines, that screening tests be performed within one month of birth for all babies, confirmatory hearing tests within three months of birth for the referred cases, and intervention within six months of birth for the confirmed babies ,10. For This study aimed to investigate the status and the predicted referral rates of NHS in Korea after the implementation of Korean national health insurance coverage of NHS by analyzing the National Health Insurance Service (NHIS) database.This study was conducted by analyzing the nationwide big data from the Korean NHIS information database , which cSince the NHS received support from the national health insurance in October 2018, this study analyzed newborns born between 1 January 2019 and 31 December 2020. Those who performed the 1st NHS within one year of their birth date and those reviewed by 31 August 2021, were enrolled in this study.The number of infants who underwent the NHS test under the national health insurance, NHS method , the number of infants who performed the diagnostic hearing test , and the age (date) of the 1st screening and diagnostic ABR were evaluated based on the national test and surgical claim code classification.We chose two methods for estimating the number of referred infants from the NHS test because the actual result of the NHS is not known from the NHIS information database. First, we estimated infants who did not pass NHS  by the duplicated cases in NHS (supposed to have performed 2nd NHS due to “refer” result in 1st NHS). As a second method, the number of ABR performed cases was used (supposed to have performed ABR due to the “refer” result in NHS).Statistical analyses were performed using SAS 9.3 software . The categorical data were presented by number (%). As a representative hospital, the study protocol was approved by the Institutional Review Board of Hallym University Kangnam Sacred Heart Hospital. The Institutional Review Board waived the need for informed consent because of the retrospective database nature of this study. All methods were performed according to the relevant guidelines and regulations.The total number of live births in Korea was 306,273 in 2019 and 276,105 in 2020 . The finThe NHS performance was evaluated for the 17 major cities and provinces in Korea. Jeju island revealed the lowest NHS performance rate, while Seoul, the most populated city in Korea, showed the third lowest figure in 2019 . HoweverDuplicated cases that had performed NHS both at inpatient and outpatient clinics were 2957 in 2019 and 2510 in 2020 . The dupTotal number of infants who conducted diagnostic ABR tests each year was 4400 (inpatient 546 vs. outpatient 3854) in 2019 and 3948 (inpatient 519 vs. outpatient 3429) in 2020. The predicted referral rate calculated by the ABR’s performance, i.e., ABR’s number divided by the total NHS number, was 1.57% in 2019 and 1.55% in 2020, respectively.Most NHS were performed within one day of birth . More thThe two representative screening tests for newborns in Korea are the NHS and the congenital metabolic disease screening tests . The KorThe insurance coverage of NHS is beneficial not only to achieving EHDI but also to acknowledging the status of neonatal hearing for the national population. No national statistics on NHS in Korea could be reported before any project was performed by the government. While the national rate of actual neonatal hearing loss is not available, the estimated value can be achieved by analyzing the national database in this study. The mean national referral rate (1.5%) could be reported for the first time in Korea from the pilot project from 2014 to 2018 ,13. HoweMost 1st NHS was performed at the inpatient clinic when the babies were born, and most 2nd NHS and ABR were performed at the outpatient clinic after discharge from the hospital. Only 91.5% of NHS were performed at the inpatient clinic in the pilot project [For diagnostic hearing loss tests, most ABRs 82.3%) in Korea were performed after six months of birth, showing delayed newborn hearing diagnostic tests in these patients. The 2.9% value within one day of birth group is considered as the misuse of ABR instead of the NHS in these cases. At the national level, it is necessary to provide guidance and education for the immediate performance of confirmatory testing for ‘refer babies’ in the NHS because early detection and intervention are crucial for conserving neonatal hearing loss .3% in Ko,14. For The clinical significance of this study is that the government’s financial support of NHS offered not only the conservation of neonatal hearing but also enlightenment on the status and the statistics of the national NHS. The delayed time of ABR revealed in this study necessitates an effort to advance the timing of ABR tests for the NHS referred infants, such as parent education or financial support from the government. One limitation of this study is that the actual referral rate could be higher than the results presented here, considering those who missed ABR despite a refer result in NHS. Additionally, the COVID-19 situation that started in December 2019 may have affected the performance of the hearing tests.Since Korea’s NHS test was applied to the national health insurance in 2018, the government’s NHS coverage rate has increased compared to the previous low-income class support project, but all newborns are yet to be screened. The status of NHS and predicted referral rate of NHS in Korea were discovered by analyzing the national data after the insurance coverage, for the first time to be reported in the literature. The analysis revealed that the referred babies in the NHS test were not adequately followed-up, and the diagnostic ABR test for confirming hearing loss was not implemented promptly. In Korea, where low fertility is a social problem, national support such as hearing loss coordinators, follow-up tracking, and data management systems are needed for the EHDI of all newborns and infants.


Chronic inflammatory demyelinating polyneuropathy (CIDP) is the most common, heterogeneous, immune-mediated neuropathy, characterized by predominant demyelination of motor and sensory nerves. CIDP follows a relapsing–remitting or a progressive course and causes substantial disability. The pathogenesis of CIDP involves a complex interplay of multiple aberrant immune responses, creating a pro-inflammatory environment, subsequently inflicting damage on the myelin sheath. Though the exact triggers are unclear, diverse immune mechanisms encompassing cellular and humoral pathways are implicated. The complement system appears to play a role in promoting macrophage-mediated demyelination. Complement deposition in sural nerve biopsies, as well as signs of increased complement activation in serum and CSF of patients with CIDP, suggest complement involvement in CIDP pathogenesis. Here, we present a comprehensive overview of the preclinical and clinical evidence supporting the potential role of the complement system in CIDP. This understanding furnishes a strong rationale for targeting the complement system to develop new therapies that could serve the unmet needs of patients affected by CIDP, particularly in those refractory to standard therapies.The online version contains supplementary material available at 10.1007/s13311-022-01221-y. Chronic inflammatory demyelinating polyneuropathy  is the most common and heterogeneous, immune-mediated peripheral neuropathy, typically characterized by predominant demyelination of motor and sensory nerves. CIDP manifests typically as an insidious onset of weakness, hypo- or areflexia, numbness, paresthesia, and sensory ataxia with a progressive or relapsing–remitting pattern –3. The “The current standard treatments for CIDP include intravenous immunoglobulins (IVIg), corticosteroids, and plasma exchange . HoweverA diverse and complex interplay of different immunopathological mechanisms, involving cellular, humoral and complement pathways, culminates in a highly variable pattern of peripheral nerve damage in different clinical variants of CIDP. Humoral factors and macrophage-mediated demyelination appear to play a crucial role in CIDP ; howeverIn particular, the complement system appears to play an important role in CIDP pathogenesis. Studies have shown deposition of complement components, such as C3d and C9 neoantigen, as well as complement-fixing IgG and IgM on the myelin sheath in patients with CIDP . IncreasMost of the review articles discussing CIDP pathophysiology are focused on macrophage infiltration and demyelination and examined the role of autoantibodies directed to peripheral nerve components. However, there are no reviews centered on the role of the complement system in the pathogenesis of CIDP. Considering the current emergence and success of complement-targeted therapies in other autoimmune disorders, it is important to critically assess the role of the complement system and the suitability of complement-targeted therapeutics in CIDP patients.Cellular and humoral components, including the complement system, are variably involved in peripheral nerve damage in CIDP Fig. . HoweverThe involvement of cell-mediated immune mechanisms in CIDP is substantiated by evidence of T cell activation, the passage of activated T cells through the BNB, and the expression of pro-inflammatory cytokines . ConventIncreased permeability of the BNB is the primary precursor for nerve inflammation and damage. While the exact mechanisms remain unknown, adhesion of activated T cells and upregulation of activation biomarkers and proinflammatory cytokines facilitate the enhanced permeability of the BNB, thus allowing transfer of macrophages and soluble factors, such as antibodies and complement including the C5b-9 membrane attack complex (MAC), to cross the BNB and exert nerve damage . FurtherMacrophages are involved throughout the pathogenic process in autoimmune neuropathies and play an instrumental role in CIDP pathophysiology . They arH17) cells and expression of retinoic-acid-receptor-related orphan nuclear receptor gamma (RORγt) have been reported in the peripheral blood and CSF of patients with active CIDP, compared to those with remitting disease [+ T cells with monoclonal and oligoclonal restrictions in sural nerve biopsies and peripheral blood [Sural nerve biopsies from CIDP patients also show signs of T cell infiltration . Increas disease . Additioal blood , 23, indal blood . Becauseal blood , 25.The presence of cytokines may have implications in damaging the BNB and setting up a chemo-attractive environment for immune cell recruitment and subsequent neural insult . Higher Although direct evidence for a myelin antigen-specific auto-reactive T-cell population is currently lacking , a combiThe involvement of T cells in CIDP pathogenesis is well established; however, the use of T cell-based therapies/inhibitors has not, to date, been shown to be efficacious in the management of CIDP. Fingolimod, a first in class sphingosine 1-phosphate receptor modulator that reduces the circulating naïve T- and B-cell population, has shown efficacy in multiple sclerosis. However, in the FORCIDP study, fingolimod did not demonstrate clinical benefit in CIDP, and the study was terminated prematurely for futility . There aWhile myelin phagocytosis by macrophages is instrumental in CIDP pathogenesis, the triggers underlying this process are not clearly understood. It is postulated that deposition of autoantibodies specific to peripheral nerve antigens may be involved, via recognition of the immunoglobulin fragment crystallizable (Fc) portion or activation of the complement system .The response to plasma exchange therapy indicates the important role of circulating factors in CIDP pathogenesis ; howeverAntibodies targeting nodes of Ranvier structures have recently been identified –42. AntiActivation of the complement system by autoantibodies may contribute to various mechanisms inflicting damage to the myelin sheath. One is to facilitate macrophage-mediated myelin phagocytosis, , an obseThe efficacy of plasma exchange in CIDP further substantiates the role of circulating factors, and potentially that of the complement system, in the disease pathogenesis . A studyThe complement system is an integral part of innate immune defense and plays an important physiological role in tissue remodeling . The proComplement activation occurs through three different pathways: classical (C1q), lectin (mannose-binding lectins or ficolin), and alternative (C3 autoactivation or properdin), which converge at C3, the most abundant complement protein found in the blood . SubsequCampylobacter jejuni infection-induced autoantibodies have been found to activate complement and MAC deposition at the nodes of Ranvier [Complement is a complex system, and disruption of its delicate regulation triggers several autoimmune neurological disorders . The rol Ranvier .These data suggest that complement-mediated processes in peripheral neuropathies are important mediators in their pathobiology. Hence, the therapeutic approach of targeting the complement system seems to be viable , 63. In Complement plays a role in various autoimmune neuropathies, but much of this data comes from studies on various animal models of peripheral nerve inflammation . The EANEvidence is also available on the role of complement in other animal studies of peripheral neuropathies. In a mice model of AMAN, inhibition of C1q (with an anti-C1q antibody) resulted in attenuation of complement cascade activation and complement deposition, as demonstrated by absence of C3c staining or C5b-9 components at the nerve terminals. There was also evidence of reduced immune cell recruitment and reduced axonal injury .A novel “Human-in-a-chip” in vitro model, using a microelectrode array with directed axonal outgrowth over the electrodes, was recently developed. A human CIDP model was created by treatment of Schwann cell and motoneuron cultures with CIDP patient-derived serum, which triggered classical complement pathway activation and, subsequently, the deposition of C3b and C5b-9. Furthermore, treatment with a test monoclonal antibody inhibitor of C1s, TNT005, resulted in abrogation of complement deposition and rescued the electrophysiological deficits .Complement involvement in CIDP pathogenesis is also substantiated by several lines of histopathological, serological and clinical evidence.The study of CIDP-related genetic factors remains limited due to the rarity of the disease and heterogeneity in the CIDP phenotypes . Cases oBased on these observations, a hypothesis that de novo CD59 mutations may be present in patients with sporadic CIDP was tested. A study performed direct sequencing of all coding exons of CD59 in 35 adult patients with sporadic CIDP. However, the p.Cys89Tyr mutation was not identified in any of the patients. Only one variant, a heterozygous guanine to adenine substitution (c.18G > A), was noted but was not shown to have any deleterious effect . ConsideEvidence of complement involvement is supported by the demonstration of complement deposition in sural nerve biopsies from CIDP patients. In an early study, sural nerve biopsy specimens from seven patients with chronic relapsing demyelinating polyneuropathy showed deposition of IgM in intraneural blood vessels in all patients and deposition of C3 in six of the seven patients. Deposits of IgM, but not of C3, were also noted on the Schwann cell plasmalemma of even non-demyelinated nerve fibers in CIDP patients Fig. A. HoweveA recent case report of a patient with CIDP demonstrated presence of a circulating IgG antibody to LM1, but not to NF-155, CNTN-1, GM1, and GD1b. An immunohistochemical analysis revealed deposition of C9 neoepitope, a component of MAC, on the compact myelin sheath. Macrophage infiltration was evident with the presence of several CD68-positive cells in each fascicle. Furthermore, deposition of complement was noted at the internodes, which comprise the majority of the length of myelinated fibers. This case highlights the role of complement-dependent cytotoxicity in the pathogenesis of CIDP with anti-LM1 antibodies . in 21 newly diagnosed, treatment-naïve CIDP patients, with that of 29 control patients with noninflammatory neurological diseases. CIDP patients had significantly higher mean serum and CSF levels of C5a and TCC than control patients . This isComplement components also appear to correlate with CIDP disease activity. In CIDP patients, systemic terminal complement activation was associated with residual disease severity after 4 weeks of IVIg treatment. In 80% of these patients, inflammatory neuropathy cause and treatment (INCAT) disability scores were reduced after IVIg treatment for 4 weeks. CIDP patients with higher disability at the time of diagnosis tended to have higher serum C5a and soluble TCC levels; these levels reduced and significantly correlated with the reduction in INCAT scores at the week 4 follow-up visit after initiation of IVIg treatment. Larger studies are warranted to examine terminal complement activation as a potential surrogate marker for clinical disease activity, progression, and response to immunomodulatory therapies in patients with major phenotypic variants of CIDP .As noted previously, both GBS and CIDP exhibit similar patterns of macrophage-mediated demyelination originating from similar, pathogenic mechanisms , 20, 31 Complement involvement has been implicated among one of the diverse mechanisms of GBS pathogenesis and is validated by adequate clinical evidence. The presence of C3d was reported on the Schwann cell plasma membrane in myelinated fibers, but not in unmyelinated fibers, in autopsy studies of three GBS patients. Immunocytochemistry revealed that in many nerve fibers, there was a rim of C3d and C5b-9 along the outer surface of the Schwann cells. Further analysis of such C3d-positive fibers revealed mild vesicular changes in the outermost myelin lamellae . IncreasThe accumulating evidence regarding complement involvement in many autoimmune neuropathies has generated great interest and encouraged clinical research focused on complement-targeted therapies in several neurological disorders . Complem therapies in CIDP, though effective, have several limitations, such as variable treatment response, side effects with chronic use, need for venous access, dependency on plasma donations (IVIg) leading to potentially limited availability, and high patient burden. Various clinical phenotypes of CIDP appear to have similar but not identical, pathogenic causes, which may explain the variability in treatment response to the SOC therapies. There is, therefore, a substantial unmet need in CIDP treatment that requires the development of alternative therapies that reflect the various disease phenotypes and thereby offers treatments targeted to individual patients.The lack of efficacy of fingolimod, a T-cell-based therapy , has encSeveral complement inhibitors, each targeting a specific step in the complement pathway, are under development, and clinical studies are ongoing to explore their potential clinical benefits , 84. EcuPre-clinical research in CIDP remains limited due to lack of disease-specific animal models. Conducting clinical trials is cumbersome due to difficulties in patient recruitment, disease heterogeneity, and the lack of objective biomarkers to characterize the disease and evaluate response to therapies. Hence, further clinical research in CIDP can benefit from the use of novel approaches in study design to overcome these challenges and ensure solid conclusions when testing new treatment options in this field.In summary, considering the drawback of the current SOC therapies, the preclinical and clinical evidence supporting the role of complement in CIDP pathogenesis, and the plausible success of early stage complement inhibitors in diseases other than CIDP, exploring the benefits of complement pathway inhibitors in CIDP is an attractive proposition.Supplementary file1 (PDF 527 KB)Supplementary file2 (PDF 2081 KB)Supplementary file3 (PDF 517 KB)Supplementary file4 (PDF 1225 KB)Supplementary file5 (PDF 517 KB)Supplementary file6 (PDF 535 KB)Supplementary file7 (PDF 509 KB)Supplementary file8 (PDF 534 KB)Below is the link to the electronic supplementary material.