Modern Micro and Nanoparticle-Based Imaging Techniques

Sensors 2012, 12, 14792-14820; doi:10.3390/s121114792 sensors ISSN 1424-8220 www.mdpi.com/journal/sensors Review Modern Micro and Nanoparticle-Based Imaging Techniques Marketa Ryvolova 1,2, Jana Chomoucka 1, Jana Drbohlavova 1, Pavel Kopel 1,2, Petr Babula 1, David Hynek 1,2, Vojtech Adam 1,2, Tomas Eckschlager 3, Jaromir Hubalek 1, Marie Stiborova 4, Jozef Kaiser 1 and Rene Kizek 1,2,* 1 Central European Institute of Technology, Brno University of Technology, Technicka 3058/10, CZ-616 00 Brno, Czech Republic; E-Mails: [email protected] (M.R.); [email protected] (J.C.); [email protected] (J.D.); [email protected] (P.K.); [email protected] (P.B.); [email protected] (D.H.); [email protected] (V.A.); [email protected] (J.H.); [email protected].cz (J.K.) 2 Department of Chemistry and Biochemistry, Faculty of Agronomy, Mendel University in Brno, Zemedelska 1, CZ-613 00 Brno, Czech Republic 3 Department of Paediatric Haematology and Oncology, 2nd Faculty of Medicine Charles University in Prague and University Hospital Motol, Prague, Czech Republic; E-Mail: [email protected] 4 Department of Biochemistry, Faculty of Science, Charles University, Albertov 2030, CZ 128 40 Prague 2, Czech Republic; E-Mail: [email protected] * Author to whom correspondence should be addressed; E-Mail: [email protected]. Received: 17 September 2012; in revised form: 18 October 2012 / Accepted: 19 October 2012 / Published: 2 November 2012 Abstract: The requirements for early diagnostics as well as effective treatment of insidious diseases such as cancer constantly increase the pressure on development of efficient and reliable methods for targeted drug/gene delivery as well as imaging of the treatment success/failure. One of the most recent approaches covering both the drug delivery as well as the imaging aspects is benefitting from the unique properties of nanomaterials. Therefore a new field called nanomedicine is attracting continuously growing attention. Nanoparticles, including fluorescent semiconductor nanocrystals (quantum dots) and magnetic nanoparticles, have proven their excellent properties for in vivo imaging techniques in a number of modalities such as magnetic resonance and fluorescence imaging, respectively. In this article, we review the main properties and applications of nanoparticles in various in vitro imaging techniques, including microscopy and/or laser breakdown spectroscopy and in vivo methods such as magnetic resonance imaging and/or OPEN ACCESS Sensors 2012, 12 14793 fluorescence-based imaging. Moreover the advantages of the drug delivery performed by nanocarriers such as iron oxides, gold, biodegradable polymers, dendrimers, lipid based carriers such as liposomes or micelles are also highlighted. Keywords: imaging techniques; nanoparticles; fluorescence; magnetic resonance imaging 1. Introduction Imaging techniques play an important role in the medical care of all organ systems and better and increased research in medical imaging may be beneficial for the complete health and disease management process. Medical imaging covers many different imaging modalities: X-ray-based methods such as radiography and Computed Tomography (CT), Magnetic Resonance Imaging (MRI), Ultrasound (US), Nuclear medicine with Positron Emission Tomography (PET) and Single Photon Emission Computed Tomography (SPECT), and several optical imaging methods (Figure 1). Figure 1. Main in vivo imaging techniques. The important and exciting progress in biotechnology, nanomedicine and new innovative therapies is in many cases highly dependent on the integration of the medical imaging into standard clinical practice. The development of new materials of nanometer dimensions for biomedical applications has been in the focus in the last few years. Their applications became important in medicine, targeted therapies and diagnostics. Modern materials like nanowires [1], quantum dots [2] carbon nanotubes [3], nanoparticles [4,5] or nanomaterials [6] (Figure 2) are in the centre of attention due to the fact that the mechanical, chemical, electrical, optical, magnetic, electro-optical and magneto-optical properties of these particles are different from their bulk properties and depend on the particle size. Nanoparticles Sensors 2012, 12 14794 (NPs) have been developed as an important approach for delivery of conventional drugs, recombinant proteins, vaccines and more recently, nucleotides. NPs and other colloidal drug-delivery systems modify the kinetics, body distribution and drug release of an associated drug. Figure 2. Published items each year containing ―imaging and nanoparticle*‖ in title. 2. Magnetic Resonance Imaging (MRI) 2.1. Magnetic Resonance Imaging by Nanoparticles Magnetic nanoparticles (MNPs) for medical and biological applications are being of great interest due to their unique properties. MNPs can be divided into paramagnetic, ferromagnetic and superparamagnetic particles. Superparaand ferromagnetic particles are composed of a magnetic core and a surface coating. Paramagnetic particles are mainly based on chelates of paramagnetic ions with no explicit core and surface coating. Thus, their influence on magnetic resonance imaging (MRI) contrast is rather different from that of superparaand ferromagnetic particles [7,8]. The switchable magnetic properties of superparamagnetic nanoparticles (SPIONs) make these materials useful for magnetic drug targeting [9], cell tracking [10], hyperthermia [11] and medical imaging [12,13]. In addition, MNPs are efficiently used for DNA, protein or cell labeling and their subsequent purification in a magnetic field [14]. MRI on its own already offers outstanding soft tissue contrast. The ongoing development of MNPs as contrast agents (CAs) further enhances image contrast. New magnetic contrast agents can be size-tailored to accumulate in specific organs or their surface can be specifically functionalized to target cells (tumor, transplanted cells). Hence, the contrast of MRI is significantly enhanced and diseases can be potentially detected at an earlier stage. Most commonly, a paramagnetic CA, usually a gadolinium-based compound, is used [15]. Gadolinium-doped tissues and fluids appear extremely bright in MR images, and for this reason paramagnetic CAs are called positive CAs. Sensors 2012, 12 14795 2.2. Biological Application of Superparamagnetic Particles More recently, superparamagnetic CAs based on iron oxide MNPs have become commercially available. The regions where such agents are delivered appear darker, and therefore, they are called negative CAs. The big advantage of this type of CAs is their higher sensitivity that is expected to reach single cell level [16]. Iron oxides with core/shell structures are the most widely used as sources of magnetic materials [17]. Iron oxides have several crystalline polymorphs known as α-Fe2O3 (hematite), β-Fe2O3, γ-Fe2O3 (maghemite), ε-Fe2O3, Fe3O4 (magnetite) and some others (amorphous and high pressure forms) [18]. Nevertheless, only maghemite and magnetite have found the greatest interest of bioapplications [19]. Readily, carbonyl iron, which is well-known material with a unique form of elemental iron because of its small particle size, was also used as magnetic core [20]. The surface of MNPs may be coated with number of different functionalities, depending upon the coating material and the reactive groups presented on the targeting ligand. It is desirable that MNPs retain sufficient hydrophilicity and, with coating not exceed 100 nm in size, to avoid rapid clearance by the reticuloendothelial system (RES) [21]. It was found that the surface functionalization also plays a key role in nanoparticle toxicity [17]. Jain et al. investigated MNPs consisting of an iron-oxide magnetic core coated with oleic acid and stabilized with a Pluronic® or Tetronic® block copolymer as an effective cancer theranostic (therapy and diagnostic) agent, i.e., an agent with combined drug delivery and imaging properties [22]. The authors revealed that these block copolymers, mainly their molecular structures and weight, significantly influence the physical properties of MNPs, their interactions with biological environment following intravenous administration, and ability to localize to tumors. Schweiger et al. investigated γ-Fe2O3 MNPs for application as CAs for MRI and as switchable drug delivery vehicles [23]. Namely, the authors studied colloidal stability and cytotoxic effects of these MNPs in adenocarcinomic epithelial cells. They prepared poly (ethylene imine)-g-poly (ethylene glycol) (PEGPEI) as stabilizer for iron oxide MNPs in comparison to branched PEI. Amiri et al. presented in their work a series of maghemite/polymer composite ferrofluids with variable magnetic core size, which showed a good efficiency as MRI CA [16]. These ferrofluids were biocompatible and can be proposed as possible platforms for multifunctional biomedical applications. They contain anchoring groups for biofunctionalization, can incorporate fluorescent dyes, and have shown low cellular toxicity. Kumagai et al. developed block copolymer-coated magnetite nanoparticles for pancreatic cancer imaging, by means of a chelation between the carboxylic acid groups in PEG-poly (aspartic acid) block copolymer and Fe on the surface of the iron oxide nanoparticles [24]. Masoudi et al. synthesized ex situ PEG coated iron oxide NPs to be used as potential MRI contrast agents for early detection of lymph nodes and their metastases [25]. According to the important role of nanoparticle size in biodistribution, magnetite nanoparticles coated with different PEG concentrations up to 10/1 PEG/iron oxide weight ratio in an ex situ manner incomplete sentence—has no conclusion. To predict the PEG-coated nanoparticle behavior in biological media, such as the blood stream or tissues, colloidal stability was evaluated to estimate the coating endurance under different conditions. Martelli and coworkers synthesized a simple and stable nanoprobe exhibiting magnetic and fluorescent properties for detection of lymph node homing of tumor-specific dendritic cells in a murine breast cancer model [26]. The mechanism of synthesis relies on the covalent coupling of modified organic fluorophores with chitosan, which strongly interact with the surface of the ferric oxide NPs. Jiang et al. Sensors 2012, 12 14796 investigated targeted MR CA transferrin-conjugated SPIONs (Tf-SPIONs) for brain glioma detection [27]. They observed the obvious contrast change of brain glioma before and after administration of Tf-SPIONs in C6 glioma rat model in vivo on T2 weighted imaging. Among the wide variety of iron oxide nanoparticles which are routinely used as MRI CAs, magnetoliposomes (MLs) may be included. The flexibility of the lipid coating allows an efficient functionalization, enabling bimodal imaging or the use of MLs as theranostic agents. Especially the large MLs containing several magnetite cores encapsulated within a single large liposome were found to be highly efficient. A special type of MLs, biogenic MLs, can also be efficiently used in this regard, with potential applications in cancer treatment and imaging. In small MLs the lipid bilayer is immediately attached onto a solid magnetite core. The flexibility of the lipid bilayer allows the incorporation of poly(ethylene glycol)–lipid conjugates to increase blood circulation times and be used as bone marrow contrast agents. Cationic lipids can also be incorporated, leading to high cell uptake and associated strong contrast generation in MRI of implanted cells [28]. 3. Fluorescence-Based Imaging Techniques In the group of fluorescence-based techniques fluorescence microscopy, flow cytometry, cell sorting fluorescence correlation spectroscopy as well as particle tracking velocimetry may be included. Historically, biological research is based on optical imaging, in particular microscopy, but also several chromophoric assays and photographic approaches. With the recent emergence of methods appropriate for bio-marker in vivo staining, such as bioluminescence, fluorescent molecular probes and proteins, as well as nanoparticle-based targeted agents, significant attention has been shifted toward in vivo interrogations of different dynamic biological processes at the molecular level. This progress has been largely supported by the development of advanced tomographic imaging technologies suitable for obtaining volumetric visualization of bio-marker distributions in small animals at a whole-body or whole-organ scale, an imaging frontier that is not accessible by the existing tissue-sectioning microscopic techniques due to intensive light scattering beyond the depth of a few hundred microns [29]. In Vivo Imaging by Fluorescent Nanoparticles Semiconductor quantum dots (QDs) are of special interest as tiny light-emitting particles on the nanometer scale that belong to new class of fluorescent probes for in vivo biomolecular and cellular imaging [30–33]. In comparison with organic dyes and fluorescent proteins, QDs have unique optical and electronic properties. QDs have molar extinction coefficients that are 10–50 times larger than that of organic dyes, which make them much brighter in photon-limited in vivo conditions. The long lifetime in the order of 10–40 ns increases the probability of absorption at shorter wavelengths, and produces a broad absorption spectrum. Further, QD emission wavelengths are size tunable. For example, CdSe/ZnS QDs of approximately 2 nm in diameter produce a blue emission, whereas QDs approximately 7 nm in diameter emit red light [34,35]. The emission wavelength was extended into the near infrared region (650 nm to 950 nm), to take advantage of the improved tissue penetration depth and reduced background fluorescence at these wavelengths. A key property for in vivo imaging is the unusual QD Stokes shift, which can be as large as 300–400 nm, depending on the wavelength of the excitation light [32]. In conjunction with broadband absorption and narrow emission peaks of QDs, Sensors 2012, 12 14797 this property allows multiplexed imaging applications in which one light source is used to simultaneously excite multicolor QDs without the need for complicated instrumentation. Another important feature is the long-term photostability of QD imaging probes, which opens the possibility of investigating the dynamics of cellular processes over time, such as continuously tracking cell migration, differentiation, and metastasis. These properties have made QDs a topic of intensive interest in cancer biology, molecular imaging, and molecular profiling [36–44]. As expected, in vitro investigation of QDs is of a great interest [34,45–51]. Some examples of application of QDs in imaging are given hereinafter. A specific advantage of quantum dots for in vivo applications is their photostability; QDs allow images to be recorded over a longer period of time than possible with the use of fluorescent dyes or proteins due to their resistance to photobleaching. Maysinger et al. visualized CdSe and CdTe QDs detectable at one hour-, one day-, three days-, and seven days post intracortical injection using in vivo imaging techniques [52]. Mice were injected subcutaneously and scanned for fluorescence, in particular in the brain, where peak fluorescence was observable at three days post injection and persisted for seven days. Sub-cellular resolution was achieved and allowed the identification of the location of the conjugates. The researchers demonstrated internalization of their QD conjugates, with varied rate and efficacy of internalization for different cell-types. Neuronal internalization is especially challenging, and in vivo imaging in neural cells has important relevance to quantum dot toxicity, as this approach would allow the investigation of the neuronal immune response in real time. This approach was novel not only in the in vivo imaging aspects but the animal model was transgenic, and was monitored for an astrocyte-responsive luciferase reporter in addition to the injection of quantum dot conjugates. The activation of the astrocyte response was observable as an increase in bioluminescence in response to foreign nanoparticles in mice. Zimmer et al. have employed NIR emitting QDs due to their ability to act as a reporter at a wavelength minimally absorbed by biological species [53]. They prepared a series of InAs/ZnSe core/shell QDs. The small core size, along with variation to the shell thickness and composition offered a range of size tunable emission wavelengths, between 750 and 920 nm. The conjugation of dihydrolipoic acid to the quantum dots allowed for the observations within the interstitial fluid in rats, where the quantum dot conjugates exited the blood vessels. This visualization of the extravasations sites is important, and has potential to interrogate the delivery mechanism of QDs to tumor cells. Tumor growth requires a supply of nutrients from the blood stream. Angiogenesis is the process by which new vasculature establishes a blood supply to a growing tumor. Receptors, such as integrins, are highly expressed in tumor cells during angiogenesis, and diagnostics targeting such receptors can lend insight into the type and extent of diseases, including cancer. The work by Cai et al. detailed the use of a tri-peptide QD conjugate which specifically binds to integrin [54]. The conjugation of arginine-glycine-aspartic acid to a quantum dot with emission at 705 nm yielded a diagnostic tool specific for the integrin receptor that emits in the NIR region. In vitro analysis was performed on human glioblastoma and human breast cancer cells, showing specificity for integrin-positive cells only. In addition, binding was inhibited by the presence of an integrin antagonist. Injections of both the conjugates and non-specific controls through the tail vein of mice also demonstrated the specificity, with a maximum fluorescence at six hours post-injection. Sensors 2012, 12 14798 Smith et al. showed how QDs allow for the non-invasive visualization of blood vessel development over time [55]. The conjugation of biotinylated fibrinogen to QDs showed specificity towards the membrane of blood vessels during angiogenesis. The conjugates had robust biocompatibility, staying in circulation for days without noticeable toxicity. The authors employed QDs with longer emission wavelengths to avoid native autofluorescence [56]. Jaiswal et al. investigated slime mold behavior, a characteristic feature of single-celled organisms in response to starvation. Four distinct cultures of AX2 amoebae cells were starved for different periods of time, each labeled with a different color QDs. The cells were then mixed with a 10-fold excess of unlabeled, non-starved cells and the QD fluorescence from the labeled cells was tracked for extended observation times. Five melanoma tumor cell populations were labeled with distinct color QDs, using cationic (Lipofectamine 2000) encapsulation, injected them into the tail vein of mice, and tracked the QD emission by multi-photon fluorescence microscopy as the cells extravasated into lung tissues [57]. An alternative to two-photon fluorescence imaging, which requires high excitation powers and a pulsed laser source, deep tissue imaging could rely on NIR fluorescence using nanocrystals that emit at 650–900 nm, a region of the optical spectrum that overlaps with tissue transparency window. NIR fluorescence could allow deep tissue imaging in animals with increased depth and with reduced background [58,59]. Krishnan et al. reported in vivo NIR imaging using epidermal growth factor (EGF) conjugated NIR emitting QDs to image the over-expression of epidermal growth factor receptor (EGFR) in tumors compared to normal expression levels in the surrounding normal tissues [60]. They found that following initial incubation, both unconjugated and EGF-coupled QDs exhibited comparable nonspecific uptake into tumor. However, the concentration of free QDs in the tumors gradually decreased, while EGF-QDs progressively accumulated in tumors up to 6 h after injection. Recently, Kim et al. explored the targeting and imaging of lymphatic vessels using QD-conjugated to hyaluronic acid (QD-HA conjugates). They prepared conjugates using electrostatic attractions between anionic HA and amine functionalized DHLA-QDs, and utilized the specific properties of hyaluronic acid to target and visualize lymphatic vessels [61]. Hyaluronic acid (HA) is found in the extracellular matrix and contributes significantly to an array of processes, including cell proliferation, wound healing, metastasis, and prognosis of some malignant tumors. Authors injected HA-QDs into ears of nude mice, and showed that endothelial cells (in lymphatic vessels) could be clearly visualized for up to several days. Choi et al. explored renal clearance of an intravenously administered CdSe-ZnS QDs in mice as a model system [62]. They used several size QDs capped with various hydrophilic small ligands via cap exchange and measured their half-life circulation before eventually being collected in the bladder or other organs (liver, spleen). More recently they followed up on those findings and reported on the correlation between the stabilizing ligands and the biodistribution and clearance of the hydrophilic QDs. They stabilized InAs-ZnS QDs (using InAs instead of a CdSe core) with DHLA and DHLA-PEG ligands with varying PEG chain-length. These results suggested that the biodistribution and clearance of QDs were dependent not only on the core size of the QDs but the state of the stabilizing ligands. Ultra short PEG with two units of ethylene glycol produced QDs with small hydrodynamic diameter (HD) but retained a hydrophobic character and these QDs were rapidly captured by the liver. Conversely, increasing PEG chain length from two to 22 units produced larger HD and uptake into the liver, kidney, pancreas, and lymph nodes, though with varying degrees of uptake. On the other hand, Sensors 2012, 12 14799 long PEG chains with larger than 22 units are highly hydrophilic and QDs remain in the vasculature for long periods of time. These findings showed that PEG, usually considered as a bio-inert polymer, affected the physical properties of QDs and their biological distribution [63,64]. QD-conjugates with overall small size could also be excreted by the kidney. These studies confirmed that proper surface functionalization and size control of QDs are essential to controlling targeting, biodistribution and excretion from the body. Growing interest in study on QDs can be further demonstrated by preparation and application of different composition of QDs such as CdPbS [65], CdSe, CdSe/CdS [66], CdSeS/ZnS [67], CdTe [68], CdSe [69], Ag2S [70,71], Ag2Se [72] or GdS/Eu [73]. Due to the materials used for QDs synthesis, concerns regarding their toxicity have been raised and numerous studies have been carried out [74,75]. At the same time, methods of synthesis of nonor low toxic QDs have been developed [76]. The excitation of traditional fluorescent labels such as organic dyes, fluorescent proteins and QDs usually requires the use of UV or short-wavelength radiation, which results in serious obstacles including low light-penetration depth inherent to the short-wavelength excitation light and/or low signal-to-noise ratio due to significant tissue autofluorescence. Therefore, it is highly essential to develop more efficient biolabels to overcome these limitations. One of such tags is represented by the ―upconversion‖ paricles. In particular lanthanide doped rare-earth nanocrystals, are characterized by the conversion of long-wavelength radiation, for instance infrared or near infrared radiation, to short-wavelength radiation, usually in the visible range [77–79]. Multimodal Imaging As mentioned above various imaging modalities have been exploited and are successfully employed to investigate the anatomic or functional dissemination of tissues in the body. However, no single imaging modality allows overall structural, functional, and molecular information as each imaging modality has its own unique strengths and weaknesses. The combination of two popular paging modalities that investigates the strengths of different methods might offer the prospect of improved diagnostic and therapeutic monitoring abilities. Multimodal molecular imaging can offer a synergistic improvement of diagnostic ability over a single imaging modality. Inorganic nanoparticles such as magnetic iron oxide nanoparticles are studied as MRI contrast agents, QDs or upconversion nanoparticles serve as optical probes and/or metallic gold nanoparticles exhibit unique light scattering patterns for surface-enhanced Raman spectroscopy. Most popular nanostructured multimodal imaging probes are combinations of MRI and optical imaging modalities [76,80], however also trimodal imaging probes have also been designed by adding another imaging modality. In particular, positron emission tomography isotopes such as 111In or 64Cu, which emit gamma rays from their decay, have been most frequently used as the third probes [81]. 4. Imaging by Microscopic Techniques 4.1. Light and Fluorescence Microscopy Despite the fact that this technique is the well-known and the well-used in biology, its application in the detection of NPs is relatively limited. The first field is based on the histochemical detection of Sensors 2012, 12 14800 components of NPs, usually oxides of metal ions. Newer and newer procedures are being proposed and developed [82]. The second possibility is based on the immunohistochemistry, the process based on the detection of antigens in cells and tissues on the principle of monoclonal or polyclonal antibodies binding specifically to antigens [83,84]. However, this technique is usable only for functionalized NPs carrying antigenic structure. Chromogenic reporters are used for the detection of target, in our case NPs. The most common chromogenic reporters are enzymes, such as alkaline phosphatase (AP) and horseradish peroxidase (HRP). In addition, they can be detected not only by chromogenic substrates, such as 3,3ʹ-diaminobenzidine (DAP) or 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium (BCIP/NBT), but also by fluorogenic and chemiluminiscent substrates usable in fluorescence microscopy. Light microscopy is used for the visualization of iron oxide-based NPs, which is based mostly on the processing of the semi thin section and Prussian blue/Berlin blue reaction method staining. This technique was used in the visualization of SPIONs in the human cochleae and mouse inner ear cell culture [85]. Incorporation of peroxidase into or onto NPs, which is widely used in the construction of hydrogen peroxide biosensors, may represent useful tool in the visualization of nanoparticles [86,87]. Like this modified NPs may catalyze the oxidation of peroxidase substrates to produce a color reaction at the site of NPs. Based on this NPs-peroxidase approach, Zhuang et al. determined the biodistribution and organ clearance of magnetic NPs in mice by visualizing and quantifying the localization of MNPs within the main organs [88]. This approach has significantly increased the sensitivity of NPs detection compared to convenient Prussian blue staining. Light microscopy may be suitably combined with next imaging techniques, such as magnetic resonance. For example, Neuwelt et al. compared histochemical and magnetic resonance detection of Ferumoxtran-10, a dextran-coated iron oxide nanoparticles, in patients suffering from primary metastatic malignant brain tumors [5]. Histochemistry revealed presence of Ferumoxtran-10 NPs in the reactive cells, predominantly in reactive astrocytes and macrophages, and not in tumor cells. This work demonstrates possible benefit of combination of magnetic resonance with histochemistry, which may improve our knowledge about distribution of NPs suffering from malignant disease. Compared to convenient light microscopy, fluorescence microscopy brings some advantages, especially significantly higher quality of images. Samples must be fluorescently stained despite the fact that many compounds are responsible for the intrinsic autofluorescence of sample. Autofluorescence may be used for detection of nanoparticles, as NPs are able to affect autofluorescence of cells. These effects are probably caused by the high surface area of the NPs and ability to catalyze different processes leading to the chemical modification of autofluorescent biomolecules. El-Sayed et al. treated two oral squamous carcinoma cell lines and one immortalized benign epithelial cell line with gold nanoparticles of different shapes, both spheres and rods and monitored changes in autofluorescence caused by soluble NADH and collagen. They significantly quenched the autofluorescence of both compounds based on the oxidation of NADH to NAD+ catalyzed by NPs surface and photonic absorption of light by collagen [89]. This knowledge suggests new potential of NPs in cell and tissue imaging. Fluorescence microscopy enables combination of differently labeled secondary antibodies and multifunctional cell/tissue labeling. Sensors 2012, 12 14807 of QDs to produce ROI induced by direct photoactivation is lower [177]. Therefore conjugates between QDs and conventional photosensitising drugs are subjects of intensive research [178]. Other application of nanoparticules is hyperthermia. Thermal ablation involves heating cells at temperatures above 55 °C, which causes coagulative necrosis and protein denaturation that kill targeted cells. The temperature of carbon nanotubes can rise to over 60 °C within 2 min when they are exposed to near infrared wavelengths of 700–1,100 nm [179]. Research has been focused on development of nanostructures that produce heat on activation by near infrared optical excitation. Such nanoparticules target specifically the tumor and selectively ablate it, leaving surrounding healthy tissue undamaged. This therapeutic modality has been shown to be efficacious for the treatment of several tumors, e.g., lung, liver, and prostate carcinoma [180]. Magnetic nanoparticles can also be used for hyperthermia. Magnetic nanoparticles can be localized in deep tissue and fixed by external magnetic field and alteration of the AC field increase the temperature of the magnetic nanoparticles [179]. Radiosensitizers are drugs that enhance the effects of radiotherapy, and some of them are metallic nanoparticles. Such nanoparticles should provide enhanced interaction cross-section with gamma photons. They increase effect of radiotherapy by scattering and/or absorbing X and γ rays causing localized damage of cancer cells and thus decreasing total radiation dose to minimize side effects [181]). This approach of using nanoparticules as radiosensitizers is termed Nanoparticle Enhanced X-ray Therapy (NEXT). It is planned to test CaF, LaF, ZnS or ZnO QD, carbon dots, liposomes with some radiosensitizers or antioxidants, nanoparticle-photosensitizer conjugates and silicon nanoparticles [182]. Radiosensitization by 1–12 nm gold nanoparticles irradiated by high energy electrons generated low energy electrons that caused increased DNA damage [183]. In another study, gold microspheres of 1.5–3.0 μm size have been tested both in vitro and in vivo studied for enhancement of X-rays that resulted in radiosensitization by gold particles [184]. Furthermore, the application of gold nanorods for therapeutic purposes has been investigated [185–187]. Contrast nanoparticules may also increase efficiency and/or safety of radiotherapy by improvement of planning by MRI [188]. Nanotechnology may also be applied in diagnostics for surgical oncology. Nanoparticles can be accumulated in solid tumors that enable one to localize tumors, detect margins, identify important adjacent structures, map sentinel lymph nodes, and detect residual tumor cells or micrometastases. Intraoperative imaging overcomes the tissue penetration problem of optical methods [189]. 7. Outlook Imaging techniques are progressing rapidly due to the development of new types of contrast agents as well as progress in the development of instrumentation, electronics and software. This is a multi/transdisciplinary approach combining the knowledge of chemists, biologists, physicists, and mathematicians as well as engineers and IT specialists. It is highly improbable that QDs will fully replace the well-established technologies using fluorophores or fluorescent protein-fusion, but will rather complement them for applications needing better photostability, sensitivity and wide variability. Magnetic nanoparticles will also broaden the area of already employed contrast agents and find more and more biomedical applications for their unique properties. The advantages of these types of nanoparticles are their low cost synthesis, huge modification possibilities and easy application. 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