There are numerous challenges to exciting and observing fluorescence within the tissue of animals used for biomedical research and of humans for diagnostic purposes. Tissues strongly absorb and scatter visible light, especially light in the shorter wavelength blue region of the spectrum. That makes it difficult to excite and detect fluorescence at significant tissue depths beyond a few millimeters. Living tissue also fluoresces when an external light source is used to excite fluorescent labels. This autofluorescence causes a lack of contrast in images or washes the image out of the fluorescence emanating from the labeled material of interest. Figure 1. Sensitivity and multicolor capability of quantum dot (QD) imaging in live animals. Adapted from Int J Mol Sci 2009, 10(2), 441-491. Courtesy of Creative Commons Attribution License (CC BY), original work Anal Chem 2004, 76, 2406-2410. Fortunately, tissue has two windows of relative transparency in the near-infrared (NIR) wavelength regions of 700 to 900 nm (NIR-I) and 1000 to 1400 nm (NIR-II). When the wavelengths of fluorescent light and/or excitation light employed are within a transparency window, higher-contrast images and greater tissue penetration are possible. Obtaining excitation and/or fluorescence in either NIR region has been difficult with traditional organic dye fluorescent agents because only a relatively few dyes that emit in the NIR are available. Further, those that are available generally have low fluorescent intensities, or quantum yields. This need for greatly improved fluorescent probes in the NIR regions for in vivo and tissue imaging has driven demand for quantum dots (QDs). QD advantages Due to their optical properties, photostability and suitability for the application of spatial superresolution methods, QDs have numerous advantages over organic dyes for in vivo imaging. Often cited is the fact that QDs are strongly resistant to photobleaching in comparison to organic dyes, which can quickly degrade under excitation from a light source, especially intense laser sources. Also, QDs can fluoresce more brightly in the 700- to 900-nm wavelength range optimal for in vivo imaging. Importantly, QDs are ideal for multispectral imaging where different types of cells or tissues can be imaged simultaneously by labeling each with QDs that fluoresce at distinctly different wavelengths (Figure 1). Because QDs absorb light for excitation over a wide range of wavelengths, a single light source can be used to generate simultaneous fluorescence from QDs emitting at many different wavelengths. This is a distinct advantage over fluorescent agents based upon organic dyes. Additionally, the narrow emission bandwidths of QDs allow observation of fluorescence at multiple wavelengths without significant spectral overlap. The high fluorescence levels of QDs are very well-suited to the implementation of several recently developed methods that improve the spatial resolution of in vivo imaging. These techniques allow spatial resolutions that exceed the diffraction limit of light, which is typically 200 nm or greater. Many of these methods require multiple fluorophores within a microscope’s field of view and include stimulated emission depletion (STED) microscopy, structured illumination microscopy, photoactivated localization microscopy (PALM), fluorescence photoactivated localization microscopy (FPALM) and stochastic optical reconstruction microscopy (STORM). Lateral resolutions of 100 nm or less are possible with these methods. Superresolution optical fluctuation imaging (SOFI) and independent component analysis (ICA) are two advantageous superresolution techniques that can be achieved with the use of QDs. These methods need only one fluorophore in the field of view, and they take advantage of the fact that single QDs blink fluorescence. SOFI has been shown to provide superresolution in all three dimensions using confocal microscopy. The development of QDs QDs are nanocrystals of semiconductor materials that typically have diameters ranging from 2 to 10 nm. Semiconductor materials have a characteristic bandgap between the valence and conduction bands of the material that establishes the wavelength of fluorescence. In the case of nanocrystals, their small size changes the energy difference of the bandgap and hence fluorescence wavelength due to an effect known as quantum confinement, which is a function of the diameter of the nanocrystal. By growing semiconductor nanocrystals to different sizes, the wavelength of fluorescence can be tuned over a significant range of values with larger nanocrystals yielding fluorescence at longer wavelengths. Zinc sulfide (ZnS), zinc selenide (ZnSe) and cadmium sulfide (CdS) are commonly used as shell materials and result in achievement of quantum yields in the range of 40 to 80 percent, yielding bright fluorophores. The outer shell must have a final coating of surfactant that is either hydrophobic to allow dispersion in organic solvents or is hydrophilic to allow dispersion in aqueous solutions (Figure 2). Figure 2. Typical quantum dot structure. Courtesy of NanoOptical Materials Inc. QDs are made by mixing reactive forms of the individual composition elements in a suitable solvent, most often a high boiling point organic liquid. In most cases, the elements are mixed using a rapid injection technique at high temperature to achieve rapid nucleation followed by a slower growth of the nanocrystals. Production methods can be based on batch preparation or continuous flow designs. Extended wavelength ranges and heavy-metal-free QDs Two more recent and very significant developments in QD manufacturing have been the extension of the range of fluorescence wavelengths further into the NIR and the availability of less toxic QDs that do not contain heavy metals such as cadmium (Cd) or lead (Pb). The first simple, binary QDs found to fluoresce in the NIR-I region and well-suited for in vivo imaging were made of cadmium telleride (CdTe) and lead sulfide (PbS) QDs with shells such as ZnS or CdS. PbS-based QDs also can be prepared to emit at wavelengths in the NIR-II region. However, imaging at wavelengths longer than 900 nm requires considerably more expensive cameras sensitive to the longer wavelength light. More recently, heavy-metal-free QDs have been demonstrated as fluorophores that emit light in both the visible and NIR regions of the spectrum. In the visible region, QDs with cores consisting of indium phosphide (InP), silver sulfide (Ag2S), copper indium sulfide (CuInS), copper indium zinc sulfide (CuInZnS) and other semiconductors can be prepared with shells to brighten fluorescence and in water soluble forms. In the NIR regions most important for in vivo imaging, fluorescence can be obtained from a variety of QDs having cores of copper indium selenide (CuInSe), CuInZnS, Ag2S, silver selenide (Ag2Se) and other semiconductors. Cd-based QDs offer a number of advantages for in vivo imaging. They are commercially available over a wide range of visible and NIR wavelengths in water soluble forms with a variety of coatings for bioconjugation and/or to minimize nonspecific adsorption. The narrower fluorescence bandwiths of Cd-based QDs can yield more vivid images and allow more distinct colors to be imaged in multispectral applications where different cells or tissues are simultaneously labeled. Toxicity concerns The major disadvantage of Cd- and Pb-based QDs is the potential toxicity or cytotoxicity associated with their use. Experimental studies have indicated that all QDs introduced into a living organism will slowly degrade and their elements eventually will be transported to the liver and spleen. Heavy metal QDs do have some cytotoxic effects on exposed cells, and such cytotoxicity could complicate interpretation of the results of research studies utilizing in vivo imaging. Figure 3. In vivo fluorescence images of CdSe/ZnS QDs, ICG (NIR-I), and Ag2S QDs (NIR-II) in nude mice. (a) CdSe/ZnS QDs, ICG and Ag2S QDs were injected intravenously into mice. The green-yellow signal of the mouse injected with CdSe/ZnS QDs indicates the strong autofluorsecence of tissues in the visible emission window. The red signal concentrated in the liver of the mouse injected with ICG indicates the short blood circulation half-time. The red signal widely distributed in the whole body of mouse injected with Ag2S QDs indicates the long blood circulation half-time. (b) The photoluminesence spectra of CdSe@ZnS QDs, ICG and Ag2S QDs. Courtesy of Creative Commons Attribution License (CC BY), original work Biomaterials 35 (2014) 393-400. Toxicity concerns also prevent the use of heavy-metal-containing QDs for human diagnostic procedures. The only FDA-approved fluorophores are organic dyes that have limited sensitivity and stability. Research studies have shown the use of QD labeling can enable or improve the sensitivity of such imaging, but FDA approval is highly unlikely for heavy-metal-based QDs. Future approval by the agency is more likely for heavy-metal-free QDs. Figure 4. NIR-II fluorescence imaging of a xenograft tumor with high uptake of 6PEG-Ag2S QDs. Time course of NIR-II fluorescence images of the mouse injected with 6PEG-Ag2S QDs. Adapted from Colloids and Surfaces B: Biointerfaces 124 (2014) 118–131. Courtesy of Creative Commons Attribution License (CC BY), original work, Angew. Chem Int Ed 51 (2012) 9818–9821. Bioimaging applications made possible by the use of quantum dots Numerous biomedical studies have been enabled by the use of QDs for in vivo and cell imaging. One area that has benefited greatly is vasculature imaging. The tracing of blood flow and visualization of blood vessels in experiments on animals is important to the study of many pathogenic processes including tumor growth and cancer metastasis. In the past, the NIR fluorescent dye indocyanine green (ICG) was used to trace blood flow, but its fluorescence intensity is low (Figure 3). Figure 5. RGD-labeled paramagnetic quantum dots (RGD-pQDs) specifically target tumor blood vessels in in vivo microscopy of microvessels in tumor-bearing mice (C57Bl6) after intravenous injection of RGDpQDs (a, b). Bright-field microscopy (left panels) was used to select blood vessels, indicated by the yellow lines. Fluorescence microscopy (right panels) revealed labeling of endothelial cells in tumor blood vessels of mice that were injected with RGD-pQDs, as indicated by the yellow arrows (a,b). Courtesy of Creative Commons Attribution License (CC BY), original work, Angiogenesis (2009) 12:17–24. When QDs are linked to biorecognition molecules it is possible to specifically label different tissue types, especially cancer tumors. Suitably coated QD probes can be injected into test animals where they will distribute throughout the animal and be retained within the target tissues (Figure 4). The bright fluorescence of QDs can be used to actually image and track individual cells within tissues such as tumors and blood vessels (Figure 5). Figure 6. Methylene blue dye injected around a colorectal tumor to map lymph nodes. Adapted from Symbiosis, May 2015, Courtesy of Creative Commons Attribution License (CC BY), original work Gastroenterol Pancreatol Liver Disord 2(1): 1-8. Currently, surgeons can use FDA-approved florescent dyes to label sentinel (nearby) lymph nodes during surgeries intended to remove cancerous cells and tumors, so rapid biopsies can be done to determine how extensively cancer cells may have spread. Animal studies have shown that the sensitivity of real-time imaging of lymph nodes can be significantly improved by the use of QDs (Figure 6). If heavy-metal-free QDs ultimately gain FDA approval, significant enhancements to sensitivity and contrast will be possible. Meet the author Glenn J. Bastiaans is co-founder and president of NanoOptical Materials in Carson, Calif.; email: glennb@nomcorp.com Picking the Right QD for In Vivo Imaging There is a wider variety of quantum dots (QDs) with heavy metals, such as cadmium (Cd) and lead (Pb) for all applications, but the choice of heavy-metal-free QDs is much more limited for in vivo imaging. Deciding which QD to select for a specific in vivo imaging application largely depends on the solution the specimen will be dissolved in and the wavelengths of fluorescence light to be employed. Currently, commercially available heavy-metal-free QDs only fluoresce within a small segment of the preferred NIR regions. This limitation is expected to change in the near future as the utility of these QDs becomes more widely recognized. QDs dispersible in aqueous solutions often are commercially available with a choice of chemical function at the end of the outer surfactant coating in the form of either a carboxylic acid group, a primary amine (for attachment to other probe molecules) or a polyethylene glycol polymer (for improved biocompatibility). Additionally, the overall diameters of water-soluble QDs are larger for QDs produced by overcoating with a polymer versus thiol monolayer coating and may limit tissue diffusion in some cases. There is also limited availability for water-soluble QDs attached to proteins. Table 1. A comparison of heavy metal and heavy-metal-free quantum dots for in vivo imaging.