Semiconductor fluorescent nanocrystals (quantum dots) in biological biochips

Comprehension of biological processes in cells, tissues and organisms requires identification and analysis of numerous biological objects, mechanisms of their action and regulation. Microarray (biochips) technology is a rare tool to solve this problem. It is based on high-throughput recognition of a target to the probe and has the potential to measure simultaneously the presence of numerous molecules in multiplexed testes, all contained in a small drop of test fluid. Biochips allow the parallel analysis of genomic or proteomic content in healthy versus disease-affected or altered tissues or cells. The signals read-out from the biochips is done with organic dyes which often suffer from photobleaching, low brightness and background fluorescence. Recent data show that the use of fluorescent nanocrystals "quantum dots" (QDs) allows push away these restrictions. The QDs are sufficiently bright to be detected as individual particles, extremely resistant to photobleaching and provide unique possibilities for multiplexing thus supplying the microarray technology with the novel read-out option enabling the sensitivity of detection reaching the single molecule level. This paper is aimed at the development of the approaches to the QDs application in microarray-based detection. Possibilities of QDs application both in solid state (planar) biochips as well as intensively developing technique of suspension biochips (bead-based assays or liquid biochips) are demonstrated. The latter are more and more applied for simultaneous identification of very large numbers of molecules in proteomics, genomics, drug screening and clinical diagnostics. This assays base on spectral encoded elements (as a rule polymer microbeads). The benefits of using optically encoded microbeads (instead of the solid-state two-dimensional arrays) are derived from the freedom of bead to move in three dimensions. Polymeric beads optically encoded with organic dyes allow for a limited number of unique codes, whereas the use of semiconductor nanocrystals as fluorescent tags improves the beads multiplexed imaging capabilities, photostability and sensitivity of the biological objects detection. Additionally, an employment in suspension biochips of Frster resonance energy transfer (FRET) allows improving detection specificity. The absence of fluorescent background from non-interacting with the beads dye-labelled antibodies additionally increases the sensitivity of detection and further facilitates the multiplexing capabilities of nanocrystals-based detection and diagnostics. So the combination of the biochips and QDs techniques allow increasing detection sensitivity and significantly raising the number of detected objects (multiplexing capacities). Such combination should provide the breakthrough in proteomics, particularly in new drugs development, clinical diagnostics, new disease markers identification, better understanding of intracellular mechanisms.     

Porphyrin-magnetite nanoconjugates for biological imaging

Background  

The use of silica coated magnetic nanoparticles as contrast agents has resulted in the production of highly stable, non-toxic solutions that can be manipulated via an external magnetic field. As a result, the interaction of these nanocomposites with cells is of vital importance in understanding their behaviour and biocompatibility. Here we report the preparation, characterisation and potential application of new "two-in-one" magnetic fluorescent nanocomposites composed of silica-coated magnetite nanoparticles covalently linked to a porphyrin moiety.     

The use of nanocrystals in biological detection

In the coming decade, the ability to sense and detect the state of biological systems and living organisms optically, electrically and magnetically will be radically transformed by developments in materials physics and chemistry. The emerging ability to control the patterns of matter on the nanometer length scale can be expected to lead to entirely new types of biological sensors. These new systems will be capable of sensing at the single-molecule level in living cells, and capable of parallel integration for detection of multiple signals, enabling a diversity of simultaneous experiments, as well as better crosschecks and controls.     

Biocompatible fluorescent silicon nanocrystals for single-molecule tracking and fluorescence imaging

Fluorescence microscopy is used extensively in cell-biological and biomedical research, but it is often plagued by three major problems with the presently available fluorescent probes: photobleaching, blinking, and large size. We have addressed these problems, with special attention to single-molecule imaging, by developing biocompatible, red-emitting silicon nanocrystals (SiNCs) with a 4.1-nm hydrodynamic diameter. Methods for producing SiNCs by simple chemical etching, for hydrophilically coating them, and for conjugating them to biomolecules precisely at a 1:1 ratio have been developed. Single SiNCs neither blinked nor photobleached during a 300-min overall period observed at video rate. Single receptor molecules in the plasma membrane of living cells (using transferrin receptor) were imaged for ≥10 times longer than with other probes, making it possible for the first time to observe the internalization process of receptor molecules at the single-molecule level. Spatial variations of molecular diffusivity in the scale of 1–2 µm, i.e., a higher level of domain mosaicism in the plasma membrane, were revealed.     

Semiconductor quantum dots as contrast agents for whole animal imaging

Recent developments in quantum dot (QD) technology have paved the way for using QDs as optical contrast agents for in vivo imaging. Pioneering papers showed the use of QDs as luminescent contrast agents for imaging cancer and guiding cancer surgery. The possible future use of QDs for clinical applications is expected to have a significant impact, however many challenges in this field have yet to be overcome.     

Semiconductor quantum dots for in vitro diagnostics and cellular imaging

The need for companion diagnostics, point-of-care testing (POCT) and high-throughput screening in clinical diagnostics and personalized medicine has pushed the need for more biological information from a single sample at extremely low concentrations and volumes. Optical biosensors based on semiconductor quantum dots (QDs) can answer these requirements because their unique photophysical properties are ideally suited for highly sensitive multiplexed detection. Many different biological systems have been successfully scrutinized with a large variety of QDs over the past decade but their future as widely applied commercial biosensors is still open. In this review, we highlight recent in vitro diagnostic and cellular imaging applications of QDs and discuss milestones and obstacles on their way toward integration into real-life diagnostic and medical applications.     

FeCo/graphite nanocrystals for multi-modality imaging of experimental vascular inflammation

Background

FeCo/graphitic-carbon nanocrystals (FeCo/GC) are biocompatible, high-relaxivity, multi-functional nanoparticles. Macrophages represent important cellular imaging targets for assessing vascular inflammation. We evaluated FeCo/GC for vascular macrophage uptake and imaging in vivo using fluorescence and MRI.

Methods and Results

Hyperlipidemic and diabetic mice underwent carotid ligation to produce a macrophage-rich vascular lesion. In situ and ex vivo fluorescence imaging were performed at 48 hours after intravenous injection of FeCo/GC conjugated to Cy5.5 (n = 8, 8 nmol of Cy5.5/mouse). Significant fluorescence signal from FeCo/GC-Cy5.5 was present in the ligated left carotid arteries, but not in the control (non-ligated) right carotid arteries or sham-operated carotid arteries (p = 0.03 for ligated vs. non-ligated). Serial in vivo 3T MRI was performed at 48 and 72 hours after intravenous FeCo/GC (n = 6, 270 µg Fe/mouse). Significant T2* signal loss from FeCo/GC was seen in ligated left carotid arteries, not in non-ligated controls (p = 0.03). Immunofluorescence staining showed colocalization of FeCo/GC and macrophages in ligated carotid arteries.

Conclusions

FeCo/GC accumulates in vascular macrophages in vivo, allowing fluorescence and MR imaging. This multi-functional high-relaxivity nanoparticle platform provides a promising approach for cellular imaging of vascular inflammation.     

Rhodamine-based fluorogenic probe for imaging biological thiol

We have developed a new fluorescent probe for biological thiol. The probe was synthesized by the modification of the 2,4-dinitrobenzenesulfonyl group with rhodamine 110. The selective detection of thiol species such as cysteine or glutathione was achieved in biological conditions. Moreover, the probe was successfully applied to the imaging of thiol species in living human cells.     

Image-adaptive deconvolution for three-dimensional deep biological imaging

Deconvolution algorithms are widely used in conventional fluorescence microscopy, but they remain difficult to apply to deep imaging systems such as confocal and two-photon microscopy, due to the practical difficulty of measuring the system's point spread function (PSF), especially in biological experiments. Since a separate PSF measurement performed under the design optical conditions of the microscope cannot reproduce the true experimental conditions prevailing in situ, the most natural approach to solve the problem is to extract the PSF from the images themselves. We investigate here the approach of cropping an approximate PSF directly from the images, by exploiting the presence of small structures within the samples under study. This approach turns out to be practical in many cases, allowing significantly better restorations than with a design PSF obtained by imaging fluorescent beads in gel. We demonstrate the advantages of this approach with a number of deconvolution experiments performed both on artificially blurred and noisy test images, and on real confocal images taken within an in vitro preparation of the mouse hearing organ.     

Optimization for continuous-wave terahertz reflection imaging for biological tissues

Continuous-wave terahertz reflection imaging is a potential tool for biological tissues. Based on our home-made continuous-wave terahertz reflection imaging system, the effect of both polarization mode and reflection window on the imaging performance is studied theoretically and experimentally, showing good agreement. By taking obtaining sample information and image contrast into consideration, p-polarized terahertz waves are recommended. Moreover, considering the sample boundary identification and the image contrast, selection criteria for reflection window are proposed. This work will help to improve the performance of continuous-wave terahertz reflection imaging and accelerate the THz imaging in biological application.     

Development of biological tissue-equivalent phantoms for optical imaging

Optical characteristics of freshly isolated tissues depend on their color and composition. The surface backscattered profile, which account for the tissue compositional variation in fresh excised sheep's heart, lungs, bone and muscle, were measured by multi-probe reflectometer. Optical phantoms were prepared from paraffin wax by mixing a specific combination of wax color materials till the surface backscattered profile of these matched with that of the biological tissues. The optical parameters absorption coefficient (micro(a)), reduced scattering coefficient (micro(s)) and anisotropy factor (g) of these phantoms, are the same as that of biological tissues and are obtained by matching their surface backscattered profiles with that as simulated by Monte Carlo procedure. The maximum and minimum values of absorption coefficient are for the phantoms of lungs (1.0 cm(-1)) and muscle (0.02 cm(-1)), whereas, for scattering coefficient these values are for muscle (21.2 cm(-1)) and bone (13.08 cm(-1)).     

Photoactivatable fluorophores and techniques for biological imaging applications

Photoactivatable fluorophores (PAFs) are powerful imaging probes for tracking molecular and cellular dynamics with high spatiotemporal resolution in biological systems. Recent developments in biological microscopy have raised new demands for engineering new PAFs with improved properties, such as high two photon excitation efficiency, reversibility, cellular delivery and targeting. Here we review the history and some of the recent developments in this area, emphasizing our efforts in developing a new class of caged coumarins and related imaging methods for studying dynamic cell-cell communication through gap junction channels, and in extending the application of these caged coumarins to new areas including spatiotemporal control of microRNA activity in vivo.     

Unconventional modes for STEM imaging of biological structures

In this paper recent developments are discussed in instrumentation and methodology associated with scanning transmission electron microscopes (STEM), which are of great potential interest for solving structural and chemical problems in biological specimens. After describing the main features of the instrument, an attempt is made to define which type of signal acquisition and processing is best suited to obtain a given type of information. Starting with a definition of cross sections of interest, a discussion follows of methods using angular selection, energy selection of the transmitted beam, and several ways of signal mixing. More specific attention is devoted to two main modes of processing signals: ratio contrast, which emphasizes slight changes in scattering factors, rather independent of thickness variations; and elemental mapping, which provides semi-quantitative information on the distribution of low Z elements of great significance in biological specimens. Data relevant to typical biological objects are presented and discussed; they allow for the definition of the capabilities and limitations of these methods. These unconventional imaging modes define a new attitude for improving the efficiency of this modern generation of electron microscopes.     

Volume scanning electron microscopy for imaging biological ultrastructure

Electron microscopy (EM) has been a key imaging method to investigate biological ultrastructure for over six decades. In recent years, novel volume EM techniques have significantly advanced nanometre‐scale imaging of cells and tissues in three dimensions. Previously, this had depended on the slow and error‐prone manual tasks of cutting and handling large numbers of sections, and imaging them one‐by‐one with transmission EM. Now, automated volume imaging methods mostly based on scanning EM (SEM) allow faster and more reliable acquisition of serial images through tissue volumes and achieve higher z‐resolution. Various software tools have been developed to manipulate the acquired image stacks and facilitate quantitative analysis. Here, we introduce three volume SEM methods: serial block‐face electron microscopy (SBEM), focused ion beam SEM (FIB‐SEM) and automated tape‐collecting ultramicrotome SEM (ATUM‐SEM). We discuss and compare their capabilities, provide an overview of the full volume SEM workflow for obtaining 3D datasets and showcase different applications for biological research.     

Three-dimensional imaging of biological complexity

Over the past 5 years, thanks to advances in both instrumentation and computational speed, three-dimensional imaging techniques using the electron microscope have been greatly improved in two areas: electron tomography of cell organelles or cell sections and reconstruction of macromolecules from single particles. Ice embedment has brought a breakthrough in the degree of preservation of specimens under close-to-native conditions. The current challenge is to push the resolution of electron tomographic imaging to a point where macromolecular signatures can be recognized within the cellular context. We show first progress toward this goal by examples in two areas of application: the structure of the muscle triad junction and the architecture and fine structure of mitochondria. As techniques of cryo-microtomy are perfected, we hope to be able to apply tomography to high-pressure frozen sections of tissue.     

Luminescent quantum dots for multiplexed biological detection and imaging

Recent advances in nanomaterials have produced a new class of fluorescent labels by conjugating semiconductor quantum dots with biorecognition molecules. These nanometer-sized conjugates are water-soluble and biocompatible, and provide important advantages over organic dyes and lanthanide probes. In particular, the emission wavelength of quantum-dot nanocrystals can be continuously tuned by changing the particle size, and a single light source can be used for simultaneous excitation of all different-sized dots. High-quality dots are also highly stable against photobleaching and have narrow, symmetric emission spectra. These novel optical properties render quantum dots ideal fluorophores for ultrasensitive, multicolor, and multiplexing applications in molecular biotechnology and bioengineering.