There is a growing realization that cell-to-cell variations in gene expression have important biological consequences underlying phenotype diversity and cell fate. Although analytical tools for measuring gene expression, such as DNA microarrays, reverse-transcriptase PCR and in situ hybridization have been widely utilized to discover the role of genetic variations in governing cellular behavior, these methods are performed in cell lysates and/or on fixed cells, and therefore lack the ability to provide comprehensive spatial-dynamic information on gene expression. This has invoked the recent development of molecular imaging strategies capable of illuminating the distribution and dynamics of RNA molecules in living cells. In this review, we describe a class of molecular imaging probes known as molecular beacons (MBs), which have increasingly become the probe of choice for imaging RNA in living cells. In addition, we present the major challenges that can limit the ability of MBs to provide accurate measurements of RNA, and discuss efforts that have been made to overcome these challenges. It is envisioned that with continued refinement of the MB design, MBs will eventually become an indispensable tool for analyzing gene expression in biology and medicine.

Mitochondrial redox states provide important information about energy-linked biological processes and signaling events in tissues for various disease phenotypes including cancer. The redox scanning method developed at the Chance laboratory about 30 years ago has allowed 3D highresolution (～ 50 × 50 × 10μm3) imaging of mitochondrial redox state in tissue on the basis of the fluorescence of NADH (reduced nicotinamide adenine dinucleotide) and Fp (oxidized flavoproteins including flavin adenine dinucleotide, i.e., FAD). In this review, we illustrate its basic principles, recent technical developments, and biomedical applications to cancer diagnostic and therapeutic studies in small animal models. Recently developed calibration procedures for the redox imaging using reference standards allow quantification of nominal NADH and Fp concentrations, and the concentration-based redox ratios, e.g., Fp/(Fp+NADH) and NADH/(Fp+NADH) in tissues. This calibration facilitates the comparison of redox imaging results acquired for different metabolic states at different times and/or with different instrumental settings. A redox imager using a CCD detector has been developed to acquire 3D images faster and with a higher in-plane resolution down to 10 μm. Ex vivo imaging and in vivo imaging of tissue mitochondrial redox status have been demonstrated with the CCD imager. Applications of tissue redox imaging in small animal cancer models include metabolic imaging of glioma and myc-induced mouse mammary tumors, predicting the metastatic potentials of human melanoma and breast cancer mouse xenografts, differentiating precancerous and normal tissues, and monitoring the tumor treatment response to photodynamic therapy. Possible future directions for the development of redox imaging are also discussed.

Fluorescence molecular imaging enables the visualization of basic molecular processes such as gene expression, enzyme activity, and disease-specific molecular interactions in vivo using targeted contrast agents, and therefore, is being developed for early detection and in situ characterization of breast cancers. Recent advances in developing near-infrared fluorescent imaging contrast agents have enabled the specific labeling of human breast cancer cells in mouse model systems. In synergy with contrast agent development, this paper describes a needle-based fluorescence molecular imaging device that has the strong potential to be translated into clinical breast biopsy procedures. This microendoscopy probe is based on a gradient-index (GRIN) lens interfaced with a laser scanning microscope. Specifications of the imaging performance, including the field-of-view, transverse resolution, and focus tracking characteristics were calibrated. Orthotopic MDA-MB-231 breast cancer xenografts stably expressing the tdTomato red fluorescent protein (RFP) were used to detect the tumor cells in this tumor model as a proof of principle study. With further development, this technology, in conjunction with the development of clinically applicable, injectable fluorescent molecular imaging agents, promises to perform fluorescence molecular imaging of breast cancers in vivo for breast biopsy guidance.

Even though multispectral imaging is considered very significant in biological imaging, it is only commonly used in microscopy in a 2D approach. Here, we present a Fluorescence Molecular Tomography system capable of recording simultaneously tomographic data at several spectral windows, enabling multispectral tomography. 3D reconstructed data from several spectral windows is used to construct a linear unmixing algorithm for multispectral deconvolution of overlapping fluorescence signals. The method is applied on tomographic 3D fluorescence concentration maps in tissue-mimicking phantoms, yielding absolute quantification of the concentration of each individual fluorophore. Results are compared to the case when unmixing is performed in the raw 2D data instead of the reconstructed 3D concentration map, showing greater accuracy when unmixing algorithms are applied in the reconstructed data. Both the reflection and transmission geometries are considered.

We have imaged mitochondrial oxidation–reduction states by taking a ratio of mitochondrial fluorophores: NADH (reduced nicotinamide adenine dinucleotide) to Fp (oxidized flavoprotein). Although NADH has been investigated for tissue metabolic state in cancer and in oxygen deprived tissues, it alone is not an adequate measure of mitochondrial metabolic state since the NADH signal is altered by dependence on the number of mitochondria and by blood absorption. The redox ratio, NADH/(Fp+NADH), gives a more accurate measure of steady-state tissue metabolism since it is less dependent on mitochondrial number and it compensates effectively for hemodynamic changes. This ratio provides important diagnostic information in living tissues. In this study, the emitted fluorescence of mouse colon in situ is passed through an emission filter wheel and imaged on a CCD camera. Redox ratio images of the healthy and hypoxic mouse intestines clearly showed significant differences. Furthermore, the corrected redox ratio indicated an increase from an average value of 0.51 ± 0.10 in the healthy state to 0.92 ± 0.03 in dead tissue due to severe ischemia (N = 5). We show that the CCD imaging system is capable of displaying the metabolic differences in normal and ischemic tissues as well as quantifying the redox ratio in vivo as a marker of these changes.

The fluorescence properties of reduced nicotinamide adenine dinucleotide (NADH) and oxidized flavoproteins (Fp) including flavin adenine dinucleotide (FAD) in the respiratory chain are sensitive indicators of intracellular metabolic states and have been applied to the studies of mitochondrial function with energy-linked processes. The redox scanner, a three-dimensional (3D) low temperature imager previously developed by Chance et al., measures the in vivo metabolic properties of tissue samples by acquiring fluorescence images of NADH and Fp. The redox ratios, i.e. Fp/(Fp+NADH) and NADH/(Fp+NADH), provided a sensitive index of the mitochondrial redox state and were determined based on relative signal intensity ratios. Here we report the further development of the redox scanning technique by using a calibration method to quantify the nominal concentration of the fluorophores in tissues. The redox scanner exhibited very good linear response in the range of NADH concentration between 165–1318μM and Fp between 90–720μM using snap-frozen solution standards. Tissue samples such as human tumor mouse xenografts and various mouse organs were redox-scanned together with adjacent NADH and Fp standards of known concentration at liquid nitrogen temperature. The nominal NADH and Fp concentrations as well as the redox ratios in the tissue samples were quantified by normalizing the tissue NADH and Fp fluorescence signal to that of the snap-frozen solution standards. This calibration procedure allows comparing redox images obtained at different time, independent of instrument settings. The quantitative multi-slice redox images revealed heterogeneity in mitochondrial redox state in the tissues.

In this study, we report the fabrication of engineered iron oxide magnetic nanoparticles (MNPs) functionalized with anti-human epidermal growth factor receptor type 2 (HER2) antibody to target the tumor antigen HER2. The Fc-directed conjugation of antibodies to the MNPs aids their efficient immunospecific targeting through free Fab portions. The directional specificity of conjugation was verified on a macrophage cell line. Immunofluorescence studies on macrophages treated with functionalized MNPs and free anti-HER2 antibody revealed that the antibody molecules bind to the MNPs predominantly through their Fc portion. Different cell lines with different HER2 expression levels were used to test the specificity of our functionalized nanoprobe for molecular targeting applications. The results of cell line targeting demonstrate that these engineered MNPs are able to differentiate between cell lines with different levels of HER2 expression.

Insulin secretion is a complex and highly regulated process. Although much progress has been made in understanding the cellular mechanisms of insulin secretion and regulation, it remains unclear how conclusions from these studies apply to living animals. That few studies have been done to address these issues is largely due to the lack of suitable tools in detecting secretory events at high spatial and temporal resolution in vivo. When combined with genetically encoded biosensor, optical imaging is a powerful tool for visualization of molecular events in vivo. In this study, we generated a DNA construct encoding a secretory granule resident protein that is linked with two spectrally separate fluorescent proteins, a highly pH-sensitive green pHluorin on the intra-granular side and a red mCherry in the cytosol. Upon exocytosis of secretory granules, the dim pHluorin inside the acidic secretory granules became highly fluorescent outside the cells at neutral pH, while mCherry fluorescence remained constant in the process, thus allowing ratiometric quantification of insulin secretory events. Furthermore, mCherry fluorescence enabled tracking the movement of secretory granules in living cells. We validated this approach in insulin-secreting cells, and generated a transgenic mouse line expressing the optical sensor specifically in pancreatic β-cells. The transgenic mice will be a useful tool for future investigations of molecular mechanism of insulin secretion in vitro and in vivo.

Background and Aims: Accurate endoscopic detection of premalignant lesions and early cancers in the colon is essential for cure, since prognosis is closely related to lesion size and stage. Although it has great clinical potential, autofluorescence endoscopy has limited tumorto- normal tissue image contrast for detecting small preneoplastic lesions. We have developed a molecularly specific, near-infrared fluorescent monoclonal antibody (CC49) bioconjugate which targets tumor-associated glycoprotein 72 (TAG72), as a contrast agent to improve fluorescencebased endoscopy of colon cancer. Methods: The fluorescent anti-TAG72 conjugate was evaluated in vitro and in vivo in athymic nude mice bearing human colon adenocarcinoma (LS174T) subcutaneous tumors. Autofluorescence, a fluorescent but irrelevant antibody and the free fluorescent dye served as controls. Fluorescent agents were injected intravenously, and in vivo whole body fluorescence imaging was performed at various time points to determine pharmacokinetics, followed by ex vivo tissue analysis by confocal fluorescence microscopy and histology. Results: Fluorescence microscopy and histology confirmed specific LS174T cell membrane targeting of labeled CC49 in vitro and ex vivo. In vivo fluorescence imaging demonstrated significant tumor-to-normal tissue contrast enhancement with labeled-CC49 at three hours post injection, with maximum contrast after 48 h. Accumulation of tumor fluorescence demonstrated that modification of CC49 antibodies did not alter their specific tumor-localizing properties, and was antibody-dependent since controls did not produce detectable tumor fluorescence. Conclusions: These results show proof-of-principle that our near-infrared fluorescent-antibody probe targeting a tumor-associated mucin detects colonic tumors at the molecular level in real time, and offer a basis for future improvement of image contrast during clinical fluorescence endoscopy.

Working memory is one of the most important functions in our brain, which has been widely studied with unreal-life measured technologies. A functional near-infrared spectroscopy (fNIRS) instrument with a portable and low-cost design is developed, which is capable of providing hemodynamic measurement associated with brain function in real-life situations. Using this instrument, we performed working memory studies involved in Chinese words encoding, verbal, and spatial stem recognition, which are mainly studied with other technologies. Our results show that fNIRS can well assess working memory activities, in comparison with the reported results mainly using other methodologies. Furthermore, we find that hemodynamic change in the prefrontal cortex during all working memory tasks is highly associated with subjects’ behavioral data. fNIRS is shown to be a promising alternative to the current methodologies for studying or assessing functional brain activities in natural condition.

Doppler Optical Coherence Tomography (DOCT) is a noninvasive optical diagnostic technique, which is well suited for the quantitative mapping of microflow velocity profiles and the analysis of flow-vessel interactions. The noninvasive imaging and quantitative analysis of blood flow in the complex-structured vascular bed is required in many biomedical applications, including those where the determination of mechanical properties of vessels or the knowledge of the mechanic interactions between the flow and the housing medium plays a key role. The change of microvessel wall elasticity could be a potential indicator of cardiovascular disease at the very early stage, whilst monitoring the blood flow dynamics and associated temporal and spatial variations in vessel’s wall shear stress could help predicting the possible rupture of atherosclerotic plaques. The results of feasibility studies of application of DOCT for the evaluation of mechanical properties of elastic vessel model are presented. The technique has also been applied for imaging of sub-cranial rat blood flow in vivo.

We are pleased to present this focus issue on optical molecular imaging, which has seen new developments in novel optical imaging techniques and functional optical probes for studying molecular events in living organisms. This focus issue covers optical biosensor development, probe targeting, multispectral tomography, and new imaging and signal processing techniques that improve the study of molecular and cellular events in living cells and small animals. The ultimate goal is to use optical imaging techniques to monitor genetic expression and protein function during cellular processes and elucidate biological phenomena, leading to earlier detection of diseases, more effective evaluation of treatment effects, and new approaches to speed drug discovery and development. This issue includes two review articles and nine original papers.