多光谱成像技术在生物医学中的应用进展

多光谱成像(multispectral imaging,MSI)技术在生物医学可视化方面是一种新技术,它结合了两个已建立的光学模块:成像学和光谱学。它的原理是基于液晶可调谐滤光片,从可见光到近红外波长(400-970nm)区域获取多光谱图像。自从MSI系统加上显微镜商品化以来,MSI已经成为一种快速发展的领域,可应用于细胞生物学、临床前药物开发和临床病理学等。国外已有大量关于MSI在生物医学中应用的研究报道,但国内报道少见。本文主要对多光谱成像的基本原理,近三年内该技术在生物医学领域的应用进展作一简要综述。     

Multiplexing with multispectral imaging: from mice to microscopy

Increasing sophistication in the design and application of biological models as well as the advent of novel fluorescent probes have led to new demands on molecular imaging systems to deliver enhanced sensitivity, reliable quantitation, and the ability to resolve multiple simultaneous signals. Sensitivity is limited, especially in the visible spectral range, by the presence of ubiquitous autofluorescence signals (mostly arising from the skin and gut), which need to be separated from those of targeted fluorophores. Fluorescence-based imaging is also affected by absorbing and scattering properties of tissue in both the visible and to a lesser extent the near-infrared (NIR) regions. However, the small size of typical animal models (usually mice) often permits the detection of enough light arising even from relatively deep locations to allow the capture of signals with an acceptable signal-to-noise ratio. Multispectral imaging, through its ability to separate autofluorescence from label fluorescence, can increase sensitivity as much as 300 times compared to conventional approaches, and concomitantly improve quantitative accuracy. In the NIR region, autofluorescence, while still significant, poses less of a problem. However, the task of disentangling signals from multiple fluorophores remains. Multispectral imaging allows the separation of five or more fluorophores, with each signal quantitated and visualized separately. Preclinical small animal imaging is often accompanied by microscopic analysis, both before and after the in vivo phase. This can involve tissue culture manipulations and/or histological examination of fixed or frozen tissue. Due to the same advantages in sensitivity, quantitation, and multiplexing, microscopy-based multispectral techniques form an excellent complement to in vivo imaging.     

Back Cover: J. Biophotonics 1/2012

Hyperspectral imaging microscopy of rat lung cryoslices can be used to identify individual pulmonary microvascular endothelial cells (PMVECs) in the presence of a high lung autofluorescence of the same peak fluorescence emission wavelength. PMVECs expressing green fluorescent protein are shown in green, lung autofluorescence is shown in red, and nuclei are shown in blue. A plot of the spectral library used for linear unmixing is also shown. (Picture: S. Leavesley et al., pp. 67–84 in this issue)     

Efficient Blind Spectral Unmixing of Fluorescently Labeled Samples Using Multi-Layer Non-Negative Matrix Factorization

The ample variety of labeling dyes and staining methods available in fluorescence microscopy has enabled biologists to advance in the understanding of living organisms at cellular and molecular level. When two or more fluorescent dyes are used in the same preparation, or one dye is used in the presence of autofluorescence, the separation of the fluorescent emissions can become problematic. Various approaches have been recently proposed to solve this problem. Among them, blind non-negative matrix factorization is gaining interest since it requires little assumptions about the spectra and concentration of the fluorochromes. In this paper, we propose a novel algorithm for blind spectral separation that addresses some of the shortcomings of existing solutions: namely, their dependency on the initialization and their slow convergence. We apply this new algorithm to two relevant problems in fluorescence microscopy: autofluorescence elimination and spectral unmixing of multi-labeled samples. Our results show that our new algorithm performs well when compared with the state-of-the-art approaches for a much faster implementation.     

Microspectroscopic imaging of nodulation factor-binding sites on living Vicia sativa roots using a novel bioactive fluorescent nodulation factor.

A novel bioactive fluorescent nodulation (Nod) factor, NodRlv-IV(BODIPY FL-C16), has been synthesized by attaching a BODIPY FL-C16 acyl chain to the primary amino group of chitotetraose deacetylated at the nonreducing terminus by recombinant NodB. The binding of the fluorescent Nod factor to root systems of Vicia sativa was investigated with fluorescence spectral imaging microscopy (FSPIM) and fluorescence ratio imaging microscopy (FRIM). Spatially resolved fluorescence spectra of living and labeled Vicia sativa root systems were measured by FSPIM. Strong autofluorescence, inherent to many plant systems when excited at 488 nm, was corrected for by utilizing the difference in fluorescence emission spectra of the autofluorescence and NodRlv-IV(BODIPY FL-C16). A methodology is presented to break down the in situ fluorescence emission spectra into spatially resolved autofluorescence and BODIPY FL fluorescence spectra. Furthermore, an FRIM method was developed for correcting autofluorescence in fluorescence micrographs for this system. After autofluorescence correction it was shown that NodRlv-IV(BODIPY FL-C16) was concentrated in the root hairs, but was also bound to other parts of the root surface.     

Reduction of autofluorescence on DNA microarrays and slide surfaces by treatment with sodium borohydride

Microarray technology is currently being used extensively in functional genomics research and modern drug discovery and development. Henceforward, tremendous application potential for this technology exists in the fields of clinical diagnostics and prognostics, pathology, and toxicology for high-throughput analysis of "disease" gene expression. However, the major hurdle now in this technology is not the performance of the arrays but rather the efficient reproducibility of the hybridization signal intensity in a fluorescence-based analysis. The sensitivity of fluorescence detection on an array is to a large extent limited by the amount of background signal arising due to nonspecifically bound probes and fluorescence that is intrinsically associated with the chip substrate and/or the attached target DNA, the so-called autofluorescence. Here, we describe a simple and efficient method to reduce autofluorescence from undetermined sources on coated glass slides with and without DNA arrays. This sodium borohydride-mediated reduction process resulted in significantly lower and more even background fluorescence. This in turn extended the dynamic range of detection and reduced the average coefficient of variation of fluorescent signal ratios on DNA microarrays in addition to improving the detection of genes that are expressed at a low level.     

Fluorescence-based glucose sensors

There is an urgent need to develop technology for continuous in vivo glucose monitoring in subjects with diabetes mellitus. Problems with existing devices based on electrochemistry have encouraged alternative approaches to glucose sensing in recent years, and those based on fluorescence intensity and lifetime have special advantages, including sensitivity and the potential for non-invasive measurement when near-infrared light is used. Several receptors have been employed to detect glucose in fluorescence sensors, and these include the lectin concanavalin A (Con A), enzymes such as glucose oxidase, glucose dehydrogenase and hexokinase/glucokinase, bacterial glucose-binding protein, and boronic acid derivatives (which bind the diols of sugars). Techniques include measuring changes in fluorescence resonance energy transfer (FRET) between a fluorescent donor and an acceptor either within a protein which undergoes glucose-induced changes in conformation or because of competitive displacement; measurement of glucose-induced changes in intrinsic fluorescence of enzymes (e.g. due to tryptophan residues in hexokinase) or extrinsic fluorophores (e.g. using environmentally sensitive fluorophores to signal protein conformation). Non-invasive glucose monitoring can be accomplished by measurement of cell autofluorescence due to NAD(P)H, and fluorescent markers of mitochondrial metabolism can signal changes in extracellular glucose concentration. Here we review the principles of operation, context and current status of the various approaches to fluorescence-based glucose sensing.     

Two-photon autofluorescence microscopy and spectroscopy of Antarctic fungus: new approach for studying effects of UV-B irradiation

We combined two-photon fluorescence microscopy and spectroscopy to provide functional images of UV-B (280-315 nm) induced stress on an Antarctic fungus. Two-photon excitation microscopy was used to characterize the distribution of autofluorescence inside the spore and the hyphae of the fungus. The imaging analysis clearly shows that the autofluorescence response of spores is higher than that of hyphae. The imaging analysis at different depths shows that, strikingly enough, the spore autofluorescence originates from the cell wall and membrane fluorophores. The spectroscopic results show moreover that the fluorescence spectra of spores are redshifted upon UV-B irradiation. Tentative identification of the chromophores involved in the autofluorescence response and their biological relevance are also discussed on the basis of a previous steady-state fluorescence spectroscopic study performed on both whole spore suspension and organic-soluble extracts.     

Multiphoton excitation fluorescence microscopy and spectroscopy of in vivo human skin

Multiphoton excitation microscopy at 730 nm and 960 nm was used to image in vivo human skin autofluorescence from the surface to a depth of approximately 200 microm. The emission spectra and fluorescence lifetime images were obtained at selected locations near the surface (0-50 microm) and at deeper depths (100-150 microm) for both excitation wavelengths. Cell borders and cell nuclei were the prominent structures observed. The spectroscopic data suggest that reduced pyridine nucleotides, NAD(P)H, are the primary source of the skin autofluorescence at 730 nm excitation. With 960 nm excitation, a two-photon fluorescence emission at 520 nm indicates the presence of a variable, position-dependent intensity component of flavoprotein. A second fluorescence emission component, which starts at 425 nm, is observed with 960-nm excitation. Such fluorescence emission at wavelengths less than half the excitation wavelength suggests an excitation process involving three or more photons. This conjecture is further confirmed by the observation of the super-quadratic dependence of the fluorescence intensity on the excitation power. Further work is required to spectroscopically identify these emitting species. This study demonstrates the use of multiphoton excitation microscopy for functional imaging of the metabolic states of in vivo human skin cells.     

Applications of STED fluorescence nanoscopy in unravelling nanoscale structure and dynamics of biological systems

Fluorescence microscopy, especially confocal microscopy, has revolutionized the field of biological imaging. Breaking the optical diffraction barrier of conventional light microscopy, through the advent of super-resolution microscopy, has ushered in the potential for a second revolution through unprecedented insight into nanoscale structure and dynamics in biological systems. Stimulated emission depletion (STED) microscopy is one such super-resolution microscopy technique which provides real-time enhanced-resolution imaging capabilities. In addition, it can be easily integrated with well-established fluorescence-based techniques such as fluorescence correlation spectroscopy (FCS) in order to capture the structure of cellular membranes at the nanoscale with high temporal resolution. In this review, we discuss the theory of STED and different modalities of operation in order to achieve the best resolution. Various applications of this technique in cell imaging, especially that of neuronal cell imaging, are discussed as well as examples of application of STED imaging in unravelling structure formation on biological membranes. Finally, we have discussed examples from some of our recent studies on nanoscale structure and dynamics of lipids in model membranes, due to interaction with proteins, as revealed by combination of STED and FCS techniques.     

Development of Fern Sporangia: a Fluorescence Microscopy Study

The utility of fluorescence microscopy for studying development of fern spores is investigated. Changes in the fluorescence characteristics during the developmental stages of fern sporangia can be attributed to the changes in the chemical composition of the cell wall. Bright blue autofluorescence of the spores indicated the presence of sporopollenin. The sporan-gial walls and the spores autofluoresced yellow under blue light excitation. Fluorescence microscopy is a useful addition to light, scanning, and transmission electron microscopy because living specimens can be studied owing to their fluorescence properties.     

Gold nanoclusters as novel optical probes for in vitro and in vivo fluorescence imaging

Fluorescent probes play an important role in the development of fluorescence-based imaging techniques for life sciences research. Gold nanoclusters (AuNCs) are a novel type of fluorescent nanomaterials which have attracted great interest in recent years. Composed of only a few atoms, these ultrasmall AuNCs exhibit quantum confinement effects and molecule-like properties. Fluorescent AuNCs have an attractive set of features including ultrasmall size, good biocompatibility and photostability, and tunable emission in the red to near-infrared spectral region, which make them promising as fluorescent labels for biological imaging. Examples of their application include live cell labeling, cancer cell targeting, cellular apoptosis monitoring, and in vivo tumor imaging. Here, we present a brief overview of recent advances in utilizing these emissive ultrasmall AuNCs as optical probes for in vitro and in vivo fluorescence imaging.     

Avoiding autofluorescence problems: time-resolved fluorescence microscopy with plant and fungal cells in ectomycorrhiza

Sections of Pinus sylvestris-Suillus bovinus ectomycorrhiza were used to test the applicability of time-resolved fluorescence microscopy for studying plant and fungal material. The autofluorescence problems encountered with standard indirect immunofluorescence microscopy when studying specialized plant material were eliminated by the technique. The europium-labeled structures of both plant and fungal cells were the only visible areas in the sections. However, fine details were lost due to the coarse pixel size of the digital camera used to transfer the image on screen. Even with current resolution, time-resolved imaging can be an efficient method for detection of targets in both plant and fungal tissues with autofluorescence problems.     

Reliable online measurement of population dynamics for filamentous co-cultures

Understanding population dynamics is a key factor for optimizing co-culture processes to produce valuable compounds. However, the measurement of independent population dynamics is difficult, especially for filamentous organisms and in presence of insoluble substrates like cellulose. We propose a workflow for fluorescence-based online monitoring of individual population dynamics of two filamentous microorganisms. The fluorescent tagged target co-culture is composed of the cellulolytic fungus Trichoderma reesei RUT-C30—mCherry and the pigment-producing bacterium Streptomyces coelicolor A3(2)—mNeonGreen (mNG) growing on insoluble cellulose as a substrate. To validate the system, the fluorescence-to-biomass and fluorescence-to-scattered-light correlation of the two strains was characterized in depth under various conditions. Thereby, especially for complex filamentous microorganisms, microbial morphologies have to be considered. Another bias can arise from autofluorescence or pigments that can spectrally interfere with the fluorescence measurement. Green autofluorescence of both strains was uncoupled from different green fluorescent protein signals through a spectral unmixing approach, resulting in a specific signal only linked to the abundance of S. coelicolor A3(2)—mNG. As proof of principle, the population dynamics of the target co-culture were measured at varying inoculation ratios in presence of insoluble cellulose particles. Thereby, the respective fluorescence signals reliably described the abundance of each partner, according to the variations in the inocula. With this method, conditions can be fine-tuned for optimal growth of both partners along with natural product formation by the bacterium.     

Smartphone-based multimodal tethered capsule endoscopic platform for white-light,narrow-band,and fluorescence/autofluorescence imaging

Multimodal low-cost endoscopy is highly desirable in poor resource settings such as in developing nations. In this work, we developed a smartphone-based low-cost, reusable tethered capsule endoscopic platform that allows white-light, narrowband, and fluorescence/autofluorescence imaging of the esophagus. The ex-vivo studies of swine esophagus were performed and compared with a commercial endoscope to test the white-light imaging capabilities of the endoscope. The efficacy of the capsule for narrow-band imaging was tested by imaging the vascularization of the tongue. To determine the autofluorescence/fluorescence capability of the endoscope, fluorescein dye with different concentrations was imaged. Furthermore, swine esophagus injected with fluorescein dye was imaged using the fluorescence/autofluorescence and the white-light imaging modules, ex-vivo. The overall cost of the capsules is approximately 12 €, 15 €, and 42 € for the white light imaging, the narrow-band imaging, and the fluorescence/autofluorescence imaging respectively. In addition, the cost of the laser source module required for the narrow-band imaging and the fluorescence/autofluorescence imaging is approximately 218 €. This device will open the possibility of imaging the esophagus in underprivileged areas.     

Imaging of Streptomyces coelicolor A3(2) with Reduced Autofluorescence Reveals a Novel Stage of FtsZ Localization

Imaging of low abundance proteins in time and space by fluorescence microscopy is typically hampered by host-cell autofluorescence. Streptomycetes are an important model system for the study of bacterial development, and undergo multiple synchronous cell division during the sporulation stage. To analyse this phenomenon in detail, fluorescence microscopy, and in particular also the recently published novel live imaging techniques, require optimal signal to noise ratios. Here we describe the development of a novel derivative of Streptomyces coelicolor A3(2) with strongly reduced autofluorescence, allowing the imaging of fluorescently labelled proteins at significantly higher resolution. The enhanced image detail provided novel localization information for the cell division protein FtsZ, demonstrating a new developmental stage where multiple FtsZ foci accumulate at the septal plane. This suggests that multiple foci are sequentially produced, ultimately connecting to form the complete Z ring. The enhanced imaging properties are an important step forward for the confocal and live imaging of less abundant proteins and for the use of lower intensity fluorophores in streptomycetes.     

A trichloroacetic acid-acetone method greatly reduces infrared autofluorescence of protein extracts from plant tissue

Immunoblotting is used to determine many important characteristics of proteins. After electrophoretic separation, proteins are transferred to a membrane and reacted with a specific antibody. The antibody-protein complex is then visualized by radiographic, chromogenic, chemiluminescent, or more recently described fluorescence detection methods. Fluorescence-based detection offers some advantages over other approaches, including increased sensitivity, improved quantifiable range, and the ability to detect multiple antigens on the same blot. However, this technique is unavailable for analysis of green plant tissues by standard extraction methods because of contaminating autofluorescent pigments. We have compared 3 methods for extracting protein from plant tissue for use with infrared fluorescence-based immunoblot analysis. We report a trichloroacetic acid-acetone method that effectively eliminates autofluorescence while retaining the immunogenicity of a target protein.     

VisBio: a computational tool for visualization of multidimensional biological image data

New laser scanning microscopy techniques enable biologists to acquire larger, more complex image datasets. Emerging imaging modalities such as multispectral, harmonic, and fluorescence lifetime can generate data with six or more dimensions; however, existing software is not well suited to the visualization or analysis of such data. To address these concerns, we have developed VisBio, an application and toolkit for visualization and analysis of multidimensional, biological image data of any dimensionality.     

Enumerating viruses by using fluorescence and the nature of the nonviral background fraction

Bulk fluorescence measurements could be a faster and cheaper way of enumerating viruses than epifluorescence microscopy, flow cytometry, or transmission electron microscopy (TEM). However, since viruses are not imaged, the background fluorescence compromises the signal, and we know little about its nature. In this paper the size ranges of nucleotides that fluoresce in the presence of SYBR gold were determined for wastewater and a range of freshwater samples using a differential filtration method. Fluorescence excitation-emission matrices (FEEMs) showed that >70% of the SYBR fluorescence was in the <10-nm size fraction (background) and was not associated with intact viruses. This was confirmed using TEM. The use of FEEMs to develop a fluorescence-based method for counting viruses is an approach that is fundamentally different from the epifluorescence microscopy technique used for enumerating viruses. This high fluorescence background is currently overlooked, yet it has had a most pervasive influence on the development of a simple fluorescence-based method for quantifying viral abundance in water.     

Strategies to minimize background autofluorescence in live mice during noninvasive fluorescence optical imaging

As small-animal fluorescence imaging becomes increasingly accessible to a broad spectrum of users, many lab animal researchers are just beginning to be exposed to its challenges. One setback to fluorescence imaging is background autofluorescence generated in animal tissue and in ingested food. The authors bring this issue into focus, and show how autofluorescence can be reduced in nude mice through selection of appropriate excitation wavelength and mouse diet.