Sample preparation strategies for high‐throughput mass spectrometry imaging of primary tumor organoids

Patient‐derived 3D organoids show great promise for understanding patient heterogeneity and chemotherapy response in human‐derived tissue. The combination of organoid culture techniques with mass spectrometry imaging provides a label‐free methodology for characterizing drug penetration, patient‐specific response, and drug biotransformation. However, current methods used to grow tumor organoids employ extracellular matrices that can produce small molecule background signal during mass spectrometry imaging analysis. Here, we develop a method to isolate 3D human tumor organoids out of a Matrigel extracellular matrix into gelatin mass spectrometry compatible microarrays for high‐throughput mass spectrometry imaging analysis. The alignment of multiple organoids in the same z‐axis is essential for sectioning organoids together and for maintaining reproducible sample preparation on a single glass slide for up to hundreds of organoids. This method successfully removes organoids from extracellular matrix interference and provides an organized array for high‐throughput imaging analysis to easily identify organoids by eye for area selection and further analysis. With this method, mass spectrometry imaging can be readily applied to organoid systems for preclinical drug development and personalized medicine research initiatives.     

Microarray technology as a universal tool for high-throughput analysis of biological systems

Over the last years microarray technology has become one of the principal platform technologies for the high-throughput analysis of biological systems. Starting with the construction of first DNA microarrays in the 1990s, microarray technology has flourished in the last years and many different new formats have been developed. Peptide and protein microarrays are now applied for the elucidation of interaction partners, modification sites and enzyme substrates. Antibody microarrays are envisaged to be of high importance for the high-throughput determination of protein abundances in translational profiling approaches. First cell microarrays have been constructed to transform microarray technology from an in vitro technology to an in vivo functional analysis tool. All of these approaches share a common prerequisite: the solid support on which they are generated. The demands on this solid support are thereby as manifold as the applications themselves. This review is aimed to display the recent developments in surface chemistry and derivatization, and to summarize the latest developments in the different application areas of microarray technology.     

A recommended and verified procedure for in situ tryptic digestion of formalin‐fixed paraffin‐embedded tissues for analysis by matrix‐assisted laser desorption/ionization imaging mass spectrometry

Matrix‐assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) is a molecular imaging technology uniquely capable of untargeted measurement of proteins, lipids, and metabolites while retaining spatial information about their location in situ. This powerful combination of capabilities has the potential to bring a wealth of knowledge to the field of molecular histology. Translation of this innovative research tool into clinical laboratories requires the development of reliable sample preparation protocols for the analysis of proteins from formalin‐fixed paraffin‐embedded (FFPE) tissues, the standard preservation process in clinical pathology. Although ideal for stained tissue analysis by microscopy, the FFPE process cross‐links, disrupts, or can remove proteins from the tissue, making analysis of the protein content challenging. To date, reported approaches differ widely in process and efficacy. This tutorial presents a strategy derived from systematic testing and optimization of key parameters, for reproducible in situ tryptic digestion of proteins in FFPE tissue and subsequent MALDI IMS analysis. The approach describes a generalized method for FFPE tissues originating from virtually any source.     

化学生物学中光敏分子微阵列的表面构建与应用

分子微阵列是有机合成（特别是组合化学合成）方法应用于生物和医学研究而发展起来的高科技集成技术,通过把微电子、微加工技术和有机化学合成反应相结合,在固体基质（如硅片、玻片、瓷片等）表面构建微型的生物有机化学分子系统,以实现对细胞、蛋白质、核酸及其他生物组分进行快速、敏感、高效地处理．近年来,随着表面化学构建策略研究的不断深入和迅猛发展,分子微阵列技术的应用领域不断拓展,已从最初用于核酸分子的杂交测序延伸到基因组功能研究的各个方面．本文着重综述了光敏分子微阵列的表面化学构建策略研究及其在化学生物学分析中应用的最新进展,并展望了其发展的未来趋势．内容主要包括：小分子与多肽分子微阵列、蛋白质分子微阵列、核酸分子微阵列和糖分子微阵列等．     

MALDI tissue imaging: from biomarker discovery to clinical applications

Matrix-assisted laser desorption ionization (MALDI) imaging mass spectrometry (IMS) is a powerful tool for the generation of multidimensional spatial expression maps of biomolecules directly from a tissue section. From a clinical proteomics perspective, this method correlates molecular detail to histopathological changes found in patient-derived tissues, enhancing the ability to identify candidates for disease biomarkers. The unbiased analysis and spatial mapping of a variety of molecules directly from clinical tissue sections can be achieved through this method. Conversely, targeted IMS, by the incorporation of laser-reactive molecular tags onto antibodies, aptamers, and other affinity molecules, enables analysis of specific molecules or a class of molecules. In addition to exploring tissue during biomarker discovery, the integration of MALDI-IMS methods into existing clinical pathology laboratory practices could prove beneficial to diagnostics. Querying tissue for the expression of specific biomarkers in a biopsy is a critical component in clinical decision-making and such markers are a major goal of translational research. An important challenge in cancer diagnostics will be to assay multiple parameters in a single slide when tissue quantities are limited. The development of multiplexed assays that maximize the yield of information from a small biopsy will help meet a critical challenge to current biomarker research. This review focuses on the use of MALDI-IMS in biomarker discovery and its potential as a clinical diagnostic tool with specific reference to our application of this technology to prostate cancer.     

Combination of FTIR spectral imaging and chemometrics for tumour detection from paraffin-embedded biopsies

FTIR spectral imaging was applied on formalin-fixed paraffin-embedded biopsies from colon and skin cancerous lesions. These samples were deposited onto different substrates (zinc selenide and calcium fluoride respectively) and embedded using two types of paraffin. Formalin fixation followed by paraffin embedding is the gold standard in tissue storage. It can preserve molecular structures and it is compatible with immunohistochemistry. However, paraffin absorption bands are significant in the mid-infrared region and can mask some molecular vibrations of the tissue. Direct data processing was applied on spectral images without any chemical dewaxing of the tissues. Extended Multiplicative Signal Correction was used to correct the spectral contribution from paraffin. For this purpose, the signal of paraffin was modelled using Principal Component Analysis and paraffin spectra were removed from the raw images based on an outlier detection. Then, pseudo-colour images were computed by K-means clustering in order to highlight histological structures of interest. This robust chemometrics methodology was applied on the two samples. Tumour areas were successfully demarcated from the rest of the tissue in both colon and skin independently of the embedding material and of the substrate.     

Construction of CdSe/ZnS quantum dot microarray in a microfluidic chip

Microarray technology has been proved to be greatly helpful for biomedical and biological diagnosis. And the evaluation of its biological applications lies in the detection sensitivity, which requires high intensity and stability of the signal. Recently, several nanomaterials, especially semiconductor nanomaterials, due to their excellent fluorescence properties, have been widely used to construct microarrays for biosensors. Here, we presented an approach for constructing CdSe/ZnS quantum dot (QD) microarray in microfluidic channels on a glass slide by photolithography. The conditions for immobilizing stable and uniform QD microarray on the glass slide were optimized. Several types of QD microarrays with different emission wavelengths and modified groups were constructed using silanization and lithography technology. Based on the fluorescence quenching effect of Cu2+ on QDs, the microfluidic chip with QD microarray was applied for the determination of Cu2+. 1 nmol/L Cu2+ could be detected by this method.     

Fourier transform infrared imaging analysis in discrimination studies of squamous cell carcinoma

Oral squamous cell carcinoma (OSCC) of the oral cavity and oropharynx represents more than 95% of all malignant neoplasms in the oral cavity. Histomorphological evaluation of this cancer type is invasive and remains a time consuming and subjective technique. Therefore, novel approaches for histological recognition are necessary to identify malignancy at an early stage. Fourier transform infrared (FTIR) imaging has become an essential tool for the detection and characterization of the molecular components of biological processes, such as those responsible for the dynamic properties of tumor progression. FTIR imaging is a modern analytical technique enabling molecular imaging of a complex biological sample and is based on the absorption of IR radiation by vibrational transitions in covalent bonds. One major advantage of this technique is the acquisition of local molecular expression profiles, while maintaining the topographic integrity of the tissue and avoiding time-consuming extraction, purification, and separation steps. With this imaging technique, it is possible to obtain unique images of the spatial distribution of proteins, lipids, carbohydrates, cholesterols, nucleic acids, phospholipids, and small molecules with high spatial resolution. Analysis and visualization of FTIR imaging datasets are challenging and the use of chemometric tools is crucial in order to take advantage of the full measurement. Therefore, methodologies for this task based on the novel developed algorithm for multivariate image analysis (MIA) are often necessary. In the present study, FTIR imaging and data analysis methods were combined to optimize the tissue measurement mode after deparaffinization and subsequent data evaluation (univariate analysis and MIAs). We demonstrate that it is possible to collect excellent IR spectra from formalin-fixed paraffin-embedded (FFPE) tissue microarrays (TMAs) of OSCC tissue sections employing an optimised analytical protocol. The correlation of FTIR imaging to the morphological tissue features obtained by histological staining of the sections demonstrated that many histomorphological tissue patterns can be visualized in the colour images. The different algorithms used for MIAs of FTIR imaging data dramatically increased the information content of the IR images from squamous cell tissue sections. These findings indicate that intra-operative and surgical specimens of squamous cell carcinoma tissue can be characterized by FTIR imaging.     

Micro-immunohistochemistry using a microfluidic probe

A flexible method to extract more high-quality information from tissue sections is critically needed for both drug discovery and clinical pathology. Here, we present micro-immunohistochemistry (μIHC), a method for staining tissue sections at the micrometre scale. Nanolitres of antibody solutions are confined over micrometre-sized areas of tissue sections using a vertical microfluidic probe (vMFP) for their incubation with primary antibodies, the key step in conventional IHC. The vMFP operates several micrometres above the tissue section, can be interactively positioned on it, and even enables the staining of individual cores of tissue microarrays with multiple antigens. μIHC using such a microfluidic probe is preservative of tissue samples and reagents, alleviates antibody cross-reactivity issues, and allows a wide range of staining conditions to be applied on a single tissue section. This method may therefore find broad use in tissue-based diagnostics and in research.     

Preparation of tissue samples for X-ray fluorescence microscopy

As is well-known, trace elements, especially metals, play an important role in the pathogenesis of many disorders. The topographic and quantitative elemental analysis of pathologically changed tissues may shed some new light on processes leading to the degeneration of cells in the case of selected diseases. An ideal and powerful tool for such purpose is the Synchrotron Microbeam X-ray Fluorescence technique. It enables the carrying out of investigations of the elemental composition of tissues even at the single cell level.

The tissue samples for histopathological investigations are routinely fixed and embedded in paraffin. The authors try to verify the usefulness of such prepared tissue sections for elemental analysis with the use of X-ray fluorescence microscopy. Studies were performed on rat brain samples. Changes in elemental composition caused by fixation in formalin or paraformaldehyde and embedding in paraffin were examined.

Measurements were carried out at the bending magnet beamline L of the Hamburger Synchrotronstrahlungslabor HASYLAB in Hamburg.

The decrease in mass per unit area of K, Br and the increase in P, S, Fe, Cu and Zn in the tissue were observed as a result of the fixation. For the samples embedded in paraffin, a lower level of most elements was observed. Additionally, for these samples, changes in the composition of some elements were not uniform for different analyzed areas of rat brain.     

An improved gel-based DNA microarray method for detecting single nucleotide mismatch

3-D polyacrylamide gel-based DNA microarray platforms provide a high capacity for nucleic acids immobilization and a solution-mimicking environment for hybridization. However, several technological bottlenecks still remain in these platforms, such as difficult microarray preparation and high fluorescent background, which limit their application. In this study, two new approaches have been developed to improve the convenience in microarray preparation and to reduce the background after hybridization. To control the polymerization process, solutions containing acrylamide-modified oligonucleotide, acrylamide, glycerol and ammonium persulfate are spotted onto a functionalized glass slide, and then the slide is transferred to a vacuum chamber with TEMED, so that TEMED is vaporized and diffused into the spots to induce polymerization. By applying an electric field across a hybridized microarray to remove the nonspecifically bound labeled targets, this approach can solve the problem of high fluorescent background of the gel-based microarray after hybridization. Experimental results show that our immobilization method can be used to construct high quality microarrays and exhibits good reproducibility. Moreover, the polymerization is not affected by PCR medium, so that PCR products can be used for microarray construction without being treated by commercial purification cartridges. Electrophoresis can improve the signal-to-noise significantly and has the ability to differentiate single nucleotide variation between two homozygotes and a heterozygote. Our results demonstrated that this is a reliable novel method for high-throughput mutation analysis and disease diagnosis.     

Automated analytical microarrays: a critical review

Microarrays provide a powerful analytical tool for the simultaneous detection of multiple analytes in a single experiment. The specific affinity reaction of nucleic acids (hybridization) and antibodies towards antigens is the most common bioanalytical method for generating multiplexed quantitative results. Nucleic acid-based analysis is restricted to the detection of cells and viruses. Antibodies are more universal biomolecular receptors that selectively bind small molecules such as pesticides, small toxins, and pharmaceuticals and to biopolymers (e.g. toxins, allergens) and complex biological structures like bacterial cells and viruses. By producing an appropriate antibody, the corresponding antigenic analyte can be detected on a multiplexed immunoanalytical microarray. Food and water analysis along with clinical diagnostics constitute potential application fields for multiplexed analysis. Diverse fluorescence, chemiluminescence, electrochemical, and label-free microarray readout systems have been developed in the last decade. Some of them are constructed as flow-through microarrays by combination with a fluidic system. Microarrays have the potential to become widely accepted as a system for analytical applications, provided that robust and validated results on fully automated platforms are successfully generated. This review gives an overview of the current research on microarrays with the focus on automated systems and quantitative multiplexed applications. Figure MCR 3: A fully automated chemiluminescence microarray reader for analytical microarrays     

Fabrication of DNA microarrays on nanoengineered polymeric ultrathin film prepared by self-assembly of polyelectrolyte multilayers

Microarray-based technology is in need of flexible and cost-effective chemistry for fabrication of oligonucleotide microarrays. We have developed a novel method for the fabrication of oligonucleotide microarrays with unmodified oligonucleotide probes on nanoengineered three-dimensional thin films that are deposited on glass slides by consecutive layer-to-layer adsorption of polyelectrolytes. Unmodified oligonucleotide probes were spotted and immobilized on these multilayered polyelectrolyte thin films (PET) by electrostatic adsorption and entrapment on the porous structure of the PET film. The PET provides higher probe binding capacity and thus higher hybridization signal than that of the traditional two-dimensional aminosilane and poly-L-lysine coated slides. Immobilized probe densities of 3.4 x 10(12)/cm2 were observed for microarray spots on PET with unmodified 50-mer oligonucleotide probes, which is comparable to the immobilized probe densities of alkyamine-modified 50-mer probes end-tethered on an aldehyde-functionalized slide. The study of hybridization efficiency showed that 90% of immobilized probes on PET film are accessible to target DNA to form duplex format in hybridization. The DNA microarray fabricated on PET film has wider dynamic range (about 3 orders of magnitude) and lower detection limit (0.5 nM) than the conventional amino- and aldehyde-functionalized slides. Oligonucleotide microarrays fabricated on these PET-coated slides also had consistent spot morphology. In addition, discrimination of single nucleotide polymorphism of 16S rRNA genes was achieved with the PET-based oligonucleotide microarrays. The PET microarrays constructed by our self-assembly process is cost-effective, versatile, and well suited for immobilizing many types of biological active molecules so that a wide variety of microarray formats can be developed.     

Peptide microarrays for the determination of protease substrate specificity

A method is described for the preparation of substrate microarrays that allow for the rapid determination of protease substrate specificity. Peptidyl coumarin substrates, synthesized on solid support using standard techniques, are printed onto glass slides using DNA microarraying equipment. The linkage from the peptide to the slide is formed through a chemoselective reaction, resulting in an array of uniformly displayed fluorogenic substrates. The arrays can be treated with proteases to yield substrate specificity profiles. Standard instrumentation for visualization of microarrays can be used to obtain comparisons of the specificity constants for all of the prepared substrates. The utility of these arrays is demonstrated by the selective cleavage of preferred substrates with trypsin, thrombin, and granzyme B, and by assessing the extended substrate specificity of thrombin using a microarray of 361 different peptidyl coumarin substrates.     

Crisp and soft multivariate methods visualize individual cell nuclei in Raman images of liver tissue sections

Raman and infrared spectroscopy have been recognized to be promising tools in clinical diagnostics because they provide molecular contrast without external stains. Here, vertex component analysis (VCA) was applied to Raman and Fourier transform infrared (FTIR) images of liver tissue sections and the results were compared with K-means cluster analysis, fuzzy C-means cluster analysis and principal component analysis. The main components of VCA from three Raman images were assigned to the central vein, periportal vein, cell nuclei, liver parenchyma and bile duct. After resonant Mie scattering correction, VCA of FTIR images identified veins, liver parenchyma, cracks, but no cell nuclei. The advantages of VCA in the context of tissue characterization by vibrational spectroscopic imaging are that the tissue architecture is visualized and the spectral information is reconstructed. Composite images were constructed that revealed a high molecular contrast and that can be interpreted in a similar way like hematoxylin and eosin stained tissue sections.     

How to Make a DNA Chip

Microarrays are one of the hottest areas in biological research today. Microarrays have been mostly applied to nucleic acid analysis, specifically to the assessment of which genes are being expressed and at what level. Early microarrays were prepared by using photolithographic methods, which were more commonly used for integrated circuit (“computer chip”) production. Hence the colloquial term “DNA chip” came into being. The completion of the sequencing of the human genome and that of many other organisms makes the determination of gene function an important next step in understanding the role of DNA in the processes of life. DNA microarrays are an excellent tool to address this question because their numerous probe sites enable the analysis of many genes simultaneously. With good experience in this initial use, many further applications of microarrays are being developed, including genotyping in research and genetic diagnosis in medicine. DNA microarrays have made abundantly clear the power of vast parallelism in biological analysis, which is raising interest in other types of microarrays (small‐molecule, protein). Many applications for DNA microarrays have been developed and clearly many more will emerge through the creativity of the scientists who use them. In early studies, users produced their own microarrays. The apparent power of microarrays has demanded improvements in production methods, and technologies from physical sciences and engineering are now being applied to DNA chips. Many branches of chemistry can contribute to improved methods: from synthetic chemistry (to attach or prepare DNA), to the physical chemistry of surfaces, to analytical chemistry (to assess surface reactions).     

A robust small-molecule microarray platform for screening cell lysates

Herein we report the expanded functional group compatibility of small-molecule microarrays to include immobilization of primary alcohols, secondary alcohols, phenols, carboxylic acids, hydroxamic acids, thiols, and amines on a single slide surface. Small-molecule "diversity microarrays" containing nearly 10,000 known bioactive small molecules, natural products, and small molecules originating from several diversity-oriented syntheses were produced by using an isocyanate-mediated covalent capture strategy. Selected printed bioactive compounds were detected with antibodies against compounds of interest. The new surface of the diversity microarrays is highly compatible with approaches involving cellular lysates. This feature has enabled a robust, optimized screening methodology using cellular lysates, allowing the detection of specific interactions with a broad range of binding affinity by using epitope-tagged or chimeric fluorescent proteins without prior purification. We believe that this expanded research capability has considerable promise in biology and medicine.     

Effect of the elapsed time between sampling and formalin fixation on the N‐glycosylation profile of mouse tissue specimens

Formalin‐fixed, paraffin‐embedded (FFPE) samples are generally used for histology‐study, however, they also possess important molecular diagnostics information. While it has been reported that the N‐glycan moieties of glycoproteins is not affected by the FFPE process, no information is available about the effect of the elapsed time between sampling and fixation on the resulting N‐glycosylation profile. In this study, lung, brain, heart, spleen, liver, kidney, and intestine mouse tissue specimens were used for N‐glycan profiling analysis and the elapsed sampling time effect was investigated with the lung tissue. N‐glycan extraction from the tissue samples was performed by glycoprotein retrieval from the FFPE specimens using radioimmunoprecipitation assay (RIPA) buffer followed PNGase F digestion. The released oligosaccharides were fluorophore labeled and analyzed by capillary electrophoresis‐laser induced fluorescent detection (CE‐LIF). N‐glycosylation profiles of freshly collected lung‐tissue samples (zero time point), as well as 1 and 2 h after sampling were compared by carbohydrate profiling and exoglycosidase treatment based deep glycomic analysis. It was found that up to two hours of room temperature storage of tissue specimens apparently did not cause changes in the N‐glycosylation profiles of complex carbohydrates, but resulted in considerable decrease in the amount of linear glucose oligomers and high mannose type glycans present in the samples.     

Preparation and use of microarrays containing synthetic heparin oligosaccharides for the rapid analysis of heparin-protein interactions

Heparin is a highly sulfated, linear polymer that participates in a plethora of biological processes by interaction with many proteins. The chemical complexity and heterogeneity of this polysaccharide can explain the fact that, despite its widespread medical use as an anticoagulant drug, the structure-function relationship of defined heparin sequences is still poorly understood. Here, we present the chemical synthesis of a library containing heparin oligosaccharides ranging from di- to hexamers of different sequences and sulfation patterns. An amine-terminated linker was placed at the reducing end of the synthetic structures to allow for immobilization onto N-hydroxysuccinimide activated glass slides and creation of heparin microarrays. Key features of this modular synthesis, such as the influence of the amine linker on the glycosidation efficiency, the use of 2-azidoglucose as glycosylating agents for oligosaccharide assembly, and the compatibility of the protecting group strategy with the sulfation-deprotection steps, are discussed. Heparin microarrays containing this oligosaccharide library were constructed using a robotic printer and employed to characterize the carbohydrate binding affinities of three heparin-binding growth factors. FGF-1, FGF-2 and FGF-4 that are implicated in angiogenesis, cell growth and differentiation were studied. These heparin chips aided in the discovery of novel, sulfated sequences that bind FGF, and in the determination of the structural requirements needed for recognition by using picomoles of protein on a single slide. The results presented here highlight the potential of combining oligosaccharide synthesis and carbohydrate microarray technology to establish a structure-activity relationship in biological processes.