Intact Biological Samples Research Articles

Light-Seq: light-directed in situ barcoding of biomolecules in fixed cells and tissues for spatially indexed sequencing

We present Light-Seq, an approach for multiplexed spatial indexing of intact biological samples using light-directed DNA barcoding in fixed cells and tissues followed by ex situ sequencing. Light-Seq combines spatially targeted, rapid photocrosslinking of DNA barcodes onto complementary DNAs in situ with a one-step DNA stitching reaction to create pooled, spatially indexed sequencing libraries. This light-directed barcoding enables in situ selection of multiple cell populations in intact fixed tissue samples for full-transcriptome sequencing based on location, morphology or protein stains, without cellular dissociation. Applying Light-Seq to mouse retinal sections, we recovered thousands of differentially enriched transcripts from three cellular layers and discovered biomarkers for a very rare neuronal subtype, dopaminergic amacrine cells, from only four to eight individual cells per section. Light-Seq provides an accessible workflow to combine in situ imaging and protein staining with next generation sequencing of the same cells, leaving the sample intact for further analysis post-sequencing.

High-plex imaging of RNA and proteins at subcellular resolution in fixed tissue by spatial molecular imaging.

Resolving the spatial distribution of RNA and protein in tissues at subcellular resolution is a challenge in the field of spatial biology. We describe spatial molecular imaging, a system that measures RNAs and proteins in intact biological samples at subcellular resolution by performing multiple cycles of nucleic acid hybridization of fluorescent molecular barcodes. We demonstrate that spatial molecular imaging has high sensitivity (one or two copies per cell) and very low error rate (0.0092 false calls per cell) and background (~0.04 counts per cell). The imaging system generates three-dimensional, super-resolution localization of analytes at ~2 million cells per sample. Cell segmentation is morphology based using antibodies, compatible with formalin-fixed, paraffin-embedded samples. We measured multiomic data (980 RNAs and 108 proteins) at subcellular resolution in formalin-fixed, paraffin-embedded tissues (nonsmall cell lung and breast cancer) and identified >18 distinct cell types, ten unique tumor microenvironments and 100 pairwise ligand-receptor interactions. Data on >800,000 single cells and ~260 million transcripts can be accessed at http://nanostring.com/CosMx-dataset .

Comparing the Potential of Helmholtz and Planar NMR Microcoils for Analysis of Intact Biological Samples.

Nuclear magnetic resonance (NMR) spectroscopy has played an integral role in medical and environmental metabolic research. However, smaller biological entities, such as eggs and small tissue samples, are becoming increasingly important to better understand toxicity, biological growth/development, and diseases. Unfortunately, their small sizes make them difficult to study using conventional 5 mm NMR probes due to limited sensitivity. The use of microcoil NMR holds great potential for the analysis of such samples, where the coil can be designed to match the sample size to significantly improve NMR mass sensitivity and the filling factor. Here, we compare the potential of planar and Helmholtz microcoil designs to execute complex experiments for the analysis of intact, mass-limited biological samples. The planar coil offers the advantage of an open access design, potentially allowing flow systems to be incorporated and varying sample sizes to be studied; however, its relatively inhomogeneous B1 field leads to reduced NMR performance. The Helmholtz microcoil overcomes this drawback with its symmetrical design, improving B1 homogeneity across the sample but with the caveat that the size and shape of the sample is limited to the spacing between the two parallel coils. The line shape, sensitivity, and RF performance are compared on both coils using standard samples and biological samples. This study found that the Helmholtz microcoil used here considerably outperforms the planar coil in multipulse experiments and has great potential to study complex biological samples in the 50-200 nL range.

Paper‐MAP: A Novel Tool for Super‐resolution Imaging of Pathological Tissues by Rapidly Tissue Expansion and Clearing

Super‐resolution mapping of three‐dimensional (3D) structures in intact tissues is essential in many biological studies. Biological tissues has very complex structure of functional networking with various cell population/organization. Since most biological tissues are not transparent, and imaging processes are limited by light scatter, imaging deep within the tissues is problematic. In this work, we developed the novel method as call Paper‐MAP for rapidly and simple clearing/expansion of based on MAP (Magnified Analysis of Proteome) using very thin tissue. The Paper‐MAP can be analyzed to super‐resolution 3D imaging of hybrid tissue with rapidly 4‐fold clearing/expansion in under 14 hours, while preserving their overall architecture and protein content of subcellular details after hydrogel embedding. Using a Paper‐MAP, we performed the super‐resolution imaging of pathological samples, such as injured spinal cord, brain hippocampus, salivary gland organoid, tumor sphere, and glioblastoma tumor of human patients. This technique provides an ideal way for researchers to examine the pathologic patterns as well as various intact biological samples.

3D imaging for driving cancer discovery.

Our understanding of the cellular composition and architecture of cancer has primarily advanced using 2D models and thin slice samples. This has granted spatial information on fundamental cancer biology and treatment response. However, tissues contain a variety of interconnected cells with different functional states and shapes, and this complex organization is impossible to capture in a single plane. Furthermore, tumours have been shown to be highly heterogenous, requiring large-scale spatial analysis to reliably profile their cellular and structural composition. Volumetric imaging permits the visualization of intact biological samples, thereby revealing the spatio-phenotypic and dynamic traits of cancer. This review focuses on new insights into cancer biology uniquely brought to light by 3D imaging and concomitant progress in cancer modelling and quantitative analysis. 3D imaging has the potential to generate broad knowledge advance from major mechanisms of tumour progression to new strategies for cancer treatment and patient diagnosis. We discuss the expected future contributions of the newest imaging trends towards these goals and the challenges faced for reaching their full application in cancer research.

Evaluation of double-tuned single-sided planar microcoils for the analysis of small 13 C enriched biological samples using 1 H-13 C 2D heteronuclear correlation NMR spectroscopy.

Microcoils provide a cost-effective approach to improve detection limits for mass-limited samples. Single-sided planar microcoils are advantageous in comparison to volume coils, in that the sample can simply be placed on top. However, the considerable drawback is that the RF field that is produced by the coil decreases with distance from the coil surface, which potentially limits more complex multi-pulse NMR pulse sequences. Unfortunately, 1 H NMR alone is not very informative for intact biological samples due to line broadening caused by magnetic susceptibility distortions, and 1 H-13 C 2D NMR correlations are required to provide the additional spectral dispersion for metabolic assignments in vivo or in situ. To our knowledge, double-tuned single-sided microcoils have not been applied for the 2D 1 H-13 C analysis of intact 13 C enriched biological samples. Questions include the following: Can 1 H-13 C 2D NMR be performed on single-sided planar microcoils? If so, do they still hold sensitivity advantages over conventional 5mm NMR technology for mass limited samples? Here, 2D 1 H-13 C HSQC, HMQC, and HETCOR variants were compared and then applied to 13 C enriched broccoli seeds and Daphnia magna (water fleas). Compared to 5mm NMR probes, the microcoils showed a sixfold improvement in mass sensitivity (albeit only for a small localized region) and allowed for the identification of metabolites in a single intact D. magna for the first time. Single-sided planar microcoils show practical benefit for 1 H-13 C NMR of intact biological samples, if localized information within ~0.7mm of the 1mm I.D. planar microcoil surface is of specific interest.

Three-Dimensional X-ray Imaging of β-Galactosidase Reporter Activity by Micro-CT: Implication for Quantitative Analysis of Gene Expression.

Acquisition of detailed anatomical and molecular knowledge from intact biological samples while preserving their native three-dimensional structure is still a challenging issue for imaging studies aiming to unravel a system’s functions. Three-dimensional micro-CT X-ray imaging with a high spatial resolution in minimally perturbed naive non-transparent samples has recently gained increased popularity and broad application in biomedical research. Here, we describe a novel X-ray-based methodology for analysis of β-galactosidase (lacZ) reporter-driven gene expression in an intact murine brain ex vivo by micro-CT. The method relies on detection of bromine molecules in the product of the enzymatic β-galactosidase reaction. Enhancement of the X-ray signal is observed specifically in the regions of the murine brain where expression of the lacZ reporter gene is also detected histologically. We performed quantitative analysis of the expression levels of lacZ reporter activity by relative radiodensity estimation of the β-galactosidase/X-gal precipitate in situ. To demonstrate the feasibility of the method, we performed expression analysis of the Tsen54-lacZ reporter gene in the murine brain in a semi-quantitative manner. Human mutations in the Tsen54 gene cause pontocerebellar hypoplasia (PCH), a group of severe neurodegenerative disorders with both mental and motor deficits. Comparing relative levels of Tsen54 gene expression, we demonstrate that the highest Tsen54 expression is observed in anatomical brain substructures important for the normal motor and memory functions in mice.

In situ genome sequencing resolves DNA sequence and structure in intact biological samples.

Understanding genome organization requires integration of DNA sequence and three-dimensional spatial context; however, existing genome-wide methods lack either base pair sequence resolution or direct spatial localization. Here, we describe in situ genome sequencing (IGS), a method for simultaneously sequencing and imaging genomes within intact biological samples. We applied IGS to human fibroblasts and early mouse embryos, spatially localizing thousands of genomic loci in individual nuclei. Using these data, we characterized parent-specific changes in genome structure across embryonic stages, revealed single-cell chromatin domains in zygotes, and uncovered epigenetic memory of global chromosome positioning within individual embryos. These results demonstrate how IGS can directly connect sequence and structure across length scales from single base pairs to whole organisms.

Improvements in lipid suppression for 1 H NMR-based metabolomics: Applications to solution-state and HR-MAS NMR in natural and in vivo samples.

Proton nuclear magnetic resonance (NMR) spectra of intact biological samples often show strong contributions from lipids, which overlap with signals of interest from small metabolites. Pioneering work by Diserens et al. demonstrated that the relative differences in diffusivity and relaxation of lipids versus small metabolites could be exploited to suppress lipid signals, in high-resolution magic angle spinning (HR-MAS) NMR spectroscopy. In solution-state NMR, suspended samples can exhibit very broad water signals, which are challenging to suppress. Here, improved water suppression is incorporated into the sequence, and the Carr-Purcell-Meiboom-Gill sequence (CPMG) train is replaced with a low-power adiabatic spinlock that reduces heating and spectral artefacts seen with longer CPMG filters. The result is a robust sequence that works well in both HR-MAS as well as static solution-state samples. Applications are also extended to include in vivo organisms. For solution-state NMR, samples containing significant amount of fats such as milk and hemp hearts seeds are used to demonstrate the technique. For HR-MAS, living earthworms (Eisenia fetida) and freshwater shrimp (Hyalella azteca) are used for in vivo applications. Lipid suppression techniques are essential for non-invasive NMR-based analysis of biological samples with a high-lipid content and adds to the suite of experiments advantageous for in vivo environmental metabolomics.

Effective combined water and sideband suppression for low-speed tissue and in vivo MAS NMR.

High-resolution magic angle spinning (HR-MAS) NMR is a powerful technique that can provide metabolic profiles and structural constraints on intact biological and environmental samples such as cells, tissues and living organisms. However, centripetal force from fast spinning can lead to a loss of sample integrity. In analyses focusing on structural organization, metabolite compartmentalization or in vivo studies, it is critical to keep the sample intact. As such, there is growing interest in slow spinning studies that preserve sample longevity. In this study, for example, reducing the spinning rate from 2500 to 500Hz during the analysis of a living freshwater shrimp increased the 100% survivability threshold from ~14 to 40h. Unfortunately, reducing spinning rate decreases the intensity of the isotropic signals and increases both the intensity and number of spinning sidebands, which mask spectral information. Interestingly, water suppression approaches such as excitation sculpting and W5 WATERGATE, which are effective at higher spinning rates, fail at lower spinning rates (<2500Hz) while simpler approaches such as presaturation are not able to effectively suppress water when the ratio of water to biomass is very high, as is the case in vivo. As such there is a considerable gap in NMR approaches which can be used to suppress water signals and sidebands in biological samples at lower spinning rates. This research presents simple but practically important sequences that combine PURGE water suppression with both phase-adjusted spinning sidebands and an analogue of TOSS termed TOSS.243. The result is simple and effective water and sideband suppression even in extremely dilute samples in pure water down to ~100Hz spinning rate. The approach is introduced, described and applied to a range of samples including, ex vivo worm tissue, Daphnia magna (water fleas), and in vivo Hyalella azteca (shrimp).

Illustrated Review of the Ventral Striatum's Olfactory Tubercle.

Modern neuroscience often relies upon artistic renderings to illustrate key aspects of anatomy. These renderings can be in 2 or even 3 dimensions. Three-dimensional renderings are especially helpful in conceptualizing highly complex aspects of neuroanatomy which otherwise are not visually apparent in 2 dimensions or even intact biological samples themselves. Here, we provide 3 dimensional renderings of the gross- and cellular-anatomy of the rodent olfactory tubercle. Based upon standing literature and detailed investigations into rat brain specimens, we created biologically inspired illustrations of the olfactory tubercle in 3 dimensions as well as its connectivity with olfactory bulb projection neurons, the piriform cortex association fiber system, and ventral pallidum medium spiny neurons. Together, we intend for these illustrations to serve as a resource to the neuroscience community in conceptualizing and discussing this highly complex and interconnected brain system with established roles in sensory processing and motivated behaviors.

Photoactivated Localization Microscopy with Bimolecular Fluorescence Complementation (BiFC-PALM).

Protein-protein interactions (PPIs) are key molecular events to biology. However, it remains a challenge to visualize PPIs with sufficient resolution and sensitivity in cells because the resolution of conventional light microscopy is diffraction-limited to ~250 nm. By combining bimolecular fluorescence complementation (BiFC) with photoactivated localization microscopy (PALM), PPIs can be visualized in cells with single molecule sensitivity and nanometer spatial resolution. BiFC is a commonly used technique for visualizing PPIs with fluorescence contrast, which involves splitting of a fluorescent protein into two non-fluorescent fragments. PALM is a recent superresolution microscopy technique for imaging biological samples at the nanometer and single molecule scales, which uses phototransformable fluorescent probes such as photoactivatable fluorescent proteins (PA-FPs). BiFC-PALM was demonstrated by splitting PAmCherry1, a PA-FP compatible with PALM, for its monomeric nature, good single molecule brightness, high contrast ratio, and utility for stoichiometry measurements. When split between amino acids 159 and 160, PAmCherry1 can be made into a BiFC probe that reconstitutes efficiently at 37 °C with high specificity to PPIs and low non-specific reconstitution. Ras-Raf interaction is used as an example to show how BiFC-PALM helps to probe interactions at the nanometer scale and with single molecule resolution. Their diffusion can also be tracked in live cells using single molecule tracking (smt-) PALM. In this protocol, factors to consider when designing the fusion proteins for BiFC-PALM are discussed, sample preparation, image acquisition, and data analysis steps are explained, and a few exemplary results are showcased. Providing high spatial resolution, specificity, and sensitivity, BiFC-PALM is a useful tool for studying PPIs in intact biological samples.

Two-Dimensional J-Resolved NMR Analyses of Fish and Its Products via Spatially Encoded Intermolecular Double-Quantum Coherences

Nuclear magnetic resonance (NMR) spectroscopy provides a powerful tool for food analyses due to its high-throughput information related to component analyses and its non-invasive property without altering detected samples. Current liquid NMR approaches, however, are subject to the influence of field inhomogeneity in direct measurements of biological foods, generally resorting to techniques of tissue extractions or magic-angle spin to eliminate the inhomogeneity. Therefore, we propose an NMR approach based on intermolecular double-quantum coherences (iDQCs) and spatial encoding technique to fast recover high-resolution two-dimensional (2D) J-resolved spectra in inhomogeneous fields. Inheriting from the immunity to field inhomogeneity of iDQCs and the enhancement in acquisition efficiency of the spatial encoding technique, the proposed method can resist the field inhomogeneity in biological foods without superfluous pretreatments and time-consuming shimming procedures, and yield satisfactory 2D J-resolved information for analyses within minutes. Fish and its products, which are closely related to human daily life, capture extensive attentions with the rapid development of the aquaculture. At first, experiments on cod liver oils are implemented in a purposely deshimmed magnetic field to demonstrate the feasibility of the proposed approach. Then, caviars and an intact fish are tested to further show the applicability of the proposed approach for direct measurements on intact biological food samples. Herein, the proposed method may constitute an effective technique in analyzing fish and its products.

A high-resolution 2D J-resolved NMR detection technique for metabolite analyses of biological samples.

NMR spectroscopy is a commonly used technique for metabolite analyses. Due to the observed macroscopic magnetic susceptibility in biological tissues, current NMR acquisitions in measurements of biological tissues are generally performed on tissue extracts using liquid NMR or on tissues using magic-angle spinning techniques. In this study, we propose an NMR method to achieve high-resolution J-resolved information for metabolite analyses directly from intact biological samples. A dramatic improvement in spectral resolution is evident in our contrastive demonstrations on a sample of pig brain tissue. Metabolite analyses for a postmortem fish from fresh to decayed statuses are presented to further reveal the capability of the proposed method. This method is a previously-unreported high-resolution 2D J-resolved spectroscopy for biological applications without specialised hardware requirements or complicated sample pretreatments. It provides a significant contribution to metabolite analyses of biological samples, and may be potentially applicable to in vivo samples. Furthermore, this method also can be applied to measurements of semisolid and viscous samples.

Laser desorption postionization mass spectrometry imaging of biological targets.

Laser desorption photoionization mass spectrometry (LDPI-MS) utilizes two separate light sources for desorption and photoionization of species from a solid surface. This technique has been applied to study a wide variety of molecular analytes in biological systems, but is not yet available in commercial instruments. For this reason, a generalized protocol is presented here for the use of LDPI-MS imaging to detect small molecules within intact biological samples. Examples are provided here for LDPI-MS imaging of an antibiotic within a tooth root canal and a metabolite within a coculture bacterial biofilm.

Elemental and chemically specific X-ray fluorescence imaging of biological systems.

From the perspective of a chemist, biology confers a rich variety of roles on a number of metal ions. It is widely agreed that a large fraction of the genomic output of living things contains metal or metalloid ions, although estimates of this fraction vary widely and depend upon which metal ions are considered.1−3 Moreover, recent reports suggest that, at least in some cases, there are many uncharacterized metalloproteins.4 With inclusion of the s-block metals such as Na, K, Mg, and Ca, the proportion likely approaches 100%; recent estimates from the protein data bank indicate that the prevalence of heavier metal ions of atomic number above 20 within proteins is around 22%,5 with Zn2+ proteins alone constituting about 11%. Living organisms have an inherent and very rich physical structure, with relevant length scales ranging from the nanometer scale for subcellular structure to hundreds of micrometers and above for tissue, organ, or organism-level organization. The ability to derive the spatial distribution of elements on this diversity of length scales is a key to understanding their function. Metals play essential and central roles in the most important and chemically challenging processes required for life, with active site structures and mechanisms that, at the time of their discovery, have usually not yet been duplicated in the chemical laboratory. Furthermore, diseases of metal dysregulation can cause disruption in the distribution of metals.6 For example, Menke’s disease and Occipital Horn Syndrome,7 and Wilson’s disease,8 involve disruption in copper uptake and excretion, respectively, through mutation in the ATP7A and ATP7B Cu transporters.9 The mechanisms of action of toxic elements such as mercury and arsenic are also of interest, as are essential nonmetal trace elements, such as selenium. Likewise, an increasing number of pharmaceuticals include metals or heavier elements; such chemotherapeutic drugs include the platinum derivatives cisplatin and carboplatin,10 some promising new ruthenium drugs,11 and arsenic trioxide, which has been used to treat promyelocytic leukemia.12 Understanding the localization, speciation, and distribution of these at various length scales is of significant interest. A wide variety of heavier elements can be probed by X-ray spectroscopic methods; these are shown graphically in Figure ​Figure1.1. X-ray fluorescence imaging is a powerful technique that can be used to determine elemental and chemical species distributions at a range of spatial resolutions within samples of biological tissues. Most modern applications require the use of synchrotron radiation as a tunable and high spectral brightness source of X-rays. The method uses a microfocused X-ray beam to excite X-ray fluorescence from specific elements within a sample. Because the method depends upon atomic physics, it is highly specific and enables a wide range of chemical elements to be investigated. A significant advantage over more conventional methods is the ability to measure intact biological samples without significant treatment with exogenous reagents. The technique is capable of determining metal and nonmetal distributions on a variety of length scales, with information on chemical speciation also potentially available. Figure ​Figure22 shows examples of rapid-scan X-ray fluorescence imaging at two contrasting length scales: rapid-scan imaging13 of a section of a human brain taken from an individual suffering from multiple sclerosis and showing elemental profiles for Fe, Cu, and Zn;14 and a high-resolution image showing mercury and other elements in a section of retina from a zebrafish larva treated with methylmercury chloride.15 We will discuss both the state of the art in terms of experimental methods and some recent applications of the methods. This Review considers X-ray fluorescence imaging with incident X-ray energies in the hard X-ray regime, which we define as 2 keV and above. We review technologies for producing microfocused X-ray beams and for detecting X-ray fluorescence, as well as methods that confer chemical selectivity or three-dimensional visualization. We discuss applications in key areas with a view to providing examples of how the technique can provide information on biological systems. We also discuss synergy with other methods, which have overlapping or complementary capabilities. Our goal is to provide useful and pertinent information to encourage and enable further use of this powerful method in chemical and biochemical studies of living organisms. Figure 1 Periodic table of the elements showing elements of biological interest that can be probed using X-ray fluorescence imaging. Elements are divided into three categories, those that are physiologically important, those that are pharmacologically active, ...

High-Resolution Two-Dimensional J-Resolved NMR Spectroscopy for Biological Systems

NMR spectroscopy is a principal tool in metabolomic studies and can, in theory, yield atom-level information critical for understanding biological systems. Nevertheless, NMR investigations on biological tissues generally have to contend with field inhomogeneities originating from variations in macroscopic magnetic susceptibility; these field inhomogeneities broaden spectral lines and thereby obscure metabolite signals. The congestion in one-dimensional NMR spectra of biological tissues often leads to ambiguities in metabolite identification and quantification. We propose an NMR approach based on intermolecular double-quantum coherences to recover high-resolution two-dimensional (2D) J-resolved spectra from inhomogeneous magnetic fields, such as those created by susceptibility variations in intact biological tissues. The proposed method makes it possible to acquire high-resolution 2D J-resolved spectra on intact biological samples without recourse to time-consuming shimming procedures or the use of specialized hardware, such as magic-angle-spinning probes. Separation of chemical shifts and J couplings along two distinct dimensions is achieved, which reduces spectral crowding and increases metabolite specificity. Moreover, the apparent J coupling constants observed are magnified by a factor of 3, facilitating the accurate measurement of small J couplings, which is useful in metabolic analyses. Dramatically improved spectral resolution is demonstrated in our applications of the technique on pig brain tissues. The resulting spectra contain a wealth of chemical shift and J-coupling information that is invaluable for metabolite analyses. A spatially localized experiment applied on an intact fish (Crossocheilus siamensis) reveals the promise of the proposed method in in vivo metabolite studies. Moreover, the proposed method makes few demands on spectrometer hardware and therefore constitutes a convenient and effective manner for metabonomics study of biological systems.

NMR‐based metabolomics coupled with pattern recognition methods in biomarker discovery and disease diagnosis

Molecular biomarkers could detect biochemical changes associated with disease processes. The key metabolites have become an important part for improving the diagnosis, prognosis, and therapy of diseases. Because of the chemical diversity and dynamic concentration range, the analysis of metabolites remains a challenge. Assessment of fluctuations on the levels of endogenous metabolites by advanced NMR spectroscopy technique combined with multivariate statistics, the so-called metabolomics approach, has proved to be exquisitely valuable in human disease diagnosis. Because of its ability to detect a large number of metabolites in intact biological samples with isotope labeling of metabolites using nuclei such as H, C, N, and P, NMR has emerged as one of the most powerful analytical techniques in metabolomics and has dramatically improved the ability to identify low concentration metabolites and trace important metabolic pathways. Multivariate statistical methods or pattern recognition programs have been developed to handle the acquired data and to search for the discriminating features from biosample sets. Furthermore, the combination of NMR with pattern recognition methods has proven highly effective at identifying unknown metabolites that correlate with changes in genotype or phenotype. The research and clinical results achieved through NMR investigations during the first 13 years of the 21st century illustrate areas where this technology can be best translated into clinical practice. In this review, we will present several special examples of a successful application of NMR for biomarker discovery, implications for disease diagnosis, prognosis, and therapy evaluation, and discuss possible future improvements.

Characterization of differential Toll-like receptor responses below the optical diffraction limit.

Many membrane receptors are recruited to specific cell surface domains to form nanoscale clusters upon ligand activation. This step appears to be necessary to initiate cell signaling, including pathways in innate immune system activation. However, virulent pathogens such as Yersinia pestis (the causative agent of plague) are known to evade innate immune detection, in contrast to similar microbes (such as Escherichia coli) that elicit a robust response. This disparity has been partly attributed to the structure of lipopolysaccharides (LPS) on the bacterial cell wall, which are recognized by the innate immune receptor TLR4. It is hypothesized that nanoscale differences exist between the spatial clustering of TLR4 upon binding of LPS derived from Y. pestis and E. coli. Although optical imaging can provide exquisite details of the spatial organization of biomolecules, there is a mismatch between the scale at which receptor clustering occurs (<300 nm) and the optical diffraction limit (>400 nm). The last decade has seen the emergence of super-resolution imaging methods that effectively break the optical diffraction barrier to yield truly nanoscale information in intact biological samples. This study reports the first visualizations of TLR4 distributions on intact cells at image resolutions of <30 nm using a novel, dual-color stochastic optical reconstruction microscopy (STORM) technique. This methodology permits distinction between receptors containing bound LPS from those without at the nanoscale. Importantly, it is also shown that LPS derived from immunostimulatory bacteria result in significantly higher LPS-TLR4 cluster sizes and a nearly twofold greater ligand/receptor colocalization as compared to immunoevading LPS.

Rapid, simple, and highly sensitive analysis of drugs in biological samples using thin-layer chromatography coupled with matrix-assisted laser desorption/ionization mass spectrometry

Rapid and precise identification of toxic substances is necessary for urgent diagnosis and treatment of poisoning cases and for establishing the cause of death in postmortem examinations. However, identification of compounds in biological samples using gas chromatography and liquid chromatography coupled with mass spectrometry entails time-consuming and labor-intensive sample preparations. In this study, we examined a simple preparation and highly sensitive analysis of drugs in biological samples such as urine, plasma, and organs using thin-layer chromatography coupled with matrix-assisted laser desorption/ionization mass spectrometry (TLC/MALDI/MS). When the urine containing 3,4-methylenedioxymethamphetamine (MDMA) without sample dilution was spotted on a thin-layer chromatography (TLC) plate and was analyzed by TLC/MALDI/MS, the detection limit of the MDMA spot was 0.05 ng/spot. The value was the same as that in aqueous solution spotted on a stainless steel plate. All the 11 psychotropic compounds tested (MDMA, 4-hydroxy-3-methoxymethamphetamine, 3,4-methylenedioxyamphetamine, methamphetamine, p-hydroxymethamphetamine, amphetamine, ketamine, caffeine, chlorpromazine, triazolam, and morphine) on a TLC plate were detected at levels of 0.05-5 ng, and the type (layer thickness and fluorescence) of TLC plate did not affect detection sensitivity. In addition, when rat liver homogenate obtained after MDMA administration (10 mg/kg) was spotted on a TLC plate, MDMA and its main metabolites were identified using TLC/MALDI/MS, and the spots on a TLC plate were visualized by MALDI/imaging MS. The total analytical time from spotting of intact biological samples to the output of analytical results was within 30 min. TLC/MALDI/MS enabled rapid, simple, and highly sensitive analysis of drugs from intact biological samples and crude extracts. Accordingly, this method could be applied to rapid drug screening and precise identification of toxic substances in poisoning cases and postmortem examinations.