Imaging of atomic and molecular species in tissue with laser‐SNMS for pharmaceutical studies

Successful anticancer therapies will have the ability to selectively deliver compounds to target cells while sparing normal tissue. Currently, methods to determine the distribution of compounds with very high sensitivity and subcellular resolution are still unavailable. Laser secondary neutral mass spectrometry (laser‐SNMS) and time‐of‐flight secondary ion mass spectrometry (ToF‐SIMS) are capable of detecting atoms and molecules with high sensitivity and a spatial resolution of up to 80 nm. The use of such methods requires special preparation techniques that preserve the morphological and chemical integrity of living cells. In this paper, the ability of laser‐SNMS to study transportation processes in animals of boron‐containing compounds for boron neutron capture therapy will be discussed. The data show that with laser‐SNMS it is possible to measure the distribution of these compounds in tissues with subcellular resolution, and that laser‐SNMS is a very powerful tool for locating anticancer drugs in tissues. Copyright © 2006 John Wiley & Sons, Ltd.     

Electron Cryo‐microscopy as a Tool for Structure‐Based Drug Development

For decades, X‐ray crystallography and NMR have been the most important techniques for studying the atomic structure of macromolecules. However, as a result of size, instability, low yield, and other factors, many macromolecules are difficult to crystallize or unsuitable for NMR studies. Electron cryo‐microscopy (cryo‐EM) does not depend on crystals and has therefore been the method of choice for many macromolecular complexes that cannot be crystallized, but atomic resolution has mostly been beyond its reach. A new generation of detectors that are capable of sensing directly the incident electrons has recently revolutionized the field, with structures of macromolecules now routinely being solved to near‐atomic resolution. In this review, we summarize some of the most recent examples of high‐resolution cryo‐EM structures. We put particular emphasis on proteins with pharmacological relevance that have traditionally been inaccessible to crystallography. Furthermore, we discuss examples where interactions with small molecules have been fully characterized at atomic resolution. Finally, we stress the current limits of cryo‐EM, and methodological issues related to its usage as a tool for drug development.     

Unambiguous Intracellular Localization and Quantification of a Potent Iridium Anticancer Compound by Correlative 3D Cryo X‐Ray Imaging

The iridium half‐sandwich complex [Ir(η⁵:κ¹‐C₅Me₄CH₂py)(2‐phenylpyridine)]PF₆ is highly cytotoxic: 15–250× more potent than clinically used cisplatin in several cancer cell lines. We have developed a correlative 3D cryo X‐ray imaging approach to specifically localize and quantify iridium within the whole hydrated cell at nanometer resolution. By means of cryo soft X‐ray tomography (cryo‐SXT), which provides the cellular ultrastructure at 50 nm resolution, and cryo hard X‐ray fluorescence tomography (cryo‐XRF), which provides the elemental sensitivity with a 70 nm step size, we have located the iridium anticancer agent exclusively in the mitochondria. Our methodology provides unique information on the intracellular fate of the metallodrug, without chemical fixation, labeling, or mechanical manipulation of the cells. This cryo‐3D correlative imaging method can be applied to a number of biochemical processes for specific elemental localization within the native cellular landscape.     

A new sample substrate for imaging and correlating organic and trace metal composition in biological cells and tissues

Many disease processes involve alterations in the chemical makeup of tissue. Synchrotron-based infrared (IR) and X-ray fluorescence (XRF) microscopes are becoming increasingly popular tools for imaging the organic and trace metal compositions of biological materials, respectively, without the need for extrinsic labels or stains. Fourier transform infrared microspectroscopy (FTIRM) provides chemical information on the organic components of a material at a diffraction-limited spatial resolution of 2–10 μm in the mid-infrared region. The synchrotron X-ray fluorescence (SXRF) microprobe is a complementary technique used to probe trace element content in the same systems with a similar spatial resolution. However to be most beneficial, it is important to combine the results from both imaging techniques on a single sample, which requires precise overlap of the IR and X-ray images. In this work, we have developed a sample substrate containing a gold grid pattern on its surface, which can be imaged with both the IR and X-ray microscopes. The substrate consists of a low trace element glass slide that has a gold grid patterned on its surface, where the major and minor parts of the grid contain 25 and 12 nm gold, respectively. This grid pattern can be imaged with the IR microscope because the reflectivity of gold differs as a function of thickness. The pattern can also be imaged with the SXRF microprobe because the Au fluorescence intensity changes with gold thickness. The tissue sample is placed on top of the patterned substrate. The grid pattern’s IR reflectivity image and the gold SXRF image are used as fiducial markers for spatially overlapping the IR and SXRF images from the tissue. Results show that IR and X-ray images can be correlated precisely, with a spatial resolution of less than one pixel (i.e., 2–3 microns). The development of this new tool will be presented along with applications to paraffin-embedded metalloprotein crystals, Alzheimer’s disease, and hair composition.     

High resolution inductively coupled plasma mass spectrometry (HR-ICPMS) determination and multivariate evaluation of 10 trace elements in mussels from 7 sites in Limfjorden, Denmark

The contents of V, Cr, Co, Ni, Cu, Ga, Rb, Cd, Ba, and Pb in the soft tissue of blue mussel (Mytilus edulis) were determined by a high resolution inductively coupled plasma mass spectrometry (HR-ICPMS) method. Sample digestions were performed in closed microwave vessels using nitric acid and hydrogen peroxide. Using HR-ICPMS it is possible to resolve the analytical peaks from otherwise interfering polyatomic ions with a mass resolution setting of 4000 (Cr, Ni, Cu). The proposed method was validated using a mussel tissue reference material (NIST SRM 2974). The proposed method was applied to real samples of blue mussel from seven sites in the inlet “Limfjorden”, Denmark, and the levels of trace elements found were compared with the levels found in an earlier study. For the mussel samples large inter-regional differences in trace element concentrations in the tissues were recorded. The mussels from the different sites could be separated using principal component analysis (PCA). Comparison with the levels of trace elements in mussels found in 1982 showed that the trace elemental contamination has increased during the last 15 years. From the data obtained, mussel tissue appears to be good bio-indicator for identification of coastal areas exposed to metallic contaminants.     

The regional distribution of copper and other trace elements in the human brain with special reference to Wilson's disease

The regional distribution of copper and other trace elements was determined in the brain of a patient deceased in Wilson's disease against a control brain. The heterogeneous distribution of copper was checked by scanning electron microscope equipped with an X-ray microanalyzer.     

Imaging of metals in biological tissue by laser ablation inductively coupled plasma mass spectrometry (LA–ICP–MS): state of the art and future developments

Laser ablation inductively coupled plasma mass spectrometry (LA–ICP–MS) is well established as a sensitive trace and ultratrace analytical technique with multielement capability for bioimaging of metals and studying metallomics in biological and medical tissue. Metals and metalloproteins play a key role in the metabolism and formation of metal‐containing deposits in the brain but also in the liver. In various diseases, analysis of metals and metalloproteins is essential for understanding the underlying cellular processes. LA–ICP–MS imaging (LA–ICP–MSI) combined with other complementary imaging techniques is a sophisticated tool for investigating the regional and cellular distribution of metals and related metal‐containing biomolecules. On the basis of successful routine techniques for the elemental bioimaging of cryosections by LA–ICP–MSI with a spatial resolution between 200 and ~10 µm, the further development used online laser microdissection ICP–MSI to study the metal distribution in small biological sample sections (at the cellular level from 10 µm to the submicrometer range). The use of mass spectrometric imaging of metals and also nonmetals is demonstrated on a series of biological specimens. This article discusses the state of the art of bioimaging of metals in thin biological tissue sections by LA–ICP–MSI with spatial resolution at the micrometer scale, future developments and prospects for quantitative imaging techniques of metals in the nanometer range. In addition, combining quantitative elemental imaging by LA/laser microdissection–ICP–MSI with biomolecular imaging by matrix‐assisted laser desorption/ionization–MSI will be challenging for future life science research. Copyright © 2013 John Wiley & Sons, Ltd.     

Incubation with Cu(II) and Zn(II) salts enhances MALDI‐TOF mass spectra of amyloid‐beta and α‐synuclein toward in vivo analysis

Insoluble senile plaque aggregates are indicative of Alzheimer's disease pathology. A similar phenomenon occurs in Parkinson's disease with the build‐up of Lewy bodies. The analysis of senile plaques, and other brain samples, from Alzheimer's disease and Parkinson's disease patients by matrix‐assisted laser desorption/ionization mass spectrometry has advantages but also presents obstacles because of the nature of the processes utilized in isolation procedures and storage. Salts, buffers, and detergents necessary in the isolation of biological species may cause adducts and ion suppression that convolute the spectra obtained. We previously determined that amyloid‐beta from isolated senile plaque deposits fragment similarly to the synthetic 40 and 42 amino acid peptide when analyzed by matrix‐assisted laser desorption/ionization mass spectrometry. In addition, α‐synuclein also fragments predictably by in‐source decay. This provides information that may be applied to the identification and localization of amyloid‐beta and α‐synuclein in senile plaques and intact tissue sections. Ion suppression must still be accounted for when analyzing biological samples, which makes identifying fragments at lower abundance difficult. The addition of certain transition‐metal salts (Cu(II), Zn(II)) to the sample prior to analysis serves to “clean” the spectra and allow the peptide fragments produced to be observed with a much higher signal to noise and occasionally, improved resolution. We present a systematic study of incubation with different metal salts and their impact on the quality of the spectra, as well as the role of the binding of the metals to the model biological compounds, obtained for synthetic amyloid‐beta, synthetic α‐synuclein, and isolated senile plaques. The optimized sample preparation methods presented will provide for simpler and more thorough identification of these biologically relevant species in human‐derived samples.     

Unambiguous Intracellular Localization and Quantification of a Potent Iridium Anticancer Compound by Correlative 3D Cryo X-Ray Imaging

The iridium half-sandwich complex [Ir(η⁵:κ¹-C₅Me₄CH₂py)(2-phenylpyridine)]PF₆ is highly cytotoxic: 15–250× more potent than clinically used cisplatin in several cancer cell lines. We have developed a correlative 3D cryo X-ray imaging approach to specifically localize and quantify iridium within the whole hydrated cell at nanometer resolution. By means of cryo soft X-ray tomography (cryo-SXT), which provides the cellular ultrastructure at 50 nm resolution, and cryo hard X-ray fluorescence tomography (cryo-XRF), which provides the elemental sensitivity with a 70 nm step size, we have located the iridium anticancer agent exclusively in the mitochondria. Our methodology provides unique information on the intracellular fate of the metallodrug, without chemical fixation, labeling, or mechanical manipulation of the cells. This cryo-3D correlative imaging method can be applied to a number of biochemical processes for specific elemental localization within the native cellular landscape.     

Voltage‐programming‐based capillary gel electrophoresis for the fast detection of angiotensin‐converting enzyme insertion/deletion polymorphism with high sensitivity

A voltage‐programming‐based capillary gel electrophoresis method with a laser‐induced fluorescence detector was developed for the fast and highly sensitive detection of DNA molecules related to angiotensin‐converting enzyme insertion/deletion polymorphism, which has been reported to influence predisposition to various diseases such as cardiovascular disease, high blood pressure, myocardial infarction, and Alzheimer's disease. Various voltage programs were investigated for fast detection of specific DNA molecules of angiotensin‐converting enzyme insertion/deletion polymorphism as a function of migration time and separation efficiency to establish the effect of voltage strength to resolution. Finally, the amplified products of the angiotensin‐converting enzyme insertion/deletion polymorphism (190 and 490 bp DNA) were analyzed in 3.2 min without losing resolution under optimum voltage programming conditions, which were at least 75 times faster than conventional slab gel electrophoresis. In addition, the capillary gel electrophoresis method also successfully applied to the analysis of real human blood samples, although no polymorphism genes were detected by slab gel electrophoresis. Consequently, the developed voltage‐programming capillary gel electrophoresis method with laser‐induced fluorescence detection is an effective, rapid analysis technique for highly sensitive detection of disease‐related specific DNA molecules.     

Imaging mass spectrometry in biological tissues by laser ablation inductively coupled plasma mass spectrometry

Of all the inorganic mass spectrometric techniques, laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) plays a key role as a powerful and sensitive microanalytical technique enabling multi- element trace analysis and isotope ratio measurements at trace and ultratrace level. LA-ICP-MS was used to produce images of detailed regionally-specific element distribution in 20 microm thin sections of different parts of the human brain. The quantitative determination of copper, zinc, lead and uranium distribution in thin slices of human brain samples was performed using matrix-matched laboratory standards via external calibration procedures. Imaging mass spectrometry provides new information on the spatially inhomogeneous element distribution in thin sections of human tissues, for example, of different brain regions (the insular region) or brain tumor tissues. The detection limits obtained for Cu, Zn, Pb and U were in the ng g(-1) range. Possible strategies of LA-ICP-MS in brain research and life sciences include the elemental imaging of thin slices of brain tissue or applications in proteome analysis by combination with matrix-assisted laser desorption/ionization MS to study phospho- and metal- containing proteins will be discussed.     

Recent advances of ambient ionization mass spectrometry imaging in clinical research

The structural information and spatial distribution of molecules in biological tissues are closely related to the potential molecular mechanisms of disease origin, transfer, and classification. Ambient ionization mass spectrometry imaging is an effective tool that provides molecular images while describing in situ information of biomolecules in complex samples, in which ionization occurs at atmospheric pressure with the samples being analyzed in the native state. Ambient ionization mass spectrometry imaging can directly analyze tissue samples at a fairly high resolution to obtain molecules in situ information on the tissue surface to identify pathological features associated with a disease, resulting in the wide applications in pharmacy, food science, botanical research, and especially clinical research. Herein, novel ambient ionization techniques, such as techniques based on spray and solid‐liquid extraction, techniques based on plasma desorption, techniques based on laser desorption ablation, and techniques based on acoustic desorption were introduced, and the data processing of ambient ionization mass spectrometry imaging was briefly reviewed. Besides, we also highlight recent applications of this imaging technology in clinical researches and discuss the challenges in this imaging technology and the perspectives on the future of the clinical research.     

头发六种元素含量变化与Graves病关系的研究

为探讨Ｇｒａｖｅｓ病患者头发６种元素含量变化与临床的关系,检测并比较了３２例患者头发中微量元素Ｚｎ、Ｃｕ、Ｆｅ、Ｍｎ和常量元素Ｃａ、Ｍｇ含量。提示Ｇｒａｖｅｓ病人某些临床表现可能与体内元素异常有关。     

Molecular imaging of drug‐eluting coronary stents: method development,optimization and selected applications

A molecular imaging application was developed to characterize the drug distribution on CYPHER® and NEVO? Drug‐eluting Stents using MALDI Qq‐ToF analytical methodology. The coating matrix, laser energy, laser frequency, spatial resolution (related to rastering speed) and mass spectrometer parameters were optimized to analyze drug distribution in both durable and biodegradable polymer matrices. The developed method was extended to generate data from stents explanted from porcine coronary arteries. Due to the method's intrinsic specificity, it offers a significant advantage over other techniques in that it allows low‐level detection of the target molecule without biological interferences from the blood or tissue. The method is also capable of detecting drug‐related degradation products both from the finished stent product and from explanted stents. Copyright © 2012 John Wiley & Sons, Ltd.     

青海枸杞子中微量元素含量的测定

测定了青海枸杞子中锌、铜、锰、铅、砷、铁、硒等微量元素的含量。结果表明,青海枸杞子中含锌18.65μg/g,铜15.62μg/g,锰6.24μg/g,铅1.03μg/g,砷0.24μg/g,铁19.68μg/g,硒2.15μg/g,人体必需微量元素含量丰富,是枸杞具有极为重要的保健、防病、治病功能的重要原因之一。     

Single‐Molecule Spectroscopy,Imaging, and Photocontrol: Foundations for Super‐Resolution Microscopy (Nobel Lecture)

The initial steps toward optical detection and spectroscopy of single molecules in condensed matter arose out of the study of inhomogeneously broadened optical absorption profiles of molecular impurities in solids at low temperatures. Spectral signatures relating to the fluctuations of the number of molecules in resonance led to the attainment of the single‐molecule limit in 1989 using frequency‐modulation laser spectroscopy. In the early 90s, many fascinating physical effects were observed for individual molecules, and the imaging of single molecules as well as observations of spectral diffusion, optical switching and the ability to select different single molecules in the same focal volume simply by tuning the pumping laser frequency provided important forerunners of the later super‐resolution microscopy with single molecules. In the room temperature regime, imaging of single copies of the green fluorescent protein also uncovered surprises, especially the blinking and photoinduced recovery of emitters, which stimulated further development of photoswitchable fluorescent protein labels. Because each single fluorophore acts a light source roughly 1 nm in size, microscopic observation and localization of individual fluorophores is a key ingredient to imaging beyond the optical diffraction limit. Combining this with active control of the number of emitting molecules in the pumped volume led to the super‐resolution imaging of Eric Betzig and others, a new frontier for optical microscopy beyond the diffraction limit. The background leading up to these observations is described and current developments are summarized.     

Bioimaging mass spectrometry of trace elements – recent advance and applications of LA-ICP-MS: A review

Bioimaging using laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) offers the capability to quantify trace elements and isotopes within tissue sections with a spatial resolution ranging about 10–100 μm. Distribution analysis adds to clarifying basic questions of biomedical research and enables bioaccumulation and bioavailability studies for ecological and toxicological risk assessment in humans, animals and plants. Major application fields of mass spectrometry imaging (MSI) and metallomics have been in brain and cancer research, animal model validation, drug development and plant science. Here we give an overview of latest achievements in methods and applications. Recent improvements in ablation systems, operation and cell design enabled progressively better spatial resolutions down to 1 μm. Meanwhile, a body of research has accumulated covering basic principles of the element architecture in animals and plants that could consistently be reproduced by several laboratories such as the distribution of Fe, Cu, Zn in rodent brain. Several studies investigated the distribution and delivery of metallo-drugs in animals. Hyper-accumulating plants and pollution indicator organisms have been the key topics in environmental science. Increasingly, larger series of samples are analyzed, may it be in the frame of comparisons between intervention and control groups, of time kinetics or of three-dimensional atlas approaches.     

Mass spectrometry imaging of small molecules from live cells and tissues using nanomaterials

We review recently developed methods for analyzing live cells and tissues in ambient conditions without the use of harsh chemical fixation or physical freezing and drying. The first method is based on laser ablation in atmospheric pressure assisted by atmospheric pressure plasma and nanomaterials such as nanoparticles and graphene to enhance laser ablation. The second method is based on secondary ion mass spectrometry imaging of live cells in solution capped with single-layer graphene to preserve intact and hydrated biological samples even under ultrahigh vacuum for secondary ion mass spectrometry bio-imaging in solution with subcellular spatial resolution. Mass spectrometry imaging of small molecules from live cells and tissues can provide an innovative molecular imaging methodology for several biomedical and material research applications.     

心脑血管疾病与微量元素研究进展

心脑血管疾病发病日益增多的原因之一是机体缺乏人体必需微量元素,目前报道硒、锗、锌、钴、铜、锰、铬、钼、钒、镍等与心脑血管疾病有密切关系。用微量元素调控防治心脑血管疾病,目前是国内外研究的新课题,其目的是降低心脑血管疾病的死亡率和发病率,促进人类健康长寿。     

Comparative studies of MCs+-SIMS and e?-beam SNMS for quantitative analysis of bulk materials and layered structures

For the quantification of heterostructure depth profiles the knowledge of relative sensitivity factors (RSF) and the influence of matrix effects on the measured profiles is necessary. Matrix dependencies of the measured ion intensities have been investigated for sputtered neutral mass spectrometry (SNMS) and MCs⁺-SIMS. The use of Cs as primary ions for SNMS is advantageous compared to Ar because the depth resolution is improved without changing RSFs determined under Ar bombardment. No significant amount of molecules has been found in the SNMS spectra under Cs bombardment. Using MCs⁺-SIMS the RSFs are matrix dependent. An improvement of depth resolution can be achieved by biasing the sample against the primary ion beam for SNMS due to a reduction of the net energy of the primary ions and a resulting more gracing impact angle.