RESUMEN
We report the application of lanthanide-binding tags (LBTs) for two- and three-dimensional X-ray imaging of individual proteins in cells with a sub-15 nm beam. The method combines encoded LBTs, which are tags of minimal size (ca. 15-20 amino acids) affording high-affinity lanthanide ion binding, and X-ray fluorescence microscopy (XFM). This approach enables visualization of LBT-tagged proteins while simultaneously measuring the elemental distribution in cells at a spatial resolution necessary for visualizing cell membranes and eukaryotic subcellular organelles.
Asunto(s)
Imagenología Tridimensional/métodos , Elementos de la Serie de los Lantanoides/metabolismo , Proteínas/química , Espectrometría por Rayos X/métodos , Secuencia de Aminoácidos , Unión ProteicaRESUMEN
X-ray Fluorescence (XRF) microscopy is a growing approach for imaging the trace element concentration, distribution, and speciation in biological cells at the nanoscale. Moreover, three-dimensional nanotomography provides the added advantage of imaging subcellular structure and chemical identity in three dimensions without the need for staining or sectioning of cells. To date, technical challenges in X-ray optics, sample preparation, and detection sensitivity have limited the use of XRF nanotomography in this area. Here, XRF nanotomography was used to image the elemental distribution in individual E. coli bacterial cells using a sub-15 nm beam at the Hard X-ray Nanoprobe beamline (HXN, 3-ID) at NSLS-II. These measurements were simultaneously combined with ptychography to image structural components of the cells. The cells were embedded in small (3-20 µm) sodium chloride crystals, which provided a non-aqueous matrix to retain the three-dimensional structure of the E. coli while collecting data at room temperature. Results showed a generally uniform distribution of calcium in the cells, but an inhomogeneous zinc distribution, most notably with concentrated regions of zinc at the polar ends of the cells. This work demonstrates that simultaneous two-dimensional ptychography and XRF nanotomography can be performed with a sub-15 nm beam size on unfrozen biological cells to co-localize elemental distribution and nanostructure simultaneously.
Asunto(s)
Escherichia coli/ultraestructura , Tomografía por Rayos X/métodos , Tomografía por Rayos X/instrumentaciónRESUMEN
Fourier-transform infrared (FTIR) spectroscopic imaging is a widely used method for studying the chemistry of proteins, lipids, and DNA in biological systems without the need for additional tagging or labeling. This technique can be especially powerful for spatially resolved, temporal studies of dynamic changes such as in vivo protein folding in cell culture models. However, FTIR imaging experiments have typically been limited to dry samples as a result of the significant spectral overlap between water and the protein Amide I band centered at 1650 cm(-1). Here, we demonstrate a method to rapidly obtain high quality FTIR spectral images at submicron pixel resolution in vivo over a duration of 18 h and longer through the development and use of a custom-built, demountable, microfluidic-incubator and a FTIR microscope coupled to a focal plane array (FPA) detector and a synchrotron light source. The combined system maximizes ease of use by allowing a user to perform standard cell culture techniques and experimental manipulation outside of the microfluidic-incubator, where assembly can be done just before the start of experimentation. The microfluidic-incubator provides an optimal path length of 6-8 µm and a submillimeter working distance in order to obtain FTIR images with 0.54-0.77 µm pixel resolution. In addition, we demonstrate a novel method for the correction of spectral distortions caused by varying concentrations of water over a subconfluent field of cells. Lastly, we use the microfluidic-incubator and time-lapsed FTIR imaging to determine the misfolding pathway of mutant copper-zinc superoxide dismutase (SOD1), the protein known to be a cause of familial amyotrophic lateral sclerosis (FALS).
Asunto(s)
Superóxido Dismutasa/química , Esclerosis Amiotrófica Lateral/enzimología , Animales , Células CHO , Supervivencia Celular , Células Cultivadas , Cricetulus , Humanos , Técnicas Analíticas Microfluídicas/instrumentación , Conformación Proteica , Espectroscopía Infrarroja por Transformada de Fourier/instrumentación , Superóxido Dismutasa/genética , Superóxido Dismutasa/metabolismo , Superóxido Dismutasa-1 , Factores de TiempoRESUMEN
Microspectroscopic imaging in the infrared (IR) spectral region allows for the examination of spatially resolved chemical composition on the microscale. More than a decade ago, it was demonstrated that diffraction-limited spatial resolution can be achieved when an apertured, single-pixel IR microscope is coupled to the high brightness of a synchrotron light source. Nowadays, many IR microscopes are equipped with multipixel Focal Plane Array (FPA) detectors, which dramatically improve data acquisition times for imaging large areas. Recently, progress been made toward efficiently coupling synchrotron IR beamlines to multipixel detectors, but they utilize expensive and highly customized optical schemes. Here we demonstrate the development and application of a simple optical configuration that can be implemented on most existing synchrotron IR beamlines to achieve full-field IR imaging with diffraction-limited spatial resolution. Specifically, the synchrotron radiation fan is extracted from the bending magnet and split into four beams that are combined on the sample, allowing it to fill a large section of the FPA. With this optical configuration, we are able to oversample an image by more than a factor of 2, even at the shortest wavelengths, making image restoration through deconvolution algorithms possible. High chemical sensitivity, rapid acquisition times, and superior signal-to-noise characteristics of the instrument are demonstrated. The unique characteristics of this setup enabled the real-time study of heterogeneous chemical dynamics with diffraction-limited spatial resolution for the first time.
Asunto(s)
Microscopía/instrumentación , Espectroscopía Infrarroja por Transformada de Fourier/instrumentación , Sincrotrones/instrumentación , Animales , Diseño de Equipo , Ratones , Médula Espinal/química , Médula Espinal/ultraestructuraRESUMEN
Protein misfolding and aggregation are the hallmark of a number of diseases including Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, and the prion diseases. In all cases, a naturally-occurring protein misfolds and forms aggregates that are thought to disrupt cell function through a wide range of mechanisms that are yet to be fully unraveled. Fourier transform infrared (FTIR) spectroscopy is a technique that is sensitive to the secondary structure of proteins and has been widely used to investigate the process of misfolding and aggregate formation. This review focuses on how FTIR spectroscopy and spectroscopic microscopy are being used to evaluate the structural changes in disease-related proteins both in vitro and directly within cells and tissues. Finally, ongoing technological advances will be presented that are enabling time-resolved FTIR imaging of protein aggregation directly within living cells, which can provide insight into the structural intermediates, time scale, and mechanisms of cell toxicity associated with aggregate formation. This article is part of a Special Issue entitled: FTIR in membrane proteins and peptide studies.
Asunto(s)
Proteínas/química , Espectroscopía Infrarroja por Transformada de Fourier/métodos , Humanos , Enfermedades Neurodegenerativas/metabolismo , Enfermedades Neurodegenerativas/patología , Conformación ProteicaRESUMEN
BACKGROUND: Photodynamic therapy (PDT) is a promising treatment for superficial cancer. However, poor therapeutic results have been reported for melanoma, due to the high melanin content. Indocyanine green (ICG) has near infrared absorption (700-800 nm) and melanins do not absorb strongly in this area. This study explores the efficiency of ICG as a PDT agent for human melanoma, and its mechanistic role in the cell death pathway. METHODS: Human skin melanoma cells (Sk-Mel-28) were incubated with ICG and exposed to a low power Ti:Sapphire laser. Synchrotron-assisted Fourier transform infrared microspectroscopy and hierarchical cluster analysis were used to assess the cell damage and changes in lipid, protein, and nucleic acids. The cell death pathway was determined by analysis of cell viability and apoptosis and necrosis markers. RESULTS: In the cell death pathway, (1)O(2) generation evoked rapid multiple consequences that trigger apoptosis after laser exposure for only 15 min including the release of cytochrome c, the activation of total caspases, caspase-3, and caspase-9, the inhibition of NF-kappaB P65, and the enhancement of DNA fragmentation, and histone acetylation. CONCLUSION: ICG/PDT can efficiently and rapidly induce apoptosis in human melanoma cells and it can be considered as a new therapeutic approach for topical treatment of melanoma.
Asunto(s)
Verde de Indocianina/uso terapéutico , Melanoma/terapia , Fotoquimioterapia , Fármacos Fotosensibilizantes/uso terapéutico , Apoptosis , Línea Celular Tumoral , Fragmentación del ADN , Humanos , Estructura Molecular , Transducción de SeñalRESUMEN
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 microm 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.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Química Encefálica , Cabello/química , Metales/análisis , Compuestos Orgánicos/análisis , Espectrometría por Rayos X/métodos , Espectroscopía Infrarroja por Transformada de Fourier/métodos , Enfermedad de Alzheimer/patología , Animales , Técnicas de Cultivo de Célula/métodos , Cabello/citología , Humanos , Ratones , Microscopía/métodos , Nanoestructuras/química , Nanoestructuras/ultraestructura , Estadística como Asunto , Propiedades de Superficie , Integración de Sistemas , Técnicas de Cultivo de Tejidos/métodosRESUMEN
Alzheimer's disease (AD) is characterized by the misfolding and plaque-like accumulation of a naturally occurring peptide in the brain called amyloid beta (Abeta). Recently, this process has been associated with the binding of metal ions such as iron (Fe), copper (Cu), and zinc (Zn). It is thought that metal dyshomeostasis is involved in protein misfolding and may lead to oxidative stress and neuronal damage. However, the exact role of the misfolded proteins and metal ions in the degenerative process of AD is not yet clear. In this study, we used synchrotron Fourier transform infrared micro-spectroscopy (FTIRM) to image the in situ secondary structure of the amyloid plaques in brain tissue of AD patients. These results were spatially correlated with metal ion accumulation in the same tissue sample using synchrotron X-ray fluorescence (SXRF) microprobe. For both techniques, a spatial resolution of 5-10 microm was achieved. FTIRM results showed that the amyloid plaques have elevated beta-sheet content, as demonstrated by a strong amide I absorbance at 1625cm(-1). Using SXRF microprobe, we find that AD tissue also contains "hot spots" of accumulated metal ions, specifically Cu and Zn, with a strong spatial correlation between these two ions. The "hot spots" of accumulated Zn and Cu were co-localized with beta-amyloid plaques. Thus for the first time, a strong spatial correlation has been observed between elevated beta-sheet content in Abeta plaques and accumulated Cu and Zn ions, emphasizing an association of metal ions with amyloid formation in AD.