Randy J. Smith

FTIR spectroscopic imaging of protein aggregation in living cells.

Protein misfolding and aggregation are the hallmark of a number of diseases including Alzheimers disease, Parkinsons disease, Huntingtons 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.

In vitro efficiency and mechanistic role of indocyanine green as photodynamic therapy agent for human melanoma

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.

Dynamic Full-Field Infrared Imaging with Multiple Synchrotron Beams

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.

Characterization of Protein Structural Changes in Living Cells Using Time-Lapsed FTIR Imaging

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).

Development and applications of an epifluorescence module for synchrotron x-ray fluorescence microprobe imaging

Synchrotron x-ray fluorescence (XRF) microprobe is a valuable analysis tool for imaging trace element composition in situ at a resolution of a few microns. Frequently, epifluorescence microscopy is beneficial for identifying the region of interest. To date, combining epifluorescence microscopy with x-ray microprobe has involved analyses with two different microscopes. We report the development of an epifluorescence module that is integrated into a synchrotron XRF microprobe beamline, such that visible fluorescence from a sample can be viewed while collecting x-ray microprobe images simultaneously. This unique combination has been used to identify metal accumulation in Alzheimer’s disease plaques and the mineral distribution in geological samples. The flexibility of this accessory permits its use on almost any synchrotron x-ray fluorescence microprobe beamline and applications in many fields of science can benefit from this technology.

Technical Report: The Diversity of Infrared Programs at the NSLS

For about 20 years, synchrotron radiation (SR) has benefited the field of infrared (IR) spectroscopy in a wide range of disciplines from condensed matter physics to medicine. While a synchrotron infrared source does not typically produce more power than a conventional thermal (globar) source, its brightness (defined as the photon flux or power emitted per source area and solid angle) is 100–1000 times greater [1]. This advantage, arising from the small effective source size and narrow angular range of emission, has enabled a wide spectrum of throughput-limited experiments that were not possible with a conventional IR source.

JAST/IHAVS project overview

The Integrated Helmet Audio Visual System (IHAVS) is a joint advanced strike technology project which integrated previously but separately demonstrated audio and visual cockpit technologies into a single system. These technologies included a helmet mounted display system, a 3-D audio system with active noise reduction, voice control/speech recognition, and an imaging FLIR targeting system. A flight demonstration program on a TAV-8B aircraft is performing mission management and air-to-ground attack functions, demonstrating the operational utility of IHAVS technologies for strike missions. This paper serves to introduce the SPIE conference session and the associated papers that will describe in detail the IHAVS technologies, system development, integration and flight demonstration.

X-ray Fluorescence Nanotomography of Single Bacteria with a Sub-15 nm Beam

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.

Software development for multi-wavelength image correlation

The National Synchrotron Light Source II (NSLSII) at Brookhaven National Laboratory offers a large variety of synchrotron based imaging techniques that provide users with structural and chemical information of materials at the nanoscale. Multiple imaging techniques such as light microscopy, infrared imaging and X-ray fluorescence microscopy are commonly used to correlate information from the same sample. Correlating the important information in such images presents a large technological challenge because the various imaging techniques generate images of different sizes and spatial resolutions. To overcome this challenge, there are two goals involving software development: the first to identify a method for using fiducial markers to correlate visible light images with Xray fluorescence microscope images at the micro- to nanoscale and the second to develop software for the fusion (i.e. overlap) and correlation of these images. Numerous programs were identified to complete this project such as Matlab, Python, Photoshop, ImageJ and Fiji. After doing much research and testing a variety of these programs, it was clear that Fiji was the best and most efficient program for solving these challenges. It has the capability to align images automatically by converting the image to 8-bit gray scale, finding the maxima, i.e. darkest points, and stacking as well as corresponding these max points to align images with pin point accuracy. Moreover, it has the capability to use manually chosen fiducial point markers where the user inputs landmarks or fiducial points on the image and the program triangulates the points to align them. In addition, a user manual was written so that other synchrotron users can benefit from this methodology as well. Overall, this process will prove to be very useful in the future of the laboratory in cases where image correlation is vital. The majority of photon sciences involve correlating images and analyzing the data that come out.