This document discusses the techniques of correlated cryo-light microscopy (CLM) and soft X-ray tomography (SXT) for visualizing cell architecture and molecular location. CLM provides high-resolution fluorescent imaging of cells at cryogenic temperatures to localize molecules, while SXT uses soft X-rays to visualize internal cell structure. The two techniques are performed sequentially on the same sample and the data is merged to provide a composite 3D view of both cell structure and molecular location within cells. Specimen preparation, 3D reconstruction, alignment using fiducial markers, and segmentation based on linear absorption coefficients are discussed.
Study of magnetic and structural and optical properties of Zn doped Fe3O4 nan...Nanomedicine Journal (NMJ)
Objective(s):
This paper describes synthesizing of magnetic nanocomposite with co-precipitation
method.
Materials and Methods:
Magnetic ZnxFe3-xO4 nanoparticles with 0-14% zinc doping (x=0, 0.025, 0.05, 0.075, 0.1 and 0.125) were successfully synthesized by co-precipitation method. The prepared zinc-doped Fe3O4 nanoparticles were characterized by X-ray diffraction (XRD), transmission electron microscopy (TEM), Fourier transform infrared spectroscopy (FTIR), vibrating sample magnetometer (VSM) and UV-Vis spectroscopy.
Results:
results obtained from X-ray diffraction pattern have revealed the formation of single phase nanoparticles with cubic inverse spinal structures which size varies from 11.13 to 12.81 nm. The prepared nanoparticles have also possessed superparamagnetic properties at room temperature and high level of saturation magnetization with the maximum level of 74.60 emu/g for x=0.075. Ms changing in pure magnetite nanoparticles after impurities addition were explained based on two factors of “particles size” and “exchange interactions”. Optical studies results revealed that band gaps in all Zn-doped NPs are higher than pure Fe3O4. As doping percent increases, band gap value decreases from 1.26 eV to 0.43 eV.
Conclusion:
These magnetic nanocomposite structures since having superparamagnetic property
offer a high potential for biosensing and biomedical application.
Protein structure determinationand our software toolsMark Berjanskii
Protein structure determinationand our software tools. Presentation is related to: biochemistry, bioinformatics, biology, biophysics, mark berjanskii, molecular biology, molecular dynamics, molecular modeling, nmr spectroscopy, protein nmr, public speaking, python programming, sparse data, structural biology, structure determination, teaching, web design, web development, web programming, Wishart group, hybrid data, X-ray crystallography, CryoEM, Mass Spectrometry
Super Resolution Microscopy publications 2012Firstscientix
Localization Microscopy: Molecular Galaxies of protein transport in human blood-brain-barrier, tight junctions networks, gene transcription, single molecules, nuclear histones, viruses
Effect of Bi Content on Optical Properties of Se-Sb-Bi Chalcogenide Amorphous...ijsrd.com
Se90-xSb10Bix (x=0, 2, 4, 6, 8 & 10) chalcogenide glasses were prepared by well-established melt quenching technique. The glassy nature was verified by X-ray diffraction (XRD). Thin films of these samples were deposited on glass substrate using thermal evaporation technique at room temperature. The transmission spectra of thin films have been taken using UV-VIS-NIR spectrophotometer (Varian Cary 500) in the wavelength range 200 nm to 1500 nm. The refractive index and film thickness are calculated by using envelope method proposed by Swanepoel. The results indicate that n increases with the increasing Bi content which is related to the increased polarizability of the larger Bi atomic radius 1.46 Å compared with the Se atomic radius 1.16 Å. The value of absorption coefficient (α) and hence extinction coefficient (k) has been determined from transmission spectra. Optical band gap (Eg) is estimated using Tauc's extrapolation and is found to decrease from 1.46eV to 1.24 eV with the Bi addition. This behavior of optical band gap is interpreted in terms of electronegativity difference of the atoms involved and cohesive energy of the system. The variation of optical band gap with Bi content has been studied. This study is aiming to examine such structures if they are employed as photonic devices such as photo-detectors, LED's and optical switches.
Preparation and characterization of nimesulide loaded cellulose acetate hydro...Jing Zang
The aim of this study is to prepare nimesulide loaded cellulose acetate hydrogen phthalate nanoparticles by salting out technique. In this study Cellulose acetate Hydrogen phthalate was taken as polymer. Nimesulide was selected as a model drug. This technique is suitable for drugs and polymers that are soluble in polar solvents such as acetone or ethanol. The effect of drug concentration and polymer concentration on nanoparticle size, shape, uniform size distribution and stability was studied. Nanoparticles were evaluated for particle size, zetapotential and particle size distribution. Size of the particle was measured by SEM.(Scanning electron microscope).Surface charge and stability of the resultant nanoparticles was determined by Zetasizer. Particle size distribution was determined by Photon Correlation Spectroscopy (PCS) with a Malvern Zetasizer Nano-ZS. The cellulose acetate hydrogen phthalate concentration and nimesulide concentration was varied from 5mg/ml to 10 mg/ml. The effect of drug and polymer concentrations on nanoparticle size, shape, particle size distribution was studied. Increased drug concentration has no impact on the particle size. The size of the particle was found to be decreased with increased polymer concentration. Increased polymer concentration has resulted in uniform particle size distribution. Higher the polymer concentrations and lower the drug concentrations resulted in uniform particle size distribution.
Study of magnetic and structural and optical properties of Zn doped Fe3O4 nan...Nanomedicine Journal (NMJ)
Objective(s):
This paper describes synthesizing of magnetic nanocomposite with co-precipitation
method.
Materials and Methods:
Magnetic ZnxFe3-xO4 nanoparticles with 0-14% zinc doping (x=0, 0.025, 0.05, 0.075, 0.1 and 0.125) were successfully synthesized by co-precipitation method. The prepared zinc-doped Fe3O4 nanoparticles were characterized by X-ray diffraction (XRD), transmission electron microscopy (TEM), Fourier transform infrared spectroscopy (FTIR), vibrating sample magnetometer (VSM) and UV-Vis spectroscopy.
Results:
results obtained from X-ray diffraction pattern have revealed the formation of single phase nanoparticles with cubic inverse spinal structures which size varies from 11.13 to 12.81 nm. The prepared nanoparticles have also possessed superparamagnetic properties at room temperature and high level of saturation magnetization with the maximum level of 74.60 emu/g for x=0.075. Ms changing in pure magnetite nanoparticles after impurities addition were explained based on two factors of “particles size” and “exchange interactions”. Optical studies results revealed that band gaps in all Zn-doped NPs are higher than pure Fe3O4. As doping percent increases, band gap value decreases from 1.26 eV to 0.43 eV.
Conclusion:
These magnetic nanocomposite structures since having superparamagnetic property
offer a high potential for biosensing and biomedical application.
Protein structure determinationand our software toolsMark Berjanskii
Protein structure determinationand our software tools. Presentation is related to: biochemistry, bioinformatics, biology, biophysics, mark berjanskii, molecular biology, molecular dynamics, molecular modeling, nmr spectroscopy, protein nmr, public speaking, python programming, sparse data, structural biology, structure determination, teaching, web design, web development, web programming, Wishart group, hybrid data, X-ray crystallography, CryoEM, Mass Spectrometry
Super Resolution Microscopy publications 2012Firstscientix
Localization Microscopy: Molecular Galaxies of protein transport in human blood-brain-barrier, tight junctions networks, gene transcription, single molecules, nuclear histones, viruses
Effect of Bi Content on Optical Properties of Se-Sb-Bi Chalcogenide Amorphous...ijsrd.com
Se90-xSb10Bix (x=0, 2, 4, 6, 8 & 10) chalcogenide glasses were prepared by well-established melt quenching technique. The glassy nature was verified by X-ray diffraction (XRD). Thin films of these samples were deposited on glass substrate using thermal evaporation technique at room temperature. The transmission spectra of thin films have been taken using UV-VIS-NIR spectrophotometer (Varian Cary 500) in the wavelength range 200 nm to 1500 nm. The refractive index and film thickness are calculated by using envelope method proposed by Swanepoel. The results indicate that n increases with the increasing Bi content which is related to the increased polarizability of the larger Bi atomic radius 1.46 Å compared with the Se atomic radius 1.16 Å. The value of absorption coefficient (α) and hence extinction coefficient (k) has been determined from transmission spectra. Optical band gap (Eg) is estimated using Tauc's extrapolation and is found to decrease from 1.46eV to 1.24 eV with the Bi addition. This behavior of optical band gap is interpreted in terms of electronegativity difference of the atoms involved and cohesive energy of the system. The variation of optical band gap with Bi content has been studied. This study is aiming to examine such structures if they are employed as photonic devices such as photo-detectors, LED's and optical switches.
Preparation and characterization of nimesulide loaded cellulose acetate hydro...Jing Zang
The aim of this study is to prepare nimesulide loaded cellulose acetate hydrogen phthalate nanoparticles by salting out technique. In this study Cellulose acetate Hydrogen phthalate was taken as polymer. Nimesulide was selected as a model drug. This technique is suitable for drugs and polymers that are soluble in polar solvents such as acetone or ethanol. The effect of drug concentration and polymer concentration on nanoparticle size, shape, uniform size distribution and stability was studied. Nanoparticles were evaluated for particle size, zetapotential and particle size distribution. Size of the particle was measured by SEM.(Scanning electron microscope).Surface charge and stability of the resultant nanoparticles was determined by Zetasizer. Particle size distribution was determined by Photon Correlation Spectroscopy (PCS) with a Malvern Zetasizer Nano-ZS. The cellulose acetate hydrogen phthalate concentration and nimesulide concentration was varied from 5mg/ml to 10 mg/ml. The effect of drug and polymer concentrations on nanoparticle size, shape, particle size distribution was studied. Increased drug concentration has no impact on the particle size. The size of the particle was found to be decreased with increased polymer concentration. Increased polymer concentration has resulted in uniform particle size distribution. Higher the polymer concentrations and lower the drug concentrations resulted in uniform particle size distribution.
Different types of methods can be used for the preparation of Magnetic Nanoparticles, their advantages and disadvantages and applications of the materials in various fields are given in the presentation
Nanoparticles are solid colloidal particles ranging in size from 10 to 1000 nm.
Nanoparticles are made of a macromolecular material which can be of synthetic or natural origin.
Abstract
Terahertz spectral analysis has been conducted on epitaxially grown semiconductor structures. Epitaxially grown semiconductors are important for microelectronic and optoelectronic devices and also for integrated circuits
fabricated using semiconductors. In this paper, we report results of terahertz time-domain spectroscopy of grown
SiGe layers on Ge buffer and separately a Ge buffer that was grown on a Si <001> wafer. In particular, evolution of
the time-domain spectra as a function of thickness of both samples was investigated by the terahertz pump-probe
technique. Representative spectra were analyzed to determine the respective layers’ spectral signatures. It was found that the spectroscopic analysis uniquely identified different layers by characteristic absorbance peaks. In addition, terahertz imaging was conducted in a non-destructive, non-contact mode for detecting lattice stacking fault and dislocations. Sub-surface imaging of grown SiGe layers on Ge buffer and that of the Ge buffer grown on a Si wafer reveals interesting lattice features in both samples. A comparison with TEM images of the samples exhibits that the terahertz image reproduces the dimensions found from TEM images within the experimental error limits. In particular, 3D images of both samples were generated by the terahertz reconstructive technique. The images were analyzed by graphical means to determine the respective layer thicknesses. Thus, this technique offers a versatile tool for both semiconductor research and in-line inspections.
MODELING DEHYDRATION OF ORGANIC COMPOUNDS BY MEANS OF POLYMER MEMBRANES WITH ...ceij journal
The present study analyzes the amount of water-alcohol separation by pervaporation and use of polymer membranes with help of Artificial Neural Network and COMSOL Multiphysics. The influence of such parameters as volumetric flow rate, temperature, separation factor and permeate flux over the efficiency of dehydration process was analyzed through Artificial Neural Network. The reserarcher in this study used a Feed Forward multilayer Perceptron neural network with a back propogation algorithm and LevenbergMarquardt function with two inputs and two outputs. The Tansig transfer function was used for the hudden layer and Purelin was used for the output layer; five nerons were defined for the hidden layer. After data precessing, 70 percent of the data was allocated for learning, 15 percent was allocated for validation, and 25 percent was allocated for testing. The output values of Artificial Neural Network modelling were compard with the real values of pervaporation for separation of water from Ethanol, Acetone, and butanol. The results revealed that the proposed model had a good performance. Moreover, the output of COMSOL software for pervaporation of five different alcohols were compared with the real values, and the error percentage of the actual amount of flux was calculated with the modeling value by means of related membranes. The results of COMSOL modeling showed that the error percentages of 3.049, 3.7, 3.51, 2.88, and 3.82 were respectively achieved for dehydration process of Acetone, Butanol, Ethanol, Isopropanol and Methanol
Electron Diffraction Using Transmission Electron MicroscopyLe Scienze Web News
Electron diffraction via the transmission electron microscope is a powerful method for characterizing the structure of materials, including perfect crystals and defect structures. The advantages of elec- tron diffraction over other methods, e.g., x-ray or neutron, arise from the extremely short wavelength (≈2 pm), the strong atomic scattering, and the ability to exam- ine tiny volumes of matter (≈10 nm3). The NIST Materials Science and Engineer- ing Laboratory has a history of discovery and characterization of new structures through electron diffraction, alone or in combination with other diffraction methods. This paper provides a survey of some of this work enabled through electron mi- croscopy.
Different types of methods can be used for the preparation of Magnetic Nanoparticles, their advantages and disadvantages and applications of the materials in various fields are given in the presentation
Nanoparticles are solid colloidal particles ranging in size from 10 to 1000 nm.
Nanoparticles are made of a macromolecular material which can be of synthetic or natural origin.
Abstract
Terahertz spectral analysis has been conducted on epitaxially grown semiconductor structures. Epitaxially grown semiconductors are important for microelectronic and optoelectronic devices and also for integrated circuits
fabricated using semiconductors. In this paper, we report results of terahertz time-domain spectroscopy of grown
SiGe layers on Ge buffer and separately a Ge buffer that was grown on a Si <001> wafer. In particular, evolution of
the time-domain spectra as a function of thickness of both samples was investigated by the terahertz pump-probe
technique. Representative spectra were analyzed to determine the respective layers’ spectral signatures. It was found that the spectroscopic analysis uniquely identified different layers by characteristic absorbance peaks. In addition, terahertz imaging was conducted in a non-destructive, non-contact mode for detecting lattice stacking fault and dislocations. Sub-surface imaging of grown SiGe layers on Ge buffer and that of the Ge buffer grown on a Si wafer reveals interesting lattice features in both samples. A comparison with TEM images of the samples exhibits that the terahertz image reproduces the dimensions found from TEM images within the experimental error limits. In particular, 3D images of both samples were generated by the terahertz reconstructive technique. The images were analyzed by graphical means to determine the respective layer thicknesses. Thus, this technique offers a versatile tool for both semiconductor research and in-line inspections.
MODELING DEHYDRATION OF ORGANIC COMPOUNDS BY MEANS OF POLYMER MEMBRANES WITH ...ceij journal
The present study analyzes the amount of water-alcohol separation by pervaporation and use of polymer membranes with help of Artificial Neural Network and COMSOL Multiphysics. The influence of such parameters as volumetric flow rate, temperature, separation factor and permeate flux over the efficiency of dehydration process was analyzed through Artificial Neural Network. The reserarcher in this study used a Feed Forward multilayer Perceptron neural network with a back propogation algorithm and LevenbergMarquardt function with two inputs and two outputs. The Tansig transfer function was used for the hudden layer and Purelin was used for the output layer; five nerons were defined for the hidden layer. After data precessing, 70 percent of the data was allocated for learning, 15 percent was allocated for validation, and 25 percent was allocated for testing. The output values of Artificial Neural Network modelling were compard with the real values of pervaporation for separation of water from Ethanol, Acetone, and butanol. The results revealed that the proposed model had a good performance. Moreover, the output of COMSOL software for pervaporation of five different alcohols were compared with the real values, and the error percentage of the actual amount of flux was calculated with the modeling value by means of related membranes. The results of COMSOL modeling showed that the error percentages of 3.049, 3.7, 3.51, 2.88, and 3.82 were respectively achieved for dehydration process of Acetone, Butanol, Ethanol, Isopropanol and Methanol
Electron Diffraction Using Transmission Electron MicroscopyLe Scienze Web News
Electron diffraction via the transmission electron microscope is a powerful method for characterizing the structure of materials, including perfect crystals and defect structures. The advantages of elec- tron diffraction over other methods, e.g., x-ray or neutron, arise from the extremely short wavelength (≈2 pm), the strong atomic scattering, and the ability to exam- ine tiny volumes of matter (≈10 nm3). The NIST Materials Science and Engineer- ing Laboratory has a history of discovery and characterization of new structures through electron diffraction, alone or in combination with other diffraction methods. This paper provides a survey of some of this work enabled through electron mi- croscopy.
DEVELOPING CRYO-ELECTRON MICROSCOPY OF BIOMOLECULES IN WATERGuttiPavan
Cryo-electron microscopy (Cryo-EM) is a type of transmission electron microscopy that allows for the specimen of interest to be viewed at cryogenic temperatures (-150°C)
Following years of improvement, the cryo-electron microscope has become a valuable tool for viewing and studying the 3D structures of various biological molecules in water.
Morphological and Optical Study of Sol-Gel SpinCoated Nanostructured CdSThin ...iosrjce
Nanostructured CdS thin films of different thicknesses were deposited on a cleaned glass substrate
using sol-gel spin coating technique. CdS thin films were prepared using cadmium acetate as cadmium source
and thiourea as sulfur source. The Morphological, chemical composition, and optical properties of the spin- coated
CdS thin film were studied using field emission- scanning electron microscopy (FE-SEM), Energy dispersive X –ray
(EDX) spectroscopy, and a UV-Vis-NIR spectrophotometer.The morphological results revealed that the films consist
of agglomerated spherical CdS nanoparticles with diameter < 20 nm, which distributed uniformly on the substrate
surface.The films show high transmittance > 90% and very strong absorption edge at 295 nm.The absorption edge
shifts towards longer wavelength as the film thickness increased.
ADAPTIVE SEGMENTATION OF CELLS AND PARTICLES IN FLUORESCENT MICROSCOPE IMAGEJournal For Research
Understanding the mechanisms of cell motility and their regulation is an important challenge in biomedical research. The ability of cells to exert forces on their environment and alter their shape as they move is essential to various biological processes such as the immune response, embryonic development, or tumor genesis .Recent technological advances in con-focal fluorescence microscopy have given researchers the opportunity to investigate these complex processes in vivo. However, they also lead to a tremendous increase in the amount of image data acquired during the studies. Therefore, the analysis of time-lapse experiments relies increasingly on automated image processing techniques. Namely, there is a high demand for fast and robust methods to help biologists to quantitatively analyze time-lapse image data. The potential of the proposed tracking scheme and the advantages and disadvantages of both frameworks are demonstrated on 2-D and 3-D time-lapse series of rat adipose-derived mesenchymal stem cells and human lung squamous cell carcinoma cells, respectively. The crucial tasks are, in particular, segmenting, tracking, and evaluating movement tracks and morphological changes of cells, sub-cellular components and other particles.
this is a ppt prepared fro the partial fulfillment of MSc. in pharmaceutics, so if you are looking for detailed information on this topic its advisable to check other reputable journals.
Analysis Of Carbon Nanotubes And Quantum Dots In A Photovoltaic DeviceM. Faisal Halim
Analysis of Carbon Nanotubes and Quantum Dots in a Photovoltaic Device
A poster prepared by Francis and me; presented by Francis. I modified on of the photographs used, in this copy.
Analysis Of Carbon Nanotubes And Quantum Dots In A Photovoltaic Device
Suhel Dahiwal
1. Suhel Dahiwal 902933653 Essay II MEDSCI 703
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Visualizing Cell Architecture and Molecular Location Using Soft
X-Ray Tomography and Correlated Cryo-Light Microscopy
2. Suhel Dahiwal 902933653 Essay II MEDSCI 703
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INTRODUCTION
The visualization and location of higher order cells is necessary to understand the
environmental and/or genetic factors affecting cell phenotypes. This essay includes two
correlated cellular imaging techniques CLM (Cryo-Light Microscopy) and SXT (Soft X-ray
Tomography), which are used to provide location of specific reacting molecules and a high-
definition visual description of sub cellular architecture respectively. These two imaging
techniques are carried out serially on the same sample of cells and the data obtained from two
modalities are merged to form a composite view that is significantly greater than the sum of its
component parts. Further, this data is segmented as per the cell needs to be viewed and finally,
this essay will discuss advantages, few applications and futures scope for this correlated
imaging modalities.
CELL ARCHITECTURE
Cell biology is responsible for various chemical reactions and interconnected molecular
interactions. Cells not only carry out specific chemical reactions but also perform them in vast
numbers. Each cell performs thousands, or even millions, of chemical reactions per second.
These cells are structured to create a range of microenvironments which supports cell
functions. (McDermott et al., 2012) Cell size and sub-cellular volume (organelle) are most
important physical characteristics for chemical and molecular reactions to occur. (Uchida et al.,
2011) Cells, particularly eukaryotic cells, are higher order cells and have verycomplex
structures. (Fig. 1) However, partition of eukaryotic cells into membrane-bound, sub-cellular
volumes termed as organelles radically changes reaction kinetics. (McDermott et al., 2012)
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Fig. 1: Complex structure of higher order eukaryotic cell (Siegel, 2008)
HISTORY
Imaging modalities are primarily classified by the physical characteristics of their specimen
illumination. However, specimen illumination dictates factors such as maximum spatial
resolution and size range of specimens that can be imaged. (McDermott et al., 2012) The
imaging modalities used for imaging cell architecture is (conventional) fluorescence microscopy
and electron microscopy. However, both the modalities have their own limitations associated
with the imaging of cell biology. McDermott et al. (2012) stated that “fluorescence microscopy
is a very sensitive technique”and used to measure concentration of molecules along with the
relative molecular location. However, imaging of cells with the fluorescence microscopy is
limited up to 3µm. Electron microscopy is an imaging modality which is used to image very
small cells up to 700 nm on high resolution. (McDermott et al., 2012) However, due to low SNR
(signal to noise ratio), it is very difficult to segment the molecules after the image acquisition.
(Uchida et al., 2011)
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CLM (CRYO-LIGHT MICROSCOPY)
CLM is a powerful tool for localizing molecules in a cell. The conventional fluorescence
microscopy has a limitation of photo bleaching (excess exposure of light to fluorescent tags
damages the image quality and life of fluorescent molecules). However, In CLM, the specimen is
cooled at cryogenic temperature (below -160⁰c) with the use of cryogens such as liquid
propane as an immersion fluid. The low-temperature microscopes used in the past are
operated in air with the low numerical aperture lenses causing mismatch of refractive index,
degradation of maximum spatial resolution and fidelity of an image. (Smith et al., 2014)
However, CLM uses cryogenic immersion lens which allows frozen specimen to be imaged at
high spatial resolution by using index-matched cryogens. With the use of cryogen, cell
(specimen) is kept in its native state (original microenvironment) and hence causing constant
cumulative exposure to the cell. (Fig. 3) The frozen (amorphous ice) specimen is then labeled
using fluorescent tags such as green fluorescent protein. However, use of electron dense tag in
immune-labeling causes damage to the specimen. (McDermott et al., 2012) Fig.2 shows an
overview of CLM.
Fig.2: Overview of cryogenic fluorescent microscope (Cinquin et al., 2014)
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Fig. 3: Photo bleaching curves for yellow fluorescent protein, (YFP), expressed in E. coli. (A) The
blue curve shows fluorescence decay at cryogenic temperature; the red curve shows decay at
room temperature. The resistance to photo-bleaching at 77 K is ∼50 times that observed at
room temperature; (B) low-temperature bleaching curve plotted over a longer time period.
(LeGross et al., 2009)
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SXT (SOFT X-RAY TOMOGRAPHY)
Soft X-ray tomography is an imaging modality which is used in correlation with CLM to visualize
cell biology. SXT is a non-invasive method for imaging the internal structure of intact cells with
the use of grazing-incidence reflective optics to focus the X-rays on to specimen. (Kirkpatrick et
al., 1995) However, SXT uses Fresnel zone optical system situated into a third generation
synchrotron light source in a soft X-ray microscope. (Fig.4) (McDermott et al., 2012). SXT
produces soft X-rays of 0.28 Kev-0.53 Kev within a region called as “water window” (K-edge of
X-rays between oxygen-2.34nm and carbon-4.4nm) Soft X-rays are attenuated more strongly by
carbon and nitrogen relative to their attenuation by water. The absorption of X-rays adheres to
the Beer-Lambert law and is therefore a linear, quantitative and a function of thickness and
chemical composition of the specimen. (I.e. X-ray absorption will vary in accordance with the
thickness and concentration of the specimen)
Fig.4: The optical configuration of a bend magnet–based soft X-ray microscope. The specimen
sits between the objective and the condenser optical elements. (McDermott et al., 2012)
There are two main components of SXT; a) Synchrotron light source b) Fresnel zone plates
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a) Synchrotron source
Synchrotron light source is the optimal source of illumination photons for a soft X-ray
microscope. (McDermott et al., 2012) These light sources produce intense beam of X-
rays that can be readily collimated and focused into tiny specimens. The synchrotron
light source has many advantages including high accuracy, zero errors from beam
hardening or scattering, high spatial resolution and production of photon with energy-
selective narrow bandwidth. (Adam et al., 2009) However, synchrotron light sources are not
readily available in the laboratory. (McDermott et al., 2012) Efforts are taken to increase the
productivity and produce tabletop X-ray sources which can be used in the laboratory.
(Tuohimaa et al., 2008)
b) Fresnel zone plates
Fresnel zone plate contains radically symmetric rings known as Fresnel zones. (Andersen
et al., 2000) In a soft X-ray microscope Fresnel zones alternate between being opaque
and transparent toward X-ray photons. In operation, a soft X-ray beam diffracts around
the opaque zones (McDermott et al., 2012). The zones can be spaced so that the light
diffracted by each zone constructively interferes at the desired focus. Zones become
narrower and more closely packed towards the outer side of zone plate from the centre,
until the outermost zone is reached. The spatial resolution of SXT depends upon the
outermost zone of objective zone plate. (McDermott et al., 2012)
Fig. 5: Overview of Fresnel zone plate (Source: ESCO)
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The focal length (f) of a zone plate is,
f=OD ∆Rn /λ …………… (1)
Where,
OD = Diameter of zone plate
∆Rn = Outermost zone width
λ = X-ray wavelength
Fig.6: Scanning mechanism of the CLM-STXM as seen from the direction of the incident X-ray
beam. The cryo specimen holder is inserted into the vacuum chamber through an airlock, which
inturn is connected to the vacuum chamber through bellows. A precision lever mechanism
allows horizontal and vertical motion ofairlock and specimen holder. The tip of the cryo holder
is placed in a receptacle on the scanning stage, and held in place by anadjustable preload. For
high resolution scans, an in-vacuum flexurestage moves the specimen holder horizontally and
vertically.Coarse scans are performed by moving the whole fine stage and the specimen holder
using out-of-vacuum linear stages driven bystepping motors. (Maser et al., 2000)
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SPECIMEN PREPARATION
The specimen preparation for CLM-SXT is very minimal. Specimen preparation is an important
factor in determining the final quality and fidelity of any biological imaging
technique.(McDermott et al., 2012) Specimen is filled in a cylindrical holder usually a glass
capillary. However, use of flat surface (glass slide) for the specimen preparation increases the
specimen thickness through rotating axis while collecting the data. (Fig.7) (Cinquin et al., 2014)
However, glass capillary should be sized to match the cells being imaged since excess solution
surrounding cells adds background noise to the image. (McDermott et al., 2012)
Before loading the specimen, the glass capillary is dipped in poly-L-Lysine (0.01% Tissue culture
Grade) and immediately dipped in a solution of 100 nm gold nano-particles such as EMGC 100,
which are used as fiducial markers to align x-ray projections.(Smith et al., 2014). The glass
capillary is then heated and is then pulled to form an extended narrow tip. (McDermott et al.,
2012) The specimen is filled into a glass capillary using standard micropipette and treated with
cryogenic temperature (below -160⁰c). The frozen specimen is then cryo-transferred into
custom boxes using a home-built cryo-transfer device and stored in liquid nitrogen. (Smith et
al., 2014)
3D IMAGING
3D imaging is an important tool to visualize the internal structure of the specimen. The 2D
images captured by an imaging modality have internal structures superimposed on top of each
other causing difficulty in understanding the cell biology. However, if 2D images are captured
using different angles (over 360⁰c) around the rotation axis, a 3D tomographic reconstruction
can be calculated. (Fig.7-A) (McDermott et al., 2012) this process is done well within the
threshold of cumulative exposure required to cause observable radiation damage. (Fig.3) the
process of converting 2D projection images to 3D images is called “tomogram”.
The SXT is developed with the readily available sophisticated software packages (such as
AMIRA) for data processing and analysis. However, there are algorithms such as back-projection
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and algebraic reconstruction technique (ART) available for reconstruction of three-dimensional
volumes from two-dimensional soft X-ray projection series. (Marabini et al., 1998)
Fig. 7: Difference between (Cylindrical holder) glass capillary and (Flat surface) glass slide for
imaging isotropic data A) 3D imaging of the specimen by collecting data from rotation of
cylindrical holder by 360⁰c B) Rotation of the specimen using glass slide C) Difference in the
thickness value 5µm, 7.1µm, 16µm by rotation of 0⁰, 45⁰, 72⁰ respectively. D) Graph showing
the curve of thickness (in µm) which changes with angles (in degrees) (Cinquin et al., 2014)
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ALLIGNMENT
The alignment of CLM-SXT is very important to image the specimen with high spatial resolution.
The alignment is done using fiducial markers. (Fig. 8) The fiducial markers are used in both
ways; externally (onto the glass capillary) with the help of poly-L-Lysine, 100 nm gold
nanoparticles and internally with the help of red polystyrene microspheres (Fluro-Spheres
Carboxyl ate-Modified Microspheres, 0.2 mm, dark Red Fluorescent (660 excitation/ 680
emission), which served as fluorescence fiducials. (Smith et al., 2014) However, fiducial markers
are applied to specimen internally by fluorescently labeled organelles such as liquid droplets,
nucleus and granules. (Cinquin et al., 2014)The most common software used for alignment is
AMIRA. (Smith et al., 2014)
The alignment is done through z-stacks (data obtained by displacing glass capillary in z-axis)
using fluorescent fiducial markers. The fiducial co-ordinates are used to write as a new image
stack containing a spherically representation of fiducials which is termed as fiducial model.
Thehighest intensity values in the fiducial model correspond to the positionof the center,
surrounding voxel’s intensity values fall off as the distance from the center is increased. The
voxel dimensions of the fiducial modelswere first sampled to match the preprocessed data set
and then reduced stepwise until the errors inaligning the data sets plateaued (measured by the
distances between fiducial centers). (Smith et al., 2014)
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Fig. 8: Allignment of CLM-SXT imaging data using fiducial markers (Cinquin et al., 2014)
SEGMENTATION
Segmentation is the process of computationally isolating, visualizing and quantifying the
specific cellular components in a tomography reconstruction. In CLM-SXT, quantification is done
by segmentation of boundary regions using LAC (Linear Absorption Coefficient). LAC represents
the absorption values for each voxel in the reconstruction. Biological material attenuate soft X-
ray photons according to Beer’s law, the LAC values for identical sized voxels depends solely on
the concentration and composition of biomolecules present, with water having an order of
magnitude lower (Lower LAC) than molecules such as lipids and proteins. (Smith et al., 2014)
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Fig. 9: Detailed analysis of LAC values of X chromosome segmented from correlated CLM-SXT
reconstruction of female v-able macroH2A-EGFP transformed thymic lymphoma cells.
Representation of soft x-ray LAC values for X chromosome segmented from the SXT
reconstruction, shown from two perspectives 180⁰ apart. LAC values are categorized as high
(0.34–0.36 µm-1), medium (0.32–0.34 µm-1), or low (0.30–0.32 µm-1). From left to right the
pairs show combinations of the high, medium, and low LAC values measured in the
reconstruction. LAC color code: high, dark blue; medium, light blue; low, gold. Right-most
segmentations show all LAC values combined, together with a section of NE that makes contact
with the reconstruction (indicated by dashed ovals). (Smith et al., 2014)
Each organelle (sub-cellular volume) has a characteristic average LAC value depending upon
their chemical composition and thickness. Highly dense biomolecules such as lipid bodies,
nucleus has high LAC than the less dense molecules like vacuoles. (Fig. 10) (McDermott et al.,
2012) However, these characteristic values not only hold between cell of same type, but also
frequently are seen to hold between the cells from different species. (Uchida et al., 2011)
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Fig.10: Segmentation of organelles based on linear absorption coefficient (LAC) values in
Saccharomyces cerevisiae cell of yeast. (A) A representative diploid cell shown in an ortho-slice
(i.e. a single slice of tomographic data) and individually segmented organelles; scale bar = 1 μm.
(B) LAC values for each organelles. (C) Five different vacuolar compositions found in
tomographic data [left; the similar sizes of vacuoles were selected (i.e., 1 μm)], schematic views
(middle) and LAC values (right; indicates LAC values of structures inside vacuoles) (Uchida et al.,
2011)
Figure 10 represents the segmentation of organelles based on linear absorption coefficient
(LAC) values in yeast. Once the projection images were reconstructed, the volumes were
segmented to isolate individual cells and subsequently their component organelles (Figure
10A). The reconstructed cells were segmented into discrete volumes based on the LACs (Figure
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10B). For instance, volumes assigned as dense lipid bodies have an average LAC value of 0.55
μm-1compared with more transmissive organelles, such as nuclei, nucleoli, vacuoles and
mitochondria that have typical LAC values of 0.26, 0.33, 0.22 and 0.36 μm-1, respectively.
Assignment of organelle type to a particular segmented volume is guided by morphological
characteristics established by CLM imaging modality. For example, the nuclei/nucleoli,
mitochondria and vacuoles have distinct and very recognizable morphologies. Once vacuoles
from a number of cells had been segmented, it is categorized into five types, based on their
morphology, internal structure and densities. The LAC values for these are shown in Figure 10C.
ADVANTAGES
1) CLM-SXT produces excellent contrast without the use of contrast-enhancing stains e.g.
metals such as osmium, platinum
2) CLM-SXT increases the life of fluorescent molecules
3) CLM-SXT resolves ambiguities such as same size and same LAC (Linear Attenuation
Coefficient) over segmented regions in reconstruction
4) CLM-SXT produces impressive throughput of samples as the specimen preparation is
very minimal
5) CLM-SXT measures the volume of cell along with the high spatial resolution (up to
10nm) (Chao et al., 2005)
APPLICATION
The CLM-SXT correlated imaging modality has various applications. Due to its excellent
contrast, the CLM-SXT is used to visualize sub-cellular architecture of eukaryotic cells such as
yeast (Fig.11). (McDermott et al., 2012)The CLM-SXT is used in imaging of thymic lymphoma
cells. (Fig.12) (Smith et al., 2014) The CLM-SXT is not only used in imaging malaria parasite such
as plasmodium falciparum but also used to image mouse adenocarcinoma cells. (Cinquin et al.,
2014 and Schneider et al., 2010)
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Fig.11. Correlated soft X-ray tomography and cryo-light imaging (wide-field fluorescence). (a)
The vacuoles fluorescently labelled and imaged by cryo-light microscopy. (b, c) Slices through
the volumetric reconstruction calculated from soft X-ray tomography data, with the vacuoles
shown as segmented volumes in panel c. The segmented vacuoles correlate closely with the
locations determined from cryo-light microscopy. (d) The same cell after the major organelles
have been segmented. The nucleus is shown in blue, the nucleoli in orange, mitochondria in
grey, vacuoles in light grey, and lipid droplets in green. Scale bar = 1 μm. (McDermott et al.,
2012)
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Fig.12. Correlated CFT-SXT imaging of female v-able macroH2A-EGFP transformed thymic
lymphoma cells. (A) Four virtual sections from the de-convolved CFT reconstruction. (B) The
corresponding virtual sections from the SXT reconstruction. White arrow shows the closest
contact between Xi and the nucleolus. (C) The same sections in the combined CFT-SXT
reconstruction. (D) A 2-D projection of the Xi CFT reconstruction. (E) Cutaway of a volume
rendered SXT reconstruction. The surface of the cell is coloured light blue. LAC values are
represented in grey scale, ranging from high (dark) to low (light). (F) The CFT reconstruction
shown in (D) overlaid into the volume rendered SXT reconstruction shown in (E). (G) Surface
rendering of the inactive X chromosome segmented from the SXT reconstruction after
identification by macroH2A-EGFP CFT. The deep blue shaded areas are regions of high LAC that
contact the nuclear envelope. (Smith et al., 2014)
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FUTURE SCOPE
CLM-SXT with the Fresnel zone plate has many advantages including imaging of higher order
cells without using contrast-enhancing stains. However, ultra high resolution zone plate and
table-top synchrotron X-ray sources will allow cells to be imaged with a significantly greater
level of detail in the future. (McDermott et al, 2012 and Cinquin et al, 2014)
CONCLUSION
The correlated CLM-SXT imaging modality is an emerging modality for visualizing high-definition
sub-cellular architecture and locating specific bio-molecules. Excellent contrast, extended life of
fluorescent molecules, minimal specimen preparation, impressive throughput of samples,
solution of ambiguities (same size and same LAC) over segmented regions make CLM-SXT more
suitable for drug discovery, biomedical research and basic cell biology. However, further
improvements need to be done to increase the spatial resolution.
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