Suhel Dahiwal

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

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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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REFERENCES
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36(1), 95-106.
2. Chao, W., Harteneck, B. D., Liddle, J. A., Anderson, E. H., & Attwood, D. T. (2005). Soft X-ray
microscopy at a spatial resolution better than 15 nm. Nature, 435(7046), 1210-1213.
3. Cinquin, B. P., Do, M., McDermott, G., Walters, A. D., Myllys, M., Smith, E. A., & Larabell, C.
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10. Smith, E. A., McDermott, G., Do, M., Leung, K., Panning, B., Le Gros, M. A., & Larabell, C. A.
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12. Uchida, M., Sun, Y., McDermott, G., Knoechel, C., Le Gros, M. A., Parkinson, D., Larabell, C.
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