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Fast And Easy Method Of Lanthanoid Staining For Visualization Of Cellular Ultrastructure And Spatial Arrangement

Abstract

Considering the demand for investigating spatial cell arrangement within a scaffold, yet unsatisfied, we aimed at developing a new method of sample preparation for SEM that would, besides other things, allow visualization of cellular ultrastructure and deliver from toxic chemicals and artifacts. A combination of BSE imaging in a low-vacuum mode with supravital lanthanoid staining has yielded highly informative images that contain many of the subsurface cell structures, particularly, intercellular contacts, cell membranes, nuclei with nucleoli, mitochondria, endoplasmic reticulum, and cytoskeleton. Moreover, the developed approach provides an idea of three-dimensional arrangement of cellular elements with a greater depth of focus than that of optical methods, which is drastically important for fulfilling the needs of tissue engineering. Being fast and simple, the method is able to change the whole concept of using SEM in biomedical studies.
+
Li
+
Na
+
K
+
Rb
+
Cs
2+
Be
2+
Mg
2+
Ca
2+
Sr
2+
Ba
3+
B
3+
Al
3+
Sc
3+
Y
3+
Ln
A
C
HETEROVALENT ISOMORPHISM
INHIBITION OF BINARY Са TRANSPORT
ADPЧE'~PЧCa (ATP)ЧE'~PЧCa
2 2
(ATP) ADP
ATPЧ*E'~P ATPЧ*E'~PЧCa (ATP)Ч*E~PЧCa2
2+
Ca H+ 2+
Ca H+
2+ 3+ +
[2Ca ]®[Ln +Me ]
3+ + 3+ +
ADPЧE'~PЧ[Ln +Me ] (ATP)ЧE'~PЧ[Ln +Me ]
(ATP) ADP
3+ +
ATPЧ*E'~PЧ (ATP)Ч*E~PЧ[Ln +Me ]?
? H+
2+ 3+ +
[2Ca ]®[Ln +Me ]
x
B
EC1 EC2 EC3 EC4 EC5
EC5 EC4 EC3
EC1 EC2 EC3 EC4 EC5
EC5 EC4 EC3 EC2 EC1
EC2
EC1
2+ 3+
[3Ca ]®[2Ln ]
A
B
C
HETEROVALENT ISOMORPHISM
PASSIVE CHEMICAL SUBSTITUTION
cytoplasm extracellular space cytoplasm
A
B
IRREVERSIBLE ASSEMBLY OF MICROTUBULES
3+
IN THE PRESENCE OF Ln
3-
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Ln[PO ]Ї
4
3+
...+ Ln
Ln[PO ]Ї
4
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4Ln[PO ]Ї
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4Ln[PO ]Ї
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4Ln[PO ]Ї
4
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4
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C
a.subbot@niigb.ru
Novikov I.A., Subbot A.M., Kiryushchenkova N.P., Nesterova T.V., Gabashvili A.N., Sitnikov A.V., Bursov A.I.
Research Institute of Eye Diseases, 11 A,B Rossolimo St., 119021 Moscow, Russian Federation
FAST AND EASY METHOD OF LANTHANOID STAINING
FOR VISUALIZATION OF CELLULAR ULTRASTRUCTURE AND SPATIAL ARRANGEMENT
Lanthanoid staining method for BSE imaging Classic SEM - sophisticated sample
preparation (unchanged
from the last century)
- use of toxic
chemicals
- often produce
artifacts visualization of cellular ultrastructure and
spatial arrangement of biological objects
Binding of Ln ions
to free phosphate
anions
A) phosphate anions released
during microtubule assembly
B) phosphate anions produce
insoluble compounds with
neodymium
C) we see a halo around the
cytoskeleton that serves as
an indicator of microtubules
and statistical trends of their
assembly (bar - 5mm)
2+ 3+
Heterovalent substitution: 3Ca ↔2Ln
A) extracellular cadherin domains that mediate cell adhesion
B) three calcium ions are replaced by two lanthanide ions
C) significant increase in local brightness in a BSE-image of monolayer of corneal epithelium in
cell culture (bar - 10mm)
2+ 3+ +
Heterovalent substitution: 2Ca ↔Ln +Me
(calcium ATPases)
A) two Ca ions are transported together across the cell membrane
B) an Ln ion co-operates with an alkali metal ion causing the pump to
stop at the point when the two Ca ions were supposed to part
C) bright cell membranes in a BSE-image of corneal
epithelium in cell culture (bar - 20mm)
BACKGROUND
Ln produce soluble chlorides
with neutral pH and low toxicity
The contrast of BSE-images is
increased by several times
Principles of chemical
isomorphism
Ca ion can be easily
replaced for a Ln
BSE-image of
MSCs on
Alvetex® scaffold
stained with NdCl
shows spatial
relationships between cells
on the ribs of a porous
membrane
This biopsy of corneal epithelium contains
mostly bright cell borders. Deeper, nuclei can
also be seen.
Human
astrocytes
on culture
plastic show thin
cytoplasmic
outgrowths,
delineated with bright
cell membrane, and
rounded mitochondria in the
periphery
Ln staining allows visualization of
many of the subsurface cell structures
with minimum time required.
The developed method of lanthanoid staining has proved
not only highly effective in revealing cellular ultrastructure
and spatial arrangement within a scaffold, but also fast
and simple, and thus, is able to change the whole
concept of using SEM in biomedical studies.
BioREE staining kit (“Glaukon” LLC,
Russia) was used at all stages of
sample preparation.
The sample preparation protocol was
the following:
* First rinse
to ensure that REE ions, known for
their high affinity to phosphates, did
not produce insoluble compounds with
growth media components or the
watery ground substance adsorbed
onto the surface of the sample
* Soaking in NdCl3 solution for 15-45
min
for saturation of the sample with the
contrast agent
* Another rinse for 1-10 sec
to remove any of the remaining
contrast
* Thus prepared samples were placed
horizontally on the stage of the
microscope for BSE imaging.
Acquisition parameters and the
staining protocol considered the
differences between the objects.
To succeed in visualizing the cell interior in BSE mode,
it is necessary to saturate the structures of interest with
heavy elements.
All present
images were made on
SEM Zeiss EVO Ls10
microscope:
EP (70Pa),
LaB - cathode, 20-25kV,
6
WD < 8mm, BSE
CONCLUSION
RESULTS
MATERIAL AND METHODS
INTRODUCTION
surface topography only
Diagonal series of isomorphism
(by Goldschmidt, Fersman)
The predicted increase of contrast in BSE mode
after having Ca replaced for a Ln
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