QX-102
Applications Manual
UQX003 Issue 2.1
漏 QuantomiX Ltd. 2005. All rights reserved. November 2005
QX-102 Applications Manual, Issue 2.1 2 QX-102 Applications Manual, Issue 2.1
3
Table of Contents
This publication is the copyright of QuantomiX Ltd. and contains information
that may not be used or reproduced unless agreed in writing. QuantomiX Ltd.
reserves the right to alter without notice the specifications, design or supply of 5
Safety
any product or service. 7
Chapter 1: Introduction
Manual Scope and Contents 7
The information provided in this Application Manual is believed to be accurate.
References 7
It is the user鈥檚 responsibility to confirm the technical aspects and the suitability
Sites 7
of the technology for any particular application.
Technical Support 7
QX-102 Applications 8
Cited registered trademarks: Coulter counter is a registered trademark of
Beckman Coulter Corp, Triton is a registered trademark of Union Carbide 9
Chapter 2: Liquid Samples
Corp., and Tween of Uniquema, a business unit of ICI Americas Inc. 11
Chapter 3: Particles in Solutions
Membrane Coating 11
Poly-L-lysine coating 11
Poly (sodium-4-styrenesulfonate) coating 12
QuantomiX Ltd., 12 Hamada Street, Tamar Science Park, Rehovot
Sample Application 13
POB 4037, Nes-Ziona 70400, Israel
15
Chapter 4: Biological Applications
Tel: +972-8-9462244, Fax: +972-8-9465874
General Protocols 15
http://www.quantomix.com
QX-102 Membrane Coating Protocols 17
Fibronectin Coating 18
Gelatin Coating 19
Poly-L-lysine Coating 1
Sample Application Protocols 21
Cell Culture Growth 21
Adherent Cells 22
Cells in Suspension and Microorganisms 23
Handling Native, Non-treated Samples 25
Fixation Protocols 26
Aldehydes 26
Glutaraldehyde Fixation 27
Paraformaldehyde Fixation 28
Organic Solvents 29
Methanol Fixation 29
QX-102 Applications Manual, Issue 2.1 4 QX-102 Applications Manual, Issue 2.1
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Safety
General Staining Protocols 30
Uranyl Acetate Staining 30
PTA (Phosphotungstic Acid) Staining 32
Warnings
!
Osmium Tetroxide Staining 33
Immunogold Labeling Protocols 35
a. Appropriate safety protocols should be used when handling
Immunogold Labeling Reaction 37
biological samples, especially human or primate derived cell lines
Preparing the Samples for Imaging 40
and pathogenic microorganisms.
41
Appendix A: Glossary b. Many of the reagents used in fixation and staining protocols are
toxic. Heavy metal stains (such as Uranium and Osmium) are
43
Appendix B: Troubleshooting Instructions
long-term cumulative poisons. Observe the following cautionary
rules when handling toxic materials:
- Read carefully the labels and MSDS (Material Safety Data
List of Figures
Sheet) and proceed accordingly.
- In general, work with toxic materials should be preformed in a
Figure 1 General Protocol Flowchart 16
fume hood, wearing laboratory gloves, goggles and lab-coats.
Figure 2 Immunolabeling Protocol Flowchart 36
- All toxic waste should be disposed of according to the
guidelines of local authorities.
List of Tables
Table 1 Guidelines for Cell Dilution 22
QX-102 Applications Manual, Issue 2.1 6 Chapter 1: Introduction
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Chapter 1: Introduction
Manual Scope and Contents
This manual provides protocols for preparing material and biological samples
for imaging in QX-102 capsules.
The Applications Manual consists of the following chapters and appendices:
Chapter/
Appendix Heading Provides
1 Introduction The manual scope and contents and
introduction to QX-102 capsule
applications.
2 Liquid Samples General protocol for applying
liquid samples.
3 Particles in Solution Protocols for coating the capsule
membrane for attaching particles.
4 Biological Applications The theory and protocols for various
biological applications, including
sample handling application, fixation,
staining and immunolabeling.
A Glossary The terms and abbreviations of the
Applications Manual.
B Troubleshooting Troubleshooting instructions.
For detailed description of the QX-102 capsule technology components and
general guidelines for handling the capsules and accessories, please refer to the
QX-102 User Manual.
Technical Support
For technical support please contact tech@quantomix.com.
QX-102 Applications Manual, Issue 2.1 8 Chapter 2: Liquid Samples
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QX-102 Applications Chapter 2: Liquid Samples
The QX-102 capsules can be used for imaging various wet material and The QX-102 capsule can be used for imaging any wet sample such as:
biological samples. The applications include liquid samples (emulsions, foods,
oils, paints, inks, drugs etc.), pastes and foams (cosmetics, foods, etc.), particles Various foods
in solutions, adherent and non-adherent cultured cells, and microorganisms. Cosmetics, creams
The contrast between water and fat is especially well visualized with WETSEM Emulsions
Technology, enabling analysis of fat structure and content in samples such as Oil, grease
food and cosmetics. QX capsules can also be used for EDS (energy dispersive Paint, ink
spectroscopy) analysis of samples using a SEM equipped with an EDS system.
Samples in liquid form can be imaged directly simply by placing them inside Liquid samples can be imaged directly, and usually no treatment of the sample
the QX-102 capsule. Some material samples, such as beads in solutions, may or of the capsule membrane is required.
require coating of the capsule membrane for proper attachment.
To image liquid samples
The QX-102 capsules are designed as miniature cell culture dishes, and
are suitable for various cell biology applications. The sample preparation is
comparable to light microscopy, and no drying, coating or embedding steps are 1. Open the capsule.
required. Adherent and non-adherent cultured cells, as well as microorganisms, 2. Carefully apply 15 碌l of liquid to the liquid dish.
can be processed for imaging in the QX-102 capsules. The samples can
be imaged either following an appropriate contrast enhancement staining,
Notes
immunolabeling procedures, or without any treatment.
The sample preparation protocols provided in the Application Manual are a. Applying liquids is most conveniently done using standard
divided into the following categories: lab pipettes.
b. Care should be taken not to touch the capsule membrane with
Liquid Samples the pipette tip.
Particles in Solution c. When applying viscous solutions, creams, pastes, foams or
Biological Applications similar samples, take care that no air bubbles are trapped
between the sample and the capsule membrane.
3. Close the capsule.
4. Proceed to imaging according to guidelines provided in the QX-102 User
Manual Chapter 3.
QX-102 Applications Manual, Issue 2.1 10 Chapter 3: Particles in Solutions
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Chapter 3: Particles in Solutions
QX-102 capsules are suitable for imaging various particles such as
macromolecules, beads and fibers in solutions. The sample part imaged with
the QX capsules is the part proximal to the capsule membrane, and thus,
for optimal imaging the sample must be in close contact with the capsule
membrane. When the capsule is inverted in the SEM during imaging, some
particles, depending on their size, weight and the composition of the solution
they are suspended in, may not stay attached to the membrane. In these cases,
treating the capsule membrane by coating agents may be useful.
Membrane Coating
The coating that provides best attachment depends on the type of specimen
and the nature of the experiment. Coating protocols suitable for biological
applications are provided in chapter 4. Below are protocols for coating the
membrane with Poly-L-Lysine, a positively charged polymer, and with Poly
(sodium-4-styrenesulfonate), a negatively charged polymer. These are suitable
for attaching negatively or positively charged particles respectively. Other
coating reagents may also be used, however, one should take into account that
in order not to interfere with the imaging, the coating layer must be thin and
should not contain electron-dense materials.
For coating procedures for specific applications, please see our website
www.quantomix.com or consult tech@quantomix.com.
Poly-L-lysine coating
Poly-L-lysine is a positively charged polymer, to which negatively charged
particles can attach.
The reagents required for Poly-L-lysine coating are:
0.1% w/v Poly-L-lysine in water (for example Sigma Cat. No. P8920)
Distilled water
QX-102 Applications Manual, Issue 2.1 12 Chapter 3: Particles in Solutions
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Sample Application
To coat the capsule membrane with Poly-L-lysine
1. Apply 15 碌l of 0.1% Poly-L-lysine solution to the liquid dish and Particles can be attached to the coated membranes either by adsorption or
incubate for one hour at room temperature. Longer incubation times, by centrifugation. QX Imaging Buffer is the recommended imaging medium
up to overnight, also give good results. when applicable, since it is specially formulated to minimize damage to the
2. Remove the solution and rinse the liquid dish twice with distilled water. samples by electron beam during imaging in the SEM. In applications where
3. Keep the liquid dish filled with water until applying the sample. the particles can be firmly attached to the membrane and do not require to be
Alternatively, remove the water and dry the liquid dishes for later use. suspended in a specific solution, we recommend changing the solution to
QX-102 Imaging Buffer prior to imaging.
Poly (sodium-4-styrenesulfonate) coating (PSS)
To apply the sample on a coated membrane
PSS is a negatively charged polymer, to which positively charged particles can
attach. 1. Prepare a suspension of particles at the appropriate dilution.
2. Apply 15 碌l of the suspension into the liquid dish and incubate for
The reagents required for PSS coating are: one hour at room temperature, or centrifuge at 500 g for five minutes in a
centrifuge equipped with 96 well plate holders.
30% w/v Poly(sodium-4-styrenesulfonate) in water (for example Aldrich, 3. If applicable, wash with water several times and exchange to 15 碌l
Cat. No. 527483) QX-102 Imaging Buffer prior to imaging.
Distilled water 4. Seal the capsule.
5. Proceed to imaging according to guidelines provided in the QX-102 User
To coat the capsule membrane with PSS Manual Chapter 3.
1. Dilute the 30% stock solution to 0.3% w/v in distilled water
2. Apply 15碌l of the diluted solution to the liquid dish and incubate for one
hour at room temperature. Longer incubation times, up to overnight, also
give good results.
3. Remove the solution and rinse the liquid dish twice with distilled water.
4. Keep the liquid dish filled with distilled water until applying the sample.
Alternatively, remove the water and let the liquid dishes dry for a
later use.
QX-102 Applications Manual, Issue 2.1 14 Chapter 4: Biological Applications
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Chapter 4: Biological Applications
This chapter provides detailed protocols for preparing biological samples to
be imaged using the QX-102 capsules. The samples can be imaged either
untreated, fixed, stained or immunolabeled.
These protocols have been found to be widely applicable. However, since the
results obtained depend on the type of application, the user should optimize
the protocol for each application.
For latest protocol updates, please refer to our website: www.quantomix.com.
General Protocols
Many of the sample preparation steps, including cell attachment, fixation and
immunolabeling, are similar to protocols generally used in cytological staining
for light and fluorescent microscopy.
General staining protocols make use of heavy metals and are derived from
protocols used in electron microscopy. The protocols are fast and easy to
perform. No embedding or drying steps are needed and typical sample
preparation periods are shorter than half a day, as shown in Figure 1.
The liquid dish can serve as a miniature cell culture dish, and cell attachment,
spreading and growth on the electron-transparent capsule membrane have
characteristics and requirements similar to standard cell culture dishes. Non-
adherent cells may also be deposited on pre-coated membranes by adsorption
or centrifugation.
QX-102 Applications Manual, Issue 2.1 16 Chapter 4: Biological Applications
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QX-102 Membrane Coating Protocols
Membrane Coating (optional)
0.5 - 1 h
The capsule membrane supports growth of most cell types without additional
treatments. However in most cases, to ensure optimal growth, pre-coating
of the capsule membrane is recommended. The attachment factors that can
Cell Attachment
be used vary from extra-cellular matrix components such as Fibronectin,
1 h - overnight
Collagen or Gelatin to charged polymers such as Poly-L-lysine. The factors
that provide best attachment depend on the type of specimen and the nature
of the experiment. In choosing the most suitable coating protocol, one should
Fixation 15 min take into account that the coating layer should be thin and should not contain
electron-dense material.
The following coating protocols are provided below:
General Staining Immunolabeling
0.5 - 2 h 3-4h
Fibronectin
Gelatin
Poly-L-lysine
Imaging
Figure 1: General Protocol Flowchart
After a specimen is applied to the capsule, it can be imaged in its native form
or fixed. General staining using heavy metals can improve contrast. Labeling of
specific proteins or cellular structures can be done using immunogold labeling
protocols. Immunolabeled samples can also be counterstained to visualize
cellular details. For examples of images obtained using the technology, refer to
our website, www.quantomix.com.
The following protocols for preparing samples are described below:
QX-102 membrane coating
Sample application to the QX-102
Handling native, non-treated samples
Sample fixation
General staining
Immunogold labeling
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Gelatin Coating
Fibronectin Coating
The reagents required for Gelatin coating are:
Fibronectin supports growth of many types of cells. If Fibronectin is not
suitable, other extracellular matrix components, such as Collagen, Laminin or a
Gelatin (for example, Sigma Cat. No. G6144)
mixture of ECM molecules may be used.
Distilled water
PBS
The reagents required for Fibronectin coating are:
To coat the capsule membrane with Gelatin
0.1% Fibronectin solution (for example, Sigma F-1141)
PBS
1. Dissolve 0.1 % w/v Gelatin in distilled water at 37潞C until no lumps
are visible.
To coat the capsule membrane with Fibronectin
2. If desired, sterilize by filtration before coating or by UV irradiation of the
coated liquid dishes.
1. Dilute Fibronectin with PBS to a final concentration of 0.01%.
3. Apply 15 碌l to the liquid dish and incubate for one hour at room
2. Apply 15 碌l to the liquid dish and incubate for 30 minutes at room
temperature.
temperature.
4. Remove the solution and wash twice with PBS.
3. Remove the solution and wash twice with PBS.
5. Keep the liquid dish filled with PBS until seeding of cells.
4. Wash twice with the appropriate growth medium.
5. Keep the liquid dish filled with the medium until seeding of cells.
Notes
Note
a. It is recommended to coat the capsule membrane on the day of use.
b. Higher concentration of Gelatin may improve the attachment for
It is recommended to plate cells on fibronectin within one day from
some specimens.
coating.
QX-102 Applications Manual, Issue 2.1 20 Chapter 4: Biological Applications
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Poly-L-lysine Coating Sample Application Protocols
Poly-L-lysine is a positively charged polymer, to which cells that carry overall
Cell Culture Growth
negative charge can attach. Since it interferes with the growth and other
physiological functions of some cells, it is not the first choice to be used as a
For adherent cells, the QX-102 capsule is used as a tissue culture dish for
coating agent for growing adherent cells in the QX-102 capsule. However, it is
direct attachment and growth of cells. A large number of cell lines (for
well suited for attachment of non-adherent cells or microorganisms.
example, HeLa, CHO, A431 and NIH3T3) are grown in their regular growth
medium and no special growth conditions are required. Assays or treatments of
The reagents required for Poly-L-lysine coating are:
cells can be performed directly on the cells grown in the capsule. If desired, the
cells can be grown and treated first in tissue culture dishes and then attached
0.1% w/v Poly-L-lysine in water (for example Sigma Cat. No. P8920)
to the capsule membrane prior to imaging.
Distilled water
Cells grown in suspension can be attached to the capsule membrane by coating
To coat the capsule membrane with Poly-L-lysine
it with attachment factors such as Poly-L-lysine. Treatment of non-adherent
cells, such as staining or labeling can be done in the capsule on attached cells,
1. Apply 15 碌l of 0.1% Poly-L-lysine solution to the liquid dish and
or alternatively, cells can be first treated in sample tubes and attached to the
incubate for one hour at room temperature. Longer incubation times, up
capsule membrane prior to imaging.
to overnight, also give good results.
2. Remove the solution and wash twice with distilled water.
3. Following the wash, keep the liquid dish filled with water until applying
! Cautions
the sample. Alternatively, remove the water and dry the dishes in a sterile
environment.
a. For growing cells, it is important to use clean, sterile MP-10
multi-well plates.
b. Do not reuse multi-well plates that have been used for staining
with toxic materials.
QX-102 Applications Manual, Issue 2.1 22 Chapter 4: Biological Applications
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Adherent Cells 5. Inoculate 15 碌l of the cell suspension on the capsule membrane.
6. Fill the reservoirs along the edges of the MP-10 multi-well plate with
distilled water.
The reagents required for seeding adherent cells are:
Note
Normal growth medium
Trypsin or non-enzymatic detachment reagent
To maintain a humid atmosphere and avoid sample drying, it is important
PBS
to fill the reservoirs along the edges of the MP-10 multi-well plate. For
detailed instructions, refer to QX-102 User Manual.
To seed an adherent cells culture
7. Incubate in the normal growth environment (for example, an incubator
1. Coat the capsule membrane with Fibronectin or other suitable
with a humid, 5% CO2 atmosphere) overnight or until the cells are attached.
attachment factor prior to use. For details, refer to QX-102 Membrane
Coating Protocols.
Typical protocol outline for growing adherent cells, found to be widely
2. Detach the cells from their growth flask, for example by using Trypsin.
applicable to many cell types, is provided below:
3. Wash with PBS or a growth medium and resuspend in a fresh growth
medium. Count the cells.
Typical protocol outline for applying a sample of adherent cells
4. Dilute the cells so that 15 碌l of seeding medium in the dish contains
the desired amount of cells. For example, use the guidelines supplied
1. Coat with 0.01% Fibronectin for 30 minutes at room temperature.
in Table 1.
2. Wash twice with PBS.
3. Wash twice with culture medium.
4. Seed 2000 cells in each capsule.
Note
5. Fill each lateral reservoir of the multi-well plate with 200 碌l of water.
6. Close the lid and incubate at 37潞C until the cells are attached
Usually, seeding 1000 to 2000 cells in each dish renders a sub-confluent
(usually overnight).
density of adherent cells after overnight growth. The optimal cell density
should be adjusted according to the application.
Cells in Suspension and Microorganisms
Table 1: Guidelines for Cell Dilution
QX-102 capsules can be used for imaging cells and microorganisms grown in
suspension, such as lymphocytes, bacteria or protozoa. Since these organisms
Cells/Dish Cell Concentration Seeding Volume
usually do not attach directly onto the capsule membrane, coating is required.
6.6 x 104/ml
1000 15 碌l Preferred attachment protocols make use of Poly-L-lysine or Gelatin coating.
105/ml
1500 1x 15 碌l Cells are attached to the coated capsule membranes by incubating or by
105/ml centrifuging. The specimens can be imaged, either in their unstained form or
2000 1.3 x 15 碌l
fixed and stained according to the protocols provided below.
QX-102 Applications Manual, Issue 2.1 24 Chapter 4: Biological Applications
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The suspension of cells can be maintained in a culture medium or buffer and
Handling Native, Non-treated Samples
may be fixed to the membrane before application. The dilution factor of the
suspension depends on the type of organism and culture used and should be
The wet environment of the QX-102 capsule enables visualizing cells in their
determined experimentally. Labeling of specific antigens can be performed
native, non-fixed conditions. Even in unstained samples, the differences
using immunolabeling, either before or after attachment to the capsule
between the various constituents of the cell often generate sufficient contrast
membrane.
to distinguish some level of details. High-density materials such as salts,
phosphorous or iron concentrated in different regions of the cells may improve
The following protocol is applicable for attaching cells in suspension, such as
the contrast.
bacteria or other unicellular organisms to the capsule membrane.
The level of radiation absorbed by the cells during imaging at high
For application of a suspension of cells or microorganisms
magnification is expected to affect their viability. On short time imaging, no
obvious structural damage is apparent and several repeated scans of cells give
1. Coat the capsule membrane with either Poly-L-lysine or Gelatin. For
the same images. Thus, short time imaging of living cells may be possible. Live
details, refer to QX-102 Membrane Coating Protocols.
cells can be attached to the capsule according to the protocols provided above
2. Wash the membrane with PBS and keep it filled with PBS until use or
and imaged directly in growth medium or in PBS.
alternatively, wash with distilled water and dry overnight.
3. Prepare a suspension of cells, microorganisms or particles at the
appropriate dilution (for example, for the E.coli, 1:100 dilution of an
over-night culture renders the appropriate number of bacteria in
a capsule).
4. Apply 15 碌l of the diluted culture into the liquid dish and incubate for
one hour at room temperature, or centrifuge at 500 g for five minutes at
room temperature in a centrifuge equipped with 96 well plate holders.
! Caution
To avoid interference with cell adsorption onto the membrane, it is
recommended not to include Serum or Formaldehyde in the suspension.
5. Optional: To stabilize the binding after cell adsorption, fix the cells briefly
with 4% Formaldehyde/PBS for 10 minutes or 2.5% Glutaraldehyde/PBS
for five minutes.
6. Wash with PBS, or water if the cells are fixed, and proceed to staining
or imaging.
QX-102 Applications Manual, Issue 2.1 26 Chapter 4: Biological Applications
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fixation depend on the application and should be determined experimentally.
Fixation Protocols Samples fixed for subsequent immunolabeling require special precautions to
avoid damaging the relevant epitopes. For some applications, a mixture of
Most of the general staining and immunolabeling protocols are carried out after
Paraformaldehyde and Glutaraldehyde may render improved results.
fixation of the specimen. The purposes of the fixation are:
To preserve the cellular structures as close to the living state as possible.
To protect the sample from morphological alteration and damage during
Glutaraldehyde Fixation
the subsequent treatments.
The reagents for Glutaraldehyde fixation are:
A wide range of fixatives commonly used in cytological, immunostaining and
electron microscopy studies can be applied to the QX-102 capsule samples.
25% Glutaraldehyde solution EM grade (for example, Agar Cat. No. R1020)
Since no fixative preserves all the cellular structures, an appropriate choice
PBS
of fixatives depends on the specimen, and the cellular details of interest. For
immunolabeling, the choice of the most suitable fixation protocol is also
To fix the specimen with Glutaraldehyde
affected by the nature of the antigen and antibody.
1. Dilute the Glutaraldehyde stock solution to a 2% final concentration
in PBS.
! Warning
2. Wash the sample four times with PBS.
3. Incubate with 2% Glutaraldehyde/PBS at room temperature for 30 minutes.
Since all fixatives are toxic to some extent, all work should be performed
4. Wash four times with PBS.
in a fume hood using gloves and protective clothing. Handling and waste
5. Proceed to staining or labeling reaction.
disposal should be according to guidelines of the local authorities.
Note
For some applications, better results can be obtained with lower
Aldehydes
concentrations of Glutaraldehyde (0.2% to 1%) and longer incubation
periods.
Fixation in protein-crosslinking Aldehyde reagents, such as Paraformaldehyde
or Glutaraldehyde, is usually the first choice. Fixation with Glutaraldehyde
is permanent, whereas Paraformaldehyde may be partially reversed during
long incubation times with solutions. Glutaraldehyde penetrates slower than
Paraformaldehyde and thus requires longer fixation times.
The following two sections provide standard fixation protocols for
Paraformaldehyde and Glutaraldehyde. The optimal concentration and time of
QX-102 Applications Manual, Issue 2.1 28 Chapter 4: Biological Applications
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Paraformaldehyde Fixation Organic Solvents
The reagents required for Paraformaldehyde fixation are: Organic solvents such as alcohols are also commonly used fixatives. Since these
reagents dehydrate the cell and remove lipids, they can destroy some of the
Paraformaldehyde, EM grade (for example, a 16% solution, Electron cell architecture. These fixatives may be preferred if immunolabeling is desired,
Microscopy Sciences, Cat. No. 15710) since in some cases they may preserve target epitopes better than Aldehyde
PBS fixatives.
To fix the specimen with Paraformaldehyde
! Caution
1. Prepare 4% Paraformaldehyde solution in PBS.
2. Wash the sample four times in PBS. Please note that the QX-102 capsule is not compatible with Acetone and
3. Fix with 4% Paraformaldehyde/PBS at room temperature for 15 minutes. Toluene. For other organic solvent please consult with tech@quantomix.com.
4. Wash four times with PBS.
5. Proceed to staining or labeling reaction.
Note
Methanol Fixation
For some staining and labeling protocols, 2% Paraformaldehyde may give
better results. The reagents for Methanol fixation are:
Methanol cooled to 鈥?20掳C
PBS
To fix the specimen with Methanol
1. Wash the sample four times with PBS at room temperature.
2. Change to pre-cooled (-20掳C) 100% Methanol.
3. Incubate for five minutes at 4掳C or at room temperature.
4. Wash four times with PBS at room temperature.
5. Proceed to staining or labeling reaction.
QX-102 Applications Manual, Issue 2.1 30 Chapter 4: Biological Applications
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General Staining Protocols PBS
Distilled water
0.45 碌m syringe filters
The imaging contrast in QX-102 capsules is created from variations in atomic
numbers of the sample constituents. Thus, heavy metal stains, such as
To stain the specimen with Uranyl Acetate
Uranium and Osmium compounds, are best suited for improving the general
contrast of the biological samples, whose main constituents are Carbon,
1. Before starting, prepare fresh 1% Tannic Acid in distilled water and acidic
Hydrogen, Oxygen and Nitrogen. Heavy metal stains attach, generally non-
0.5% Uranyl Acetate (diluted in water from stock and filtered through
specifically, to cellular constituents. However, different affinities to various
0.45 碌m syringe filters).
molecules enable visualization of some cellular structures.
The following staining materials are described below:
Notes
Uranyl Acetate
a. Since Uranyl precipitates in the presence of Phosphate, samples
Phosphotungstic Acid (PTA)
must be rinsed thoroughly to remove traces of Phosphates before
Osmium Tetroxide
Uranyl staining.
b. Perform all the following steps at room temperature.
Uranyl Acetate Staining
2. Wash the sample four times with PBS.
Uranium is the heaviest metal used in staining and can be used as a general
3. Fix with 4% Paraformaldehyde/PBS for 15 minutes. For details, refer to
contrast agent. Uranyl Acetate binds to nucleic acids, to proteins and to
Fixing Protocols.
membranous structures.
4. Wash the sample four times with PBS.
5. Wash four times, five minutes each wash, with distilled water.
6. Incubate with 1% Tannic Acid for 5 minutes.
! Warning
7. Wash twice, five minutes each wash, with distilled water.
8. Incubate with 0.5 % Uranyl Acetate for 30 minutes.
Uranium compounds are toxic and radioactive. Contact your safety officer
or local authorities for appropriate handling and disposal protocols.
Note
The reagents required for Uranyl Acetate staining are:
The optimal concentration and incubation time may vary depending on
the specimen.
Uranyl Acetate (5% stock, pH 3.5 with HCl, kept at 4掳C in the dark)
Tannic Acid (2% stock in water)
4% PFA in PBS
9. Wash twice, five minutes each wash, with distilled water.
QX-102 Applications Manual, Issue 2.1 32 Chapter 4: Biological Applications
33
10. Prepare the sample for imaging according to Preparing the Samples
Notes
for Imaging Section (Page 40).
Fixation can also be done with a combination of 2% Paraformaldehyde and
1% Glutaraldehyde in PBS for 30 minutes.
PTA (Phosphotungstic Acid) Staining
3. Wash three times with PBS.
Phosphotungstic acid (PTA) is an anionic stain. PTA positively stains charged
4. Wash three times with double distilled water.
structures such as basic proteins associated with nuclear DNA and nucleoli and
5. Incubate the sample with 2% PTA for 30 minutes.
intensely stains mitochondrial matrix.
6. Wash five times with double distilled water.
7. Prepare the samples for imaging according to Preparing the Samples for
The reagents required:
Imaging (Page 40).
Phosphotungstic acid (for example Sigma Cat. No. P4006), 2% stock
solution in double distilled water, pH 1.5.
Osmium Tetroxide Staining
! Warning
Osmium Tetroxide is traditionally used in electron microscopy both as
a fixative and heavy metal stain. Osmium Tetroxide is a good fixative and
PTA solution is acidic. Appropriate personal protective clothing should be used.
excellent stain for lipids in membranous structures and vesicles. The most
prominent staining in adherent human cells (HeLa) is seen on lipid droplets
Double distilled water (see www.quantomix.com, gallery). Some intracellular structures are also
2% Glutaraldehyde in PBS visualized. Visualized cellular structures depend on the fixation protocols;
in Glutaraldehyde fixation nucleoli are visible, but overall nuclear staining is
weak. In Paraformaldehyde fixation nuclear staining becomes more prominent,
Notes but some intracellular structures are lost. As a first choice, fixating with a
combination of Glutaraldehyde and Paraformaldehyde is recommended.
The stock can be stored at room temperature for approximately one month.
Warning
!
Procedure:
Since OsO4 is toxic and volatile, all work should be performed in a fume
1. Wash the cells four times with PBS. hood using gloves and protective clothing. Handling and waste disposal
2. Fix cells with 2% Glutaraldehyde in PBS for 30 minutes. should be done according to the guidelines of the local authorities.
QX-102 Applications Manual, Issue 2.1 34 Chapter 4: Biological Applications
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Immunogold Labeling Protocols
The reagents for Osmium Tetroxide staining are:
4% OsO4 (for example, Sigma Cat. No. 75632) Colloidal gold particles are readily visualized in the QX-102 capsules.
2% Paraformaldehyde/0.1% Glutaraldehyde in PBS Gold beads conjugated to a variety of molecules, such as Protein A,
Distilled water Immunoglobulins and Streptavidin, are commercially available and can be used
PBS to immunolabel specific antigens. Since immunogold labeling combined with
QX-102 capsules allows visualization of single gold particles attached to single
To stain cells with Osmium Tetroxide molecules, the following unique advantages are offered:
Imaging receptors in the context of the cell membrane
Extremely low detection limit, allowing detection at single label sensitivity
Note Ultra-fine localization and distribution studies are possible due to the
10 nm resolution range
All the following steps should be performed at room temperature.
Labeling quantification by counting the individual particles
Immunolabeling protocols consist of four main steps:
1. Wash the cells four times with PBS.
2. Fix with 2% Paraformaldehyde/0.1% Glutaraldehyde/PBS for 30 minutes. Fixation
3. Wash four times with PBS. Blocking
4. Wash four times with distilled water. Antibody binding
5. Prepare 0.1% OsO4 solution by diluting the 4% stock solution in Detection with gold conjugate
distilled water.
6. Incubate the sample with 0.1% OsO4 for 30 minutes. For the immunolabeling protocol flowchart, refer to Figure 2.
Surface antigens can be labeled on live or fixed cells. Intracellular antigens can
Note be labeled on fixed, permeabilized cells. Since fixation protocols can mask or
change some epitopes, the optimal fixation protocol for each antibody should
The optimal incubation time may vary between samples and should be
be experimentally determined.
experimentally determined.
In addition, optimal blocking for non-specific backgrounds, concentrations
and incubation time with the primary antibody depend on the antigen and
7. Wash four times with distilled water. antibody in question. Since in some cases, specific incubation and wash buffers
8. Prepare the samples for imaging according to Preparing the Samples are required to avoid non-specific binding, there is no standard protocol that
for Imaging Section (Page 40). works for all labeling reactions. Optimal conditions may be established based
on prior experience with the particular antibody and antigen or on preliminary
experiments using immuno-fluorescence.
QX-102 Applications Manual, Issue 2.1 36 Chapter 4: Biological Applications
37
This section provides the outline and general guidelines, which should be To assess the labeling characteristics, the reactions should be compared using
modified according to the user-defined optimal conditions. appropriate controls. Control reaction, in which the primary antibody has been
omitted, should always be included.
Commercial conjugated gold particles are available in different sizes, varying from
0.8 nm to 100 nm. Smaller particles have the advantage of more efficient labeling,
but silver enhancement is required for their visualization. Larger particles can be Immunogold Labeling Reaction
visualized without further treatment. To obtain optimal conditions for each gold
conjugate, refer to the manufacturer鈥檚 recommendations. The reagents required for the immunolabeling reaction are:
PBS
Specimen
Fixative
0.2% Triton庐 X-100 in PBS (for intracellular antigens)
Blocking agent (BSA, normal Serum or other)
Fixation 0.5 h
Primary antibody
Gold particle conjugate
Distilled water
Live Surface Permeabilization Internal Silver staining kit (for example, AURION R-GENT SE-EM, Cat. No. 500.033)
cells antigens 10 min antigens
To immunogold label a sample in a QX-102 capsule
1. Coat the membrane with Fibronectin or another appropriate
Blocking 0.5 h
attachment factor.
2. Attach the cells to the membrane.
Primary antibody attachment 1h
Note
Gold conjugate attachment 1h
All the following steps should be performed at room temperature.
Silver enhancement
3. Wash four times with PBS.
4. Fix the cells, referring to Fixation Protocols. Omit the fixation step when
live cells are labeled.
Imaging
5. Wash four times with PBS.
For intracellular antigens:
Figure 2: Immunolabeling Protocol Flowchart
QX-102 Applications Manual, Issue 2.1 38 Chapter 4: Biological Applications
39
a. For Paraformaldehyde or Glutaraldehyde fixed cells, permeabilize
the cells by incubating with 0.2% Triton庐 X-100/PBS for 10 Notes
minutes. If Methanol fixation is used, no additional permeabilization
is required. For optimal dilution and conditions, refer to the manufacturer鈥檚
b. Wash twice with PBS - 5 minutes each wash. recommendations.
6. To avoid non-specific background, incubate with a blocking solution,
such as 1% BSA and 5% normal Serum from the species of the secondary 10. To remove unbound antibodies, wash four times with PBS.
antibody in PBS for 30 minutes. 11. For gold beads smaller than 30 nm, perform silver enhancement.
7. Incubate with primary antibody in 1% BSA in PBS.
In parallel, carry out the control reaction without the primary antibody.
Note
Notes The AURION R-GENT SE-EM kit is recommended. However, other
comparable kits are also available.
a. Carry out and test serial dilutions to determine the optimal
concentration of the antibody.
b. Incubation periods of 30 to 60 minutes at room temperature usually 12. Wash six to ten times with distilled water.
render good results. 13. Prepare the sample for imaging according to Preparing the Samples
c. For some antibodies, labeling can be improved by incubating at 37潞C for Imaging Section (Page 40).
or by longer incubation periods (several hours to overnight) at 4潞C.
8. Wash four times with PBS, with BSA 1%.
Notes
a. In case of background problems, a mild detergent such as 0.05% to 0.1%
Tween庐 20 can be added to the wash buffer.
b. For surface staining or labering, do not use detergents.
9. Incubate with the gold-labeled secondary reagent (gold conjugated
secondary antibody or Protein A or G) in protein containing solution,
such as 1% BSA or 5% normal Serum.
QX-102 Applications Manual, Issue 2.1 40 Appendix A: Glossary
41
Preparing the Samples for Imaging Appendix A: Glossary
Term Description
The QX Imaging Buffer is specially formulated to minimize the damage to
the samples by the electron beam during imaging in SEM and should be used
SEM Scanning electron microscope
when ever applicable, especially for biological samples. Prolonged storage of
the specimens in the imaging buffer is not recommended. For imaging live MP-10 Multi-well plate, a sterile, transparent holder for parallel
specimens, growth buffer of the samples or PBS should be used instead of the handling of up to 24 individual QX-102 capsules, serving
QX Imaging Buffer. as a cell culture apparatus and holds the capsules during
various manipulations
MA-4 Multi-well aspirator, a parallel drainage system designed
! Caution to safely aspirate liquids from the QX-102 capsules
without damaging the capsule's membrane
Solutions containing DMSO are not suitable for imaging in QX-102 capsules.
QX-102 Capsule used for SEM-imaging of a variety of liquids and
wet samples
To prepare the samples for SEM-imaging Liquid Dish QX-102 capsule base designed as miniature cell culture
dish for applying samples
1. When the sample is ready, exchange the liquid in the liquid dish to 15 碌l
Sealing Stub Part of QX-102 capsule used for sealing the capsule and
QX-102 Imaging Buffer.
for holding the capsule in SEM
2. Seal the capsule.
Calibration Capsule QX-capsule with control sample used for optimization of
imaging conditions
Notes QX Imaging Buffer Buffer optimized for imaging samples in SEM with
QX-102 capsules
For optimal imaging results, we recommend to proceed directly to imaging.
BSA Bovine Serum Albumin
If required, the closed samples can be stored at 4潞C for short periods. BSED Back-scattered electrons detector
Refrigerated capsules should be equilibrated to room temperature before
BSE Back-scattered electrons
inserting them to the SEM.
PBS Phosphate-Buffered Saline
ECM Extracellular Matrix
3. Place the QX-102 capsule in the SEM with the capsule membrane facing
PFA Paraformaldehyde
upwards and proceed to imaging according to the guidelines provided in
the QX-102 User Manual Chapter 3. GA Glutaraldehyde
w/v Weight to volume
QX-102 Applications Manual, Issue 2.1 42 Appendix B: Troubleshooting Instructions
43
Appendix B: Troubleshooting Instructions
Phase Problem Possible Cause Solution
Liquid Liquid is The capsule a. Avoid touching the capsule
Handling leaking out membrane has been membrane at any time.
of the liquid damaged. b. Always place the capsules in
dish. the MP-10 multi-well plate.
c. Do not use means other
than the MA-4 multi-well
aspirator for aspirating liquids.
Sample Vacuum used for Use weaker vacuum for liquid
particles/cells liquid handling is handling.
are detached too strong.
during liquid See Instructions for MA-4.
handling,
Sample particles/ Increase the concentration of
especially
cells are not the attachment factor or try
from the
attached well. other factors.
center of the
liquid dish.
Cell The cells do The attachment Use other attachment factors.
Growth not attach to factor in use does
the capsule not support cell
membrane. growth.
The cells do The growth Adjust the density of the cells
not grow well. conditions are not or the incubation period.
optimal. Some cells
may require specific
conditions for growth
in a QX-102 capsule.
An MP-10 multi-well plate that
Traces of toxic
has been used for staining with
materials spilled
toxic reagents should not be
onto the MP-10
used for cell growth.
multi-well plate
affect the cell
growth.
QX-102 Applications Manual, Issue 2.1 44
Phase Problem Possible Cause Solution
Cell The cells look Samples have dried Do not leave liquid dishes with
Staining damaged while being handled. low liquid levels for prolonged
after the periods of time.
staining/
labeling
procedures.
Imaging No signal is The sample is not For protocols of sample
observed. in contact with the attachment, see Chapter
capsule membrane. 3 (Particles in Solution)
or Chapter 4 (Biological
Applications).
The image is There is no The sample may require
not clear. sufficient contrast enhancement, such as
contrast between heavy metal staining.
constituents of the For staining of biological
sample. samples, see Chapter 4.
QuantomiX Ltd., 12 Hamada Street, Tamar Science Park, Rehovot
POB 4037, Nes-Ziona 70400, Israel
Tel: +972-8-9462244, Fax: +972-8-9465874
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