Frequently Asked Questions about WETSEM® Technology
This section covers the most frequently asked questions.
- SEM and imaging
- Handling the capsules
- Sample treatment in QX capsules
- Cell biology with QX-102 Capsules
SEM and imaging
Will the QX capsules fit my SEM ?
Answer: The QX capsules are designed to fit into the specimen stage of most types of SEMs. The QX capsule works with SEMs equipped with BSE (Back Scattered Electrons) detectors. Either variable pressure or high vacuum mode can be used. Adaptors are available for different stub holder dimensions. Following is a partial list of SEM models compatible with QX technology:
- FEI XL30ESEM, XL30ESEM FEG, Quanta series, Quanta FEG
- JEOL 5600, 5900, 6000 series
- LEO 1450, 1500 Series, EVO, Supra, Ultra
- Hitachi 2600, 3000 series, 4000 series
- Camscan CS3000 range, MV2300 range
For suitability of other models and availability of adaptors, please contact Electron Microscopy Sciences.
Can the capsules be used in high vacuum mode?
Answer: The QX capsules are compatible with high vacuum as well as low vacuum mode. The capsules are designed to withstand a pressure difference of up to 1 atmosphere, therefore no restrictions exist on vacuum levels, and they can also be used at 1x10-6 Torr.
Will the sample in a QX capsule remain fully wet during imaging in a SEM ?
Answer: Yes. The membrane of the QX capsule is impermeable to water and the capsule completely isolates the sample from the vacuum. No drying of the sample occurs during imaging or storage.
Why is it necessary to dehydrate the sample in conventional SEM imaging, and why is no drying needed with WETSEM® technology?
Answer: In traditional SEM , the sample is placed directly in high vacuum. A wet sample exposed to vacuum will lose its water in an uncontrolled dehydration process, which often distorts or destroys the structure of the sample. To preserve the structure in vacuum, the sample must be dehydrated in a controlled manner. The sample in the QX capsule is completely isolated from the vacuum and remains at atmospheric pressure. Therefore, there is no loss of water and no drying of the sample is needed.
What is the resolution with WETSEM® technology?
Answer: In general the resolution is about the same as dry SEM samples, depending on the specific sample and the SEM model used.
(see: Thiberge et al. PNAS Vol.101, No.10, March 9, 2004 . 3346-3351 and Review of Scientific Instruments, Vol. 75 No. 7, July 2004)
How deep below the membrane can imaging be performed?
Answer: The beam penetration depth depends on the acceleration voltage. For a sample in a water-based medium imaged with a 30 kV beam, information is retrieved from a depth of approximately 2-4 micrometer. At 10 kV this depth is reduced to a few hundred nanometers. By varying the acceleration voltage it is possible to obtain unique 3D information from your sample.
Does the membrane interfere with imaging?
Answer: The capsule membrane is very thin and flat. It is made of material transparent to electrons with energies above a few keV. Thus, the membrane induces minimal scattering of the beam and of the back scattered electrons detected by the BSE detector. The membrane itself is flat and hence does not interfere with the imaging process. In X-ray mode, the membrane produces a constant background that can be subtracted.
Does the electron beam cause damage to my sample?
Answer: Fluids are very good conductors of heat. Therefore, the damage by the electron beam is very small. The QX-102 Imaging Buffer is specially formulated to minimize the damage caused by the beam and should be used whenever applicable. Also, to minimize the damage it is recommended to work with lower beam currents (i.e. reduced spot size).
Are there any restrictions to the electron beam current?
Answer: There are some considerations to the choice of beam parameters and the recommended parameters can be found in the QX-102 User Manual. Generally, higher beam currents result in stronger signals, but increase the possibility of damage to the sample and affect the resolution. Lower beam currents minimize the damage and result in better resolution at high magnifications, but may give a signal that is not satisfactory. The optimal beam current depends on the sample and is determined empirically.
Is there a problem of charging with QX capsules?
Answer: No. Since the sample inside the QX-102 capsule is wet, and the capsule itself is conductive, there is no charging.
Are the QX-102 capsules compatible with light microscopes?
Answer: Yes. The membrane of the QX-102 capsule, as well as the MP-10 multi-well plate, are transparent to light and allow imaging of samples with a light microscope. The imaging should be done only with the QX-102 capsules properly placed in the MP-10 multi-well plate, which is especially designed for holding the capsules during various manipulations. The plate fits into a microscope stage for standard multi-well plates.
Can I perform EDS with WETSEM® technology?
Answer: Yes. QX capsules can be used for EDS (energy dispersive spectroscopy) analysis of samples using a SEM equipped with an EDS system. In fact, the QX-technology uniquely suits EDS of wet samples. Note, the presence of the metal grid and polymer membrane will be taken into account in the analysis.
What imaging parameters should be used in WETSEM® technology?
Answer: SEM imaging with QX capsules differs from standard SEM imaging in some aspects. Also, the factors that affect imaging vary among applications. Recommendation for parameters and guidelines for optimization are found in the QX-102 User Manual. Optimization of the imaging conditions is best done using the Calibration Capsule (cat no. RT-56).
Can the liquid sample leak from the capsule and contaminate the detector of my SEM ?
Answer: The membrane of the QX capsule is vacuum-tight and impermeable to liquids and gases. Thus, when the capsule is handled correctly there is no leakage of the sample and no contamination. Correct handling relates to both physical handling during sample preparation stages and to the parameters used during imaging. All care must be taken to prevent physical contact with the membrane. Any contact (e.g. with the tip of the pipette or with the hand) can potentially tear the membrane.
Imaging of capsules must begin with a low probe current (small spot size). Initial probe current should be no higher than 200 pA. If the obtained signal is not sufficiently strong, probe current can gradually be increased until the desired image is obtained. Probe current should not exceed 1nA.
Initial magnification should also be low. Magnification should be increased gradually until the desired image is obtained.
Can I image any liquid with the capsules?
Answer: QX capsules are suitable for imaging various types of wet samples and liquids, including cells, tissues, bacteria, emulsions, oils, food samples, cosmetics, inks, particles in solutions, etc. For solvents that should be avoided, please consult the QX-102 User Manual.
Are the QX capsules compatible with acidic and basic solutions?
Answer: The QX capsules have been validated for compatibility with commonly used organic and inorganic acids and bases (5 hour exposure). Results are shown in the following table:
|NaOH||0.1M- 0.1mM||13 - 10||positive|
|Tris(hydroxymethyl) aminomethane base||0.05M||10-7 (adjusted by HCl)||positive|
|HCl||1M- 0.1mM||0 - 4||positive|
Can I image live cells with QX-102 capsules?
Answer: Cells can be introduced live into the SEM and short time imaging of live cells may be possible. However, the radiation absorbed by the cells during image scanning is expected to cause structural changes and to affect the viability of the cells. Also, the contrast between different constituents of native cells may be too low for high-resolution imaging, and some staining or labeling may be required. Live cells or microorganisms can be imaged either in their growth medium or in PBS.
Can I image lipid rich structures with WETSEM® technology?
Answer: WETSEM® technology allows analysis of lipid structures in an unperturbed state. The ability to image wet samples with SEM avoids the problem of lipid extraction that occurs due to dehydration with organic solvents during lipid imaging with conventional techniques. Many lipid structures are visualized without any enhancement. If staining is required, Osmium tetroxide is well suited for lipids; for detailed protocols, see the QX-102 User Manual.
Can I image foams with WETSEM® technology?
Answer: Yes. QX-102 capsules can be used for imaging cosmetic, food or other foam samples.
I would like to image untreated food samples. What details can I see with QX-102 capsules?
Answer: The imaging contrast in QX-102 capsules is created from variations in atomic numbers of sample constituents. Thus, constituents and structures that have a significant difference in atomic numbers can be visualized without any enhancement. The contrast between water and fat is especially well visualized in QX-technology, enabling analysis of fat structure and content in food, cosmetics and other samples.
I want to look at cosmetic cream, which has a paste-like structure. Can I do this with QX-102 capsules?
Answer: Yes. Notice that the field you image is that closest to the capsule membrane. Therefore, the sample has to be in direct contact with the membrane.
If I have a sample that releases gas inside the capsule, will the membrane of the capsule withstand the pressure?
Answer: As long as the internal pressure does not increase significantly above 1 atmosphere, the membrane will withstand the pressure.
FAQ Handling the capsules
I can hear noise of loose parts inside an unopened package of capsules. Does this mean the capsules have been damaged during the shipment?
Answer: No. The packaging of the capsules is designed to prevent the capsules from moving around and to protect them from damage during shipment. To keep the capsules in a dry environment, the package contains two cylinders of desiccants, which can cause this noise.
Can I use only part of the capsules from the package and store the remaining unused capsules?
Answer: Yes. In order to maintain sterility of the capsules, open the package in a sterile environment, such as laminar flow hood. Take out the desired number of capsules, close the box and seal the top cover with tape. Store the package in a clean, dry place.
FAQ Sample treatment in QX capsules
Can I store my samples in QX capsules?
Answer: Yes. Samples can be stored in sealed QX capsules and they remain wet. The samples can also be stored in open capsules (liquid dishes), when properly sealed in the MP-10 multi-well plate. For sealing of liquid dishes and for storage of biological samples, please refer to the QX-102 User Manual.
How do I apply liquids to the QX capsules?
Answer: Liquids and samples are applied onto the liquid dishes of the capsules using standard lab pipettes. When treating multiple samples, a repetitive dispensing pipette is most convenient. It is important to apply the liquid carefully, not to touch the capsule membrane with the pipette tips.
How do I remove liquids from the QX capsules?
Answer: The liquids are removed using the MA-4 multi-well aspirator designed for safe aspiration of liquids from the capsules. Other means should not be used, since they may lead to rupturing of the capsule membrane. For detailed instructions of working with the MA-4 multi-well aspirator, refer to the instructions accompanying the product. See also the QX-102 User Manual for recommendations for proper liquid handling during staining and labeling procedures.
FAQ Cell biology with QX-102 Capsules
Will my cells grow on the QX-102 capsule membrane?
Answer: The QX-102 capsule is designed to be compatible with growth of cells, including established cell lines, as well as primary cultures. Our recommendation is to coat the membrane with a suitable attachment factor prior to cell seeding.
Can cells be grown in the QX-102 capsules for more than one day?
Answer: Yes. The MP-10 multi-well plate holder for the capsules is designed to maintain CO2 and humidity levels so that the capsules can be used as standard cell culture dishes for long-term growth of cells. During long incubations, ensure that the wells on the sides of the MP-10 plate stay filled with water. Also, it is recommended to change the growth medium of the cells to fresh medium every 2 days.
What attachment factor should I use for my cell line?
Answer: The QX-102 capsule membrane can be coated with various attachment factors such as Fibronectin, Collagen, Laminin, Gelatin, poly-L-lysine or with a combination of them. The factors that provide best attachment and growth will depend on the cell type. Fibronectin (Sigma, Cat. F-1141) has been found to support growth of many types of cells, and thus in many cases will be the recommended first choice. Please consult QX-102 User Manual for coating protocols.
Can I use QX capsules for non-adherent cells?
Answer: Yes. Cells in suspension, such as blood cells, bacteria and protozoa, can be attached to the capsule membrane coated with attachment factor such as poly-L-lysine or Gelatin. The cells can be attached to the coated membrane by incubating or by centrifuging. For detailed protocols, see the QX-102 User Manual.
Are there specific stains for different cellular compartments?
Answer: Since the imaging contrast in WETSEM® technology is based on variations in atomic numbers, heavy metal stains give the best contrast when imaging biological samples. Due to different affinities of heavy metals to various molecules, some cellular structures stain more strongly and can be visualized. For example, osmium tetroxide has a high affinity for lipids, and can be used for staining lipid vesicles. Most of the heavy metals stain the nuclei, and some of them, such as gold chloride, can be used for staining chromosomes. However, since the heavy metal stains are usually quite non-specific, for detailed localization studies immunogold labeling may be required.
Can I carry out intracellular immunolabeling with WETSEM® technology?
Answer: Yes. Colloidal gold particles are well visualized with WETSEM® technology and intracellular antigens can be labeled on fixed, permeabilized cells using commercially available gold conjugates. Generally, smaller gold particles (less than 10 nm) are better suited for intracellular labeling. Particles less than 10 nm need to be Silver enhanced for imaging.
How do I choose the fixation protocol for immunogold labeling?
Answer: The fixation protocols generally used in immunocytochemistry are also applicable to QX capsule technology. The correct choice, optimal concentration and time of fixation depend on the nature of the antigen and the antibody. Optimal conditions may be established based on your prior experience with the application or by performing preliminary experiments using immuno-fluorescence labeling.
What are WETSEM® Technology and QX-202C capsules?
WETSEM® Technology is a novel, breakthrough technology enabling the imaging of fully wet samples in a scanning electron microscope (SEM). The technology provides an opportunity to study samples in the SEM, which are isolated from the SEM’s vacuum chamber in terms of pressure and humidity.
The QX-202C capsule is a product based on WETSEM® Technology which is designed specifically for the imaging of dynamic hydration processes such as the curing of cementitious systems.
The QX-202C capsule enables the user to follow, in-situ, the dynamics of a reaction, by creating a sequence of SEM images which reflects the structural development of the sample.
For specific information for which answers do not appear on this page please contact firstname.lastname@example.org
Who could benefit from the use of the QX-202C capsules?
Any laboratory studying time dependant processes such as the curing process of cementitious or gypsum systems can obtain valuable information by using the capsules.
What type of samples is the QX-202C intended for?
The QX-202C capsules are intended for use with cementitious and other systems involving a dynamic hydration process. For more information please contact email@example.com.
What are the advantages of WETSEM® over E-SEM preparation techniques?
WETSEM® Technology can be applied in any SEM and does not require a special system (e.g. E-SEM, low-vac, variable pressure etc.)
WETSEM® Technology enables the imaging of dynamic hydration processes in their native environment. The sample is sealed in the ambient environment. The pressure, temperature and humidity conditions it is under, and its water content do not change when it is placed in the SEM vacuum chamber. Imaging with WETSEM® Technology does not require a cooling stage, and the temperature of the sample remains close to ambient temperature.
The curing process proceeds in the SEM vacuum chamber with minimal intervention – water/cement ratio does not change and imaging is not limited to the layer which is above water level. In this way samples can be probed frequently, and the true dynamics of the process can be studied in-situ. In addition, WETSEM® Technology is very easy to use, and does not require the operator’s skill and experience that E-SEM imaging does.
What are the advantages of WETSEM® over cryo-SEM techniques?
By avoiding the need to freeze samples, it becomes possible to conduct “in-situ” studies. WETSEM® Technology creates the possibility to image and study a full process rather than intermittent results. It enables the user to prepare a single sample and follow its development rather than deduce what the development process is from a jigsaw of several independent samples.
If samples are also being freeze-fractured, results will normally be obtained from the least cohesive plane – the plane where the fracture occurred. Using WETSEM® Technology, the plane observed will not be dependant on forces, internal or external, which are applied on the sample.
Preparing WETSEM® samples is quick and easy and takes only a few minutes, saving time and effort.
I am using calorimetry to study the early stages of cement hydration. Why would I need WETSEM® as well?
WETSEM® is a complementary method to calorimetry as well as to other analytical techniques such as XRD. While calorimetry provides information about the heat evolution during hydration, and XRD provides phase composition data, WETSEM® provides insight on the morphology of the developing phases and their elemental composition.
Do I need a special SEM in order to use WETSEM® Technology?
QX-202C capsules are the small devices in which wet cementitious samples can be placed and observed. The capsules fit in almost all of the currently available SEM models. Imaging is normally done in a high vacuum environment, but can also be performed in other environments (variable pressure, low-vac or E-SEM).
For best results a back-scattered detector should be used. If a back-scattered detector is not part of your system, please us at firstname.lastname@example.org.
Will I be able to obtain information about the material constituents of my sample?
For best results, one needs to use WETSEM® Technology in conjunction with a backscattered electron detector. Thus, the images obtained will correspond mainly to the average atomic number of the constituents of the sample.
Is it possible to apply EDS when using the QX-202C capsules?
Yes. The electron-transparent membrane which is the heart of the WETSEM® Technology is also transparent to X-rays, making it possible to obtain qualitative EDS spectra of the sample within it.
For how long can I follow a dynamic reaction without removing the QX-202C capsule from the SEM?
The QX-202C capsules are specifically designed to remain vacuum tight for long periods of time, and can remain in the SEM chamber, under high vacuum, for 24 hours or more. Through this whole period the samples can be continuously imaged.
Is it possible to remove a QX-202C capsule from the SEM vacuum chamber and then re-insert it?
Yes. You can conduct a dynamic study by intermittently imaging a sample. It is possible to make full use of your SEM by removing the QX-202C capsule from the SEM in the intervals between one image and the next and efficiently using this time to image other samples. This can be done through the whole length of the hydration process and will not interfere with the dynamics of the process which takes place inside the isolated capsule.
Do I need to coat the sample prior to WETSEM® imaging?
When using WETSEM® Technology there is no need to coat the sample. The purpose of sputter-coating is to create a conductive layer on the sample’s surface and thus to prevent charging artifacts. With WETSEM® Technology the presence of water in the body of the sample totally eliminates these artifacts.
What is the price of the QX-202C capsules?
The QX-202C capsules are supplied in several package sizes. One of the options is a Starter Kit, designed for first-time users, which includes all of the required tools and accessories.