Skip to main content
×
×
Home

Information:

  • Access

Actions:

      • Send article to Kindle

        To send this article to your Kindle, first ensure no-reply@cambridge.org is added to your Approved Personal Document E-mail List under your Personal Document Settings on the Manage Your Content and Devices page of your Amazon account. Then enter the ‘name’ part of your Kindle email address below. Find out more about sending to your Kindle. Find out more about sending to your Kindle.

        Note you can select to send to either the @free.kindle.com or @kindle.com variations. ‘@free.kindle.com’ emails are free but can only be sent to your device when it is connected to wi-fi. ‘@kindle.com’ emails can be delivered even when you are not connected to wi-fi, but note that service fees apply.

        Find out more about the Kindle Personal Document Service.

        ResiTAG™ – A Novel Sample Identification Solution for Microscopists and EM Facilities
        Available formats
        ×
        Send article to Dropbox

        To send this article to your Dropbox account, please select one or more formats and confirm that you agree to abide by our usage policies. If this is the first time you use this feature, you will be asked to authorise Cambridge Core to connect with your <service> account. Find out more about sending content to Dropbox.

        ResiTAG™ – A Novel Sample Identification Solution for Microscopists and EM Facilities
        Available formats
        ×
        Send article to Google Drive

        To send this article to your Google Drive account, please select one or more formats and confirm that you agree to abide by our usage policies. If this is the first time you use this feature, you will be asked to authorise Cambridge Core to connect with your <service> account. Find out more about sending content to Google Drive.

        ResiTAG™ – A Novel Sample Identification Solution for Microscopists and EM Facilities
        Available formats
        ×
Export citation

Abstract

Electron microscopy requires the use of resin-embedded samples in order to obtain ultra-thin sections for imaging. However, the identification of these resin capsules and molds can be challenging, as traditional inks will run or smear during the embedding process. This can often lead to sample mix-ups or loss of information. This article describes a novel identification solution, ResiTAG™, specifically designed for use with resin capsules and molds. The article details how this chemical-resistant label can be incorporated into the sample preparation process.

Introduction

The study of the cell, the basic unit of life, is a key point of research. As such, researchers have long sought to uncover what occurs at the cellular level within tissues to better understand how they function. Whether studying the effects of a drug treatment on a strain of bacteria or determining cellular differences between healthy and diseased tissues, it is often necessary to understand the structural biology of the cell. To this end, microscopy techniques and instruments play an important part in helping us visualize what is happening in the cell. While imaging technologies have advanced substantially, methods for sample identification have remained relatively unsophisticated. This article describes a modern approach to microscopy specimen labeling.

Microscopy

There are three main branches of microscopy: light, electron, and scanning probe microscopies. Each of the three serve a particular purpose. Advances in microscopy revolutionized the study of biology and gave rise to the histology field. Microscopy techniques have proven equally important and beneficial in the basic research laboratory as in the clinical environment.

Microscopy sample preparation

Samples used in microscopy can be collected from a variety of sources, from biopsies taken from human patients to cells grown under laboratory conditions. Regardless of the source, however, biological samples typically must be chemically fixed and embedded in a structurally supportive media prior to the production of thin sections for observation under the microscope [1]. The type and size of the tissue being studied, and the level of ultrastructural detail required, will determine the type of microscope to be employed as well as the fixation agent and embedding media needed to achieve the best imaging results. The two most common fixatives are formaldehyde and glutaraldehyde, with the latter being better suited for electron microscopy, as it offers a more rigid fixed product. There are two primary means of embedding tissue for sectioning; paraffin wax and resin. In addition, cryo-microscopy techniques are becoming quite important, where special equipment is used to section fixed and unfixed sections at cryogenic temperatures.

Light microscopy specimens

Paraffin wax embedding for light microscopy is the most commonly used medium, chosen for its versatility and relative ease of preparation. The embedding process involves multiple processing stages from dehydration, to clearing, infiltration, and finally embedding in the desired medium [2]. Following fixation in formalin or glutaraldehyde, water is removed from the tissue by slowly replacing it with ethanol. This is followed by a clearing agent, such as xylene to remove the alcohol, before the paraffin infiltration agent replaces the clearing agent. The paraffin wax blocks for histology are microtomed to 4 µm thick sections and placed on glass slides. There are few drawbacks to this popular method, although to observe the fine details of organelles, the better image resolution of electron microscopy is needed.

Electron microscopy specimens

Transmission electron microscopes (TEMs) operating at 100 kV require specimens thinner than 0.1 µm, typically 30 to 60 nm for the best image resolution [3]. In the case of electron microscopy, an epoxy resin embedding medium is recommended because it will form a hard matrix once cured at 60°C, allowing for thinner sections to be cut with an ultramicrotome. These thin specimens can then be observed in a TEM, allowing visualization of cellular structures down to the nanometer scale (Figure 1). Thicker sections can still be obtained from resin-embedded tissue for use in light microscopy if desired. The use of TEM is ideal for investigating the ultrastructure of the cell organelles.

Figure 1 Example electron micrograph of an ultrathin section of the ventricular muscle of group II (diabetic rats) showing margination of the nuclear chromatin of a nucleus of a cardiac myocyte (arrows). Myo-fibrils appear completely rarefied (r). Most mitochondria show dense matrix (m) while others show disrupted cristea (arrowhead) (original mag. 4000×). Image copyright © MM Dallak et al.[5]

Sample identification

One of the most critical aspects of any experiment is sample identification, particularly for thin specimens prepared for light and electron microscopy. This can be difficult. Most traditional labels will not adhere to the resin blocks, and those that are preserved in the capsule along with the sample are prone to smudging or smearing. This article describes a patent-pending identification solution, ResiTAGTM (GA International, Laval, Canada) and shows how to incorporate it into the microscopy laboratory workflow.

Materials and Methods

For TEM applications, ResiTAGTM is a label that is embedded in the resin capsule or mold alongside the sample (Figure 2). This tag can be printed with alphanumeric text, serialized numbering, and 2D barcodes. It works with any standard model of thermal transfer printer such as a label maker. Thermal transfer printers use ink ribbons made of wax, resin, or a blend of wax and resin to print on labels. These printers use heat and pressure to transfer the ink from the ribbon onto the label surface, producing a sharp, high-quality printout, ideal for printing serialized information and barcodes. Using a resin ribbon can provide a robust printout that can withstand extreme temperatures as well as exposure to many harsh chemicals and solvents. Moreover, printing the label instead of hand-writing the information allows the user to inscribe much more information, while ensuring the text remains readable.

Figure 2 Resin block showing identification tag and barcode within the block.

The addition of a 2D barcode tied to a database, like a laboratory information management system (LIMS), allows even more information to be encoded on the label, up to 2000 characters, while also serving to keep track of and catalog a large number of samples. A barcode scanner is required to read the 2D barcode, however there are now code-specific apps available for download that allow a smartphone to become a scanner. Furthermore, the small size of the ResiTAGTM label accommodates various sizes of embedding capsules and molds. When printed with a chemical-resistant ribbon (XAR-class, GA International), the ResiTAGTM label withstands histological resins and ovens up to 100°C. The printout can withstand up to 100% ethanol, methanol, isopropanol and other alcohols, xylene, toluene, acetone, formalin, dimethyl sulfoxide (DMSO), and methyl ethyl ketone (MEK), as well as other harsh chemicals and solvents. This has been confirmed through extensive testing, including exposing the labels to spraying, swabbing, and immersion in the various chemicals.

Procedure

Free online Word templates are available for use in generating the desired printout for ResiTAGTM labels. Furthermore, barcode labeling software, such as BarTender, can be used to design, create, and print serialized labels and scannable 2D barcodes. Once the printout has been created, the labels, supplied in roll format, can be printed in the exact number needed, whether that be one label or hundreds (Figure 3). The speed and darkness level of the thermal printer can be adjusted to optimize the appearance of the printout. Once printed, the labels can be used to label slides or containers if desired, as they come coated with a strong adhesive. The labels have been designed with small perforations down the middle, allowing the ResiTAGTM label to be folded in half, creating an adhesive-free double-sided tag. The small size and design of the label helps to ensure the correct folding of the label. This can be done prior to or while samples are being handled since these labels have a glove-friendly adhesive that will not stick to gloves (Figure 4a). The flexible tag may now be positioned as desired inside the capsule or mold containing the sample and liquid resin (Figure 4b). Additional liquid resin may be added after inserting the tag to arrive at the final volume. After baking in a histological oven to harden the resin, the completed block is permanently labeled and ready for sectioning (Figure 4c). Once the resin is solidified, the label can be easily read or scanned using a barcode scanner to identify the contained biological specimen. An ultramicrotome can then be used to trim the resin block and generate ultra-thin sections. A glass knife should be used when sectioning as a metal blade can cause severe wrinkling of the sections. Thicker sections can potentially be mounted onto glass slides, for use in light microscopy, with additional ResiTAGTM labels used to identify each new slide. This allows identification and tracking of the many generated slides, while also linking them to the original resin block. Individual labels can be identical to the one placed in the resin capsule or individualized for each new section. The labels also resist a wide range of histological stains, such as hematoxylin and eosin. This allows the labeled slides to undergo the staining process without compromising the integrity of the labels. In the case where the ultra-thin sections are then mounted onto electron microscopy grids, labels may be affixed to the grid box. However, for the small individual grids, care must be taken to maintain a manual record of their origin, processing, and observation.

Figure 3 (a) Label maker printing labels. (b) Strip of pre-printed labels.

Figure 4 (a) Folding the label. (b) Placing the label in a half-filled resin block. (c) Finished block with barcode for automated filing.

Discussion

The development of ResiTAGTM provides a reliable labeling solution for scientists, particularly those handling multiple specimens per day in large-scale microscopy facilities. It is also useful in the archiving of valuable biological specimens for research purposes. Using a thermal transfer printer allows the printing of hundreds of labels in minutes. The combination of the ability to physically store the sample data with the sample and the incorporation of laboratory information systems management software (LIMS) into the labeling process ensures the integrity of precious sample data, even during long-term storage.

The permanent identification of resin blocks is essential, as these specimen blocks serve as important archival records. In a clinical setting, tissues may be stored for long periods of time and may only be analyzed long after the sample was collected. Moreover, human biopsies are invasive and difficult to obtain for both the physician and the patient. Therefore, great care must be taken to preserve and recall the information associated with these samples, as they are not easily replaced. This is particularly true for medical testing laboratories, which often refuse samples that are not clearly identified. Furthermore, resin-embedding is often performed off-site in large microscopy facilities where hundreds or thousands of samples are processed daily, increasing the possibility for errors. The use of 2D barcoding software can reduce human error, even when many samples are processed at once, by removing the need to document specimen inventory manually.

Conclusion

The identification of resin blocks used in electron microscopy can be a challenge. It is important for proper tracking of samples and to ensure the proper conclusion is drawn from the analysis. The use of ResiTAGTM to identify resin blocks along with their associated thin sections allows for more information to be displayed, while also reducing errors and enhancing traceability.

Acknowledgments

We would like to thank the lab of Dr. Tamara Western in the Department of Biology at McGill University, along with Mike Ogden from the same department, for their help in developing and testing the product.

References

[1] Echlin, P, Handbook of Sample Preparation for Scanning Electron Microscopy and X-Ray Microanalysis, Springer, New York, 2009.
[2] Gunasegaran, JP, Textbook of Histology and A Practical Guide, Elsevier, India, New Delhi, 2016.
[3] Dallak, MM et al., Open Cardiovasc Med J 2 (2008) 7078.
[4] For more information on ResiTAG please visit: www.labtag.com.
[5] Dallak, MM et al., Open Cardiovasc Med J 2 (2008). Open Access image at https://openi.nlm.nih.gov/detailedresult.php?img=PMC2570581_TOCMJ-2-70_F6&query=MM+Dallak&it=xg&lic=by&req=4&npos=6. No changes were made, license: https://creativecommons.org/licenses/by/3.0/.