Light Microscopes (LM), Transmission electron microscopy (TEM), Scanning Electron Microscopes (SEM) and other instruments are extensively used to understand the ultrastructure of a wide variety of synthetic and biological materials in numerous areas of science and technology. For example, light microscopy samples are used for research to identify the development of different organs in animals and plant. In addition, one major use of light microscopic samples is in the histophathologic examination of biopsy samples of tissues suspected of disease. TEM is used to study biological samples, metallurgical samples and many other types of materials. TEM images can be used to investigate the atomic structure of the objects, for example to identify areas of metal fatigue and to visualize the molecular and ultrastructure of cells, such images are capable of resolution down to 0.1 nm. SEM is similar to TEMs in that it uses electrons to create an image of the target/sample. However, the resolution of the SEM is on the molecular level (e.g., 100 nm-5 μm). Due to the SEM's ability to image bulk materials they can be used to image larger samples and samples that may or may not be sliced or sectioned.
Study objects for microscopy are prepared in multiple ways depending upon the type of material to be examined, and the type of microscopy to be used. Biological materials require special handling to preserve the structure of the material when it will be examined in the electron microscope, and secondarily to enhance or enable imaging.
Both SEM and TEM instruments perform their imaging in a vacuum (the absence or partial absence of air or other gases). Since biological materials are typically 50 to 95% water, if these were placed directly within the vacuum the water would evaporate and the specimen would collapse. Consequently both SEM and TEM samples have the water removed after the structure is strengthened with chemicals such as glutaraldehyde, formaldehyde, and osmium tetroxide.
TEM samples must be very thin (typically about 40 to 100 nm) in order for the electrons used for imaging to be “transmitted” or pass through the sample. To cut specimens into such thin sections the water is replaced with plastic resin that is hardened in place. This plastic supports the sample as it is sliced very thin using a device called an ultra-microtome.
Light microscope specimens, especially those of biological origin, are also often sectioned in order to provide cross-sections for viewing, and to allow photons (light) to be transmitted through the specimen. As with TEM, LM sections are also embedded to support the specimen during sectioning, however different generally softer plastics are used as the embedding material, as are paraffin wax and simply water that is frozen with additional materials to enable the ice to be softer, provide better support of the tissue, and reduce ice crystal damage during freezing.
While SEM specimens may be imaged in the bulk, that is without slicing into thin sections, with many specimens some slices or cross-sections are desired, such as to view interior structures. When these specimens are of biological origin, it is often necessary to perform many of the same chemical treatments as with TEM specimens in order to strengthen the structure for imaging in the SEM vacuum and to obtain cross-sections when these are desired.
The spatial orientation of cross-sections for many specimens can be very important in order to obtain the desired structural information, whether the specimens are to be examined with LM and TEM, and even SEM. For especially TEM and LM imaging of specimens to imaged as many thin slices, microscopists endeavor to orient the specimen in a plastic resin or other embedding media so that the cross-sectional slices are obtained in the desired orientation. These slices are then placed on microscope slides (for LM) or TEM grids (for TEM) to facilitate imaging through these thin cross-sections.
Many microscope specimens are imaged without the need for obtaining thin cross-sectional slices, but nonetheless where it is desired to obtain cross-sections in specific orientations. This is most common for SEM imaging, but sometimes desired for other types of microscopic analysis. For such imaging, as with SEM, the specimens are not commonly embedded in a plastic resin, but are nonetheless prepared by fluidic treatments with chemicals fixatives to preserve structure, solvents to dehydrate, and then commonly air-drying from solvents, or drying by the critical point. Freeze-drying is another method, where the specimens are also commonly first preserved with fixatives.
With TEM where specimens are sectioned, the standard process to obtain the proper orientation is to place or lay the fixed, dehydrated, and resin infiltrated specimens into a flat embedding mold filled with resin, and then place the mold into an oven for curing. These flat embedding molds are generally shallow wells prepared from silicone rubber, and provide no way to hold or retain the specimens in the proper orientation. Consequently, the specimen will often move during resin solidification. This often requires that specimens that are embedded in cured resin be sawed out and then glued onto other pieces of resin to provide the desired orientation. Of course, this is a time consuming extra step. Moreover, with fixed, dehydrated and resin infiltrated specimens it is often difficult to determine what the desired orientation is since the fixation process can make all regions of a tissue sample appear the same as typically viewed with the naked eye or even through a dissecting microscope which may be used in the preparation facility. For example, osmium tetroxide or potassium permanganate fixatives, as commonly used for TEM, generally makes all specimens the same uniform black color, unlike fresh tissue, partially fixed tissue, or tissue that has been stained. Thus, obtaining the proper orientation is often not possible, hence it is often not determined until one views specimen sections with the TEM.
With LM, where specimens are sectioned, the common process to obtain the proper orientation is place or lay the fixed, dehydrated, and/or paraffin infiltrated specimens into a flat embedding mold filled with melted paraffin. While it is easy to reorient specimens embedded in paraffin, by simply warming the paraffin until it melts, this is still a time-consuming step. Moreover, with fixed, dehydrated and paraffin infiltrated specimens it is often difficult to determine what the desired orientation is since the fixation process can make all regions of a tissue sample appear the same when viewed with the naked eye or with a dissecting microscope. Thus, obtaining the proper orientation is often not possible, hence it is often not determined until one views specimen sections with the LM.
With SEM where specimens require viewing in certain orientations, there is really no standard method to obtain the proper orientation other then to view the specimen with a dissecting microscope and mount it in the desired orientation. This is usually done with biological tissue after a specimen has been fixed, dehydrated, and dried by the critical point method, or fixed, dehydrated and air dried from a solvent, or fixed and freeze-dried, or otherwise treated to maintain structure in the vacuum of the SEM. With many specimens prepared by any of these fixation and preparation methods, all or most tissue regions appear the same when viewed with the naked eye or with a dissecting microscope which may be used in the preparation facility. Thus, obtaining the proper orientation is often not possible, hence it is often not determined until one views the specimen with the SEM.
Williamson et al, in U.S. Pat. No. 7,179,424, discloses a cassette for handling and holding tissue samples during processing for LM in, especially, histology laboratories. This tissue cassette has movable pegs that are used to create slots that can hold tissue in the desired orientation for sectioning. These can enable obtaining orientation prior to chemical processing. However, as with other, similar tissue cassettes, the cassette disclosed by Williamson includes a large volume of dead-space and provides little ability to visually inspect the samples. These cassettes are not suitable for specimens with dimensions on the order of 1 mm or smaller, as typically required for TEM and sometimes for LM. Furthermore, these cassettes are too large for preparing specimens for TEM, and are too thin and large for processing most specimens for TEM or SEM since these will not fit into apartatuti such as critical point dryers and freeze dryers.
Thus, the need exists for a low-cost device and method that allows for preparation of specimens in the proper orientation, and where such orientation can be visible throughout the process, where orientation can be preformed with fresh or stained tissue when tissue structure may be discerned to enable alignment, where the orientation of the specimen can be changed during the preparation process, and where the specimen is ultimately prepared in a configuration that is appropriate for TEM, LM and SEM with little or no additional steps.
Improvements to the conventional devices and methods of preparing specimens for microscopic analysis are desirable.