Tissue For Transmission Electron Microscopy Research Articles

A new technical approach for preparing frozen biological samples for electron microscopy

BackgroundMany methodological approaches have focused so far on physiological and molecular responses of plant tissues to freezing but only little knowledge is available on the consequences of extracellular ice-formation on cellular ultrastructure that underlies physiological reactions. In this context, the preservation of a defined frozen state during the entire fixation procedure is an essential prerequisite. However, current techniques are not able to fix frozen plant tissues for transmission electron microscopy (TEM) without interrupting the cold chain. Chemical fixation by glutaraldehyde and osmium tetroxide is not possible at sub-zero temperatures. Cryo-fixation methods, such as high pressure freeze fixation (HPF) representing the state-of-the-art technique for best structural preservation, are not equipped for freezing frozen samples. In order to overcome this obstacle, a novel technical approach for maintaining the cold chain of already frozen plant samples prior and during HPF is presented.ResultsDifferent algae (Micrasterias denticulata, Klebsormidium crenulatum) and higher plant tissues (Lemna sp., Ranunculus glacialis, Pinus mugo) were successfully frozen and prepared for HPF at freezing temperatures (− 2 °C, − 5 °C, − 6 °C) within a newly developed automatic freezing unit (AFU), that we manufactured from a standard laboratory freezer. Preceding tests on photosynthetic electron transport and ability to plasmolyse show that the temperatures applied did not impair electron transport in PSII nor cell vitality. The transfer of the frozen specimen from the AFU into the HPF-device and subsequently cryo-fixation were performed without intermediate thawing. After cryo-substitution and further processing, the resulting TEM-micrographs showed excellent ultrastructure preservation of the different organisms when compared to specimens fixed at ambient temperature.ConclusionsThe method presented allows preserving the ultrastructure of plant cells in the frozen state during cryo-fixation. The resulting high quality TEM-images represent an important step towards a better understanding of the consequences of extracellular ice formation on cellular ultrastructure. It has the potential to provide new insights into changes of organelle structure, identification of intracellular injuries during ice formation and may help to understand freezing and thawing processes in plant tissues. It may be combined with analytical TEM such as electron energy loss spectroscopy (EELS), X-ray analyses (EDX) and various other electron microscopic techniques.

Sample Preparation and Stereological Methods for the Study of Glomerular Ultrastructure Using Electron Microscopy.

In this chapter we describe conventional methods used for preparing renal tissue for transmission electron microscopy. We also describe a relatively new technique, serial block face scanning electron microscopy. Protocols are given for processing, sectioning, and imaging of tissue along with methods for obtaining quantitative data from the results.

Visualizing Dark Microglia.

Dark microglia, a recently described phenotype, are found in high numbers in nonhomeostatic conditions (e.g., Alzheimer's disease pathology, aging, chronic stress). As a specific protein marker has not yet been defined, they cannot be studied using conventional cellular biology techniques. They are recognized by their unique ultrastructural features visible under electron microscopy. This nanoscale resolution imaging technique allows the identification of cells based on their ultrastructure or immunoreactivity to certain proteins. In this protocol, we describe the steps necessary for the preparation of high-quality brain tissues for transmission electron microscopy, the imaging, the identification of dark microglia, and the ultrastructural analysis of various parameters that can be studied in these cells.

Method for hydrophytic plant sample preparation for light and electron microscopy (studies on Phragmites australis Cav.)

Nowadays there are no well-established, standard methods in electron microscopy despite its 50-year history. An excessive variety of research objects prompt researchers to modify and improve methodological approaches to sample preparation. One of the difficult objects to study by electron microscopy is hydrophytic plants, for example, Phragmites australis Cav. Traditional approaches to fixation and sample preparation do not give satisfactory results due to the peculiarities in structure and physiology of hydrophytic plants. The purpose of present research is modification description of the widespread method developed for double fixation of hydrophytic plant tissue for transmission electron microscopy. Suggested approach takes into account the features of hydrophyte plants.•The developed method allows improving the quality of plant samples by additional fixatives imbibition and removing of air bubbles from aerenchyma tissue using a vacuum.•The new step of sample preparation consisting in the layer-by-layer sample mixing in a special inclined mixer is applied for the embedding media penetrate sufficiently into the sample tissue.•The process of samples inclusion in polymeric resins is carried out in the flat-bottom capsules. Compare to standard conical capsules, flat-bottom capsules allow strictly defined orientation sample pieces, that is permit to produce a semi-thin and ultra-thin slices of perpendicular to the longitudinal structures of the plant. This is especially important to conduct an adequate comparative analysis of dimensions, shape, and electron density of fragments and parts of the studying samples.

P.403 - Muscle biopsies reprocessed for electron microscopy from paraffin blocks and frozen tissue produce material of sufficient quality for diagnostic use

Reprocessed frozen tissue (FT) or formalin-fixed paraffin-embedded (FFPE) tissue for transmission electron microscopy (TEM) has been used in general pathology but there is little information on its use in diagnostic muscle pathology. A recent study of re-processed stored frozen muscle biopsies from healthy volunteers showed morphology almost comparable to that of freshly fixed muscle (FFM). This is a retrospective study of 8 muscle biopsies referred to the DNC pathology service where TEM was required to confirm/exclude diagnosis and only FFPE/FT blocks were available. A small strip parallel to the fibres was cut from each of the FT blocks in the cryo-cabinet at −25°C and immediately placed into cold 4% glutaraldehyde in cacodylate buffer and some also into a 2% glutaraldehyde/2% paraformaldehyde mixture, allowed to thaw at room temperature, then cut into smaller pieces approximately 1.5 × 0.5 mm2. After 1.5 hours the samples were thoroughly rinsed, dehydrated through an ethanol series to propylene oxide and embedded in Araldite resin. Stained slides were reviewed to select an area of interest for FFPE blocks; these were cut and de-waxed overnight in chloroform, then re-hydrated, fixed and processed as above. Resin semi-/ultra-thin sections were cut and stained as per standard procedures and viewed with a Phillips CM10 or CM12 electron microscope. Reprocessed FT/FFPE tissue morphology was somewhat poor compared to FFM, but there were better-preserved foci giving good diagnostic yield. Mitochondrial morphology was poorer in reprocessed cases. Diagnostic pathology identified included reducing bodies, granulofilamentous aggregates, nemaline rods, polyglucosan aggregates and abnormal mitochondria (5/9), non-specific pathology (2/9) and exclusion of macrophagic myofascitis (1/9). In conclusion, in instances where FFM processed tissue for TEM is not available, it is possible to produce adequate results using re-processed FT/FFPE material for diagnostic muscle biopsies.

Tandem high-pressure freezing and quick freeze substitution of plant tissues for transmission electron microscopy.

Since the 1940s transmission electron microscopy (TEM) has been providing biologists with ultra-high resolution images of biological materials. Yet, because of laborious and time-consuming protocols that also demand experience in preparation of artifact-free samples, TEM is not considered a user-friendly technique. Traditional sample preparation for TEM used chemical fixatives to preserve cellular structures. High-pressure freezing is the cryofixation of biological samples under high pressures to produce very fast cooling rates, thereby restricting ice formation, which is detrimental to the integrity of cellular ultrastructure. High-pressure freezing and freeze substitution are currently the methods of choice for producing the highest quality morphology in resin sections for TEM. These methods minimize the artifacts normally associated with conventional processing for TEM of thin sections. After cryofixation the frozen water in the sample is replaced with liquid organic solvent at low temperatures, a process called freeze substitution. Freeze substitution is typically carried out over several days in dedicated, costly equipment. A recent innovation allows the process to be completed in three hours, instead of the usual two days. This is typically followed by several more days of sample preparation that includes infiltration and embedding in epoxy resins before sectioning. Here we present a protocol combining high-pressure freezing and quick freeze substitution that enables plant sample fixation to be accomplished within hours. The protocol can readily be adapted for working with other tissues or organisms. Plant tissues are of special concern because of the presence of aerated spaces and water-filled vacuoles that impede ice-free freezing of water. In addition, the process of chemical fixation is especially long in plants due to cell walls impeding the penetration of the chemicals to deep within the tissues. Plant tissues are therefore particularly challenging, but this protocol is reliable and produces samples of the highest quality.

Tandem High-pressure Freezing and Quick Freeze Substitution of Plant Tissues for Transmission Electron Microscopy

Since the 1940s transmission electron microscopy (TEM) has been providing biologists with ultra-high resolution images of biological materials. Yet, because of laborious and time-consuming protocols that also demand experience in preparation of artifact-free samples, TEM is not considered a user-friendly technique. Traditional sample preparation for TEM used chemical fixatives to preserve cellular structures. High-pressure freezing is the cryofixation of biological samples under high pressures to produce very fast cooling rates, thereby restricting ice formation, which is detrimental to the integrity of cellular ultrastructure. High-pressure freezing and freeze substitution are currently the methods of choice for producing the highest quality morphology in resin sections for TEM. These methods minimize the artifacts normally associated with conventional processing for TEM of thin sections. After cryofixation the frozen water in the sample is replaced with liquid organic solvent at low temperatures, a process called freeze substitution. Freeze substitution is typically carried out over several days in dedicated, costly equipment. A recent innovation allows the process to be completed in three hours, instead of the usual two days. This is typically followed by several more days of sample preparation that includes infiltration and embedding in epoxy resins before sectioning. Here we present a protocol combining high-pressure freezing and quick freeze substitution that enables plant sample fixation to be accomplished within hours. The protocol can readily be adapted for working with other tissues or organisms. Plant tissues are of special concern because of the presence of aerated spaces and water-filled vacuoles that impede ice-free freezing of water. In addition, the process of chemical fixation is especially long in plants due to cell walls impeding the penetration of the chemicals to deep within the tissues. Plant tissues are therefore particularly challenging, but this protocol is reliable and produces samples of the highest quality.

Feasibility of high pressure freezing with freeze substitution after long‐term storage in chemical fixatives

Fixation of biological samples is an important process especially related to histological and ultrastructural studies. Chemical fixation was the primary method of fixing tissue for transmission electron microscopy for many years, as it provides adequate preservation of the morphology of cells and organelles. High pressure freezing (HPF) and freeze substitution (FS) is a newer alternative method that rapidly freezes non-cryoprotected samples that are then slowly heated in the FS medium, allowing penetration of the tissue to insure adequate fixation. This study addresses several issues related to tissue preservation for electron microscopy. Using mice liver tissue as model the difference between samples fixed chemically or with HPF immediately after excision, or stored before chemical or HPF fixation were tested with specific focus on the nuclear membrane. Findings are that immediate HPF is the method of choice compared to chemical fixation. Of the chemical fixatives, immediate fixation with 2.5% glutaraldehyde (GA)/formaldehyde (FA) is the best in preserving membrane morphology, 2.5% GA can be used as alternative for stored and then chemically processed samples, with 10% formalin being suitable as a storage medium only if followed by HPF fixation. Overall, storage leads to lower ultrastructural preservation, but HPF with FS can minimize these artifacts relative to other processing protocols.

Preparation of Drosophila Specimens for Examination by Transmission Electron Microscopy

There is no single, simple procedure for fixing and embedding all tissues for transmission electron microscopy (TEM). The chemistry of different cell types is to some extent unique, and this affects the way each cell type reacts to the wide array of fixatives, buffers, organic solvents, and resins used in TEM specimen preparation. A recurring theme in those organisms or cell types that are difficult to fix is the presence of a diffusion barrier that prevents the free diffusion of fixative and other chemicals in and out of the cell or tissue. This in turn means that fixation takes a relatively long time (measured in minutes or tens of minutes in some cases), during which the cells begin autolysis or are otherwise degraded from their original state. Drosophila requires specific preparation methods for TEM because most fly tissues are surrounded by significant diffusion barriers. In the embryo, it is the vitelline envelope, and in larvae and adults, it is the cuticle. In this article, we discuss methods that have evolved to cope with these barriers to achieve reasonable preservation of ultrastructure.

Microwaves and tea: new tools to process plant tissue for transmission electron microscopy

Optimizing sample processing, reducing the duration of the preparation of specimen, and adjusting procedures to adhere to new health and safety regulations, are the current challenges of plant electron microscopists. To address these issues, plant processing protocols for TEM, combining the use of polyphenolic compounds as substitute for uranyl acetate with microwave technology are being developed. In the present work, we optimized microwave-assisted processing of different types of plant tissue for ultrastuctural and immunocytochemical studies. We also explored Oolong tea extract as alternative for uranyl acetate for the staining of plant samples. We obtained excellent preservation of cell ultrastructure when samples were embedded in epoxy resin, and of cell antigenicity, when embedded in LR-White resin. Furthermore, Oolong tea extract successfully replaced uranyl acetate as a counterstain on ultrathin sections, and for in block staining. These novel protocols reduce the time spent at the bench, and improve safety conditions for the investigator. The preservation of the cell components when following these approaches is of high quality. Altogether, they offer significant simplification of the procedures required for electron microscopy of plant ultrastructure.

Preparation of White Adipose Tissue for Transmission Electron Microscopy: An Alternative Technique

An alternative fixation method for preparing white adipose tissue for transmission electron microscopy is described. The procedure uses a primary fixation mixture of glutaraldehyde and acrolein. In our hands, this protocol produces superior ultrastructural morphology and fewer artifacts than traditional immersion fixation. This technique can be used in cases where perfusion fixation is neither feasible nor possible. (The J Histotechnol 27:43, 2004)Submitted: August 28, 2003; Accepted: October 31, 2003

Morphometrical Ultrastructural Studies of the Rat's Hepatocytes Under Cooling

The aim of given investigation is to study the effect of cooling upon rat hepatocyte structure using transmission electron microscopic and computer morphometric methods. Ultrastructural and morphometrical characteristics of hepatocytes under liver cooling for various levels under in vivo and in vitro conditions were investigated. Vistar rats of 180-250 g were used in the experiment. Liver cooling (in vivo) was performed by means of original cryoapplicator with different probe temperature (1,2). Liver tissue for transmission electron microscopy was fixed in glutaraldehyde fixator on cocadylate buffer and OsO4. Dehydration was completed on acetone (3). Tissue embedding was done into the mixture of Epon/Araldite epoxy rasin. Ultrathin slices were contrasted by the method of Reinolds. Cell viewing and imaging were accomplished by electron microscope at accelerating power of 75kV.Morphometrical and stereometrical analysis was performed using the “Morpho-Tools” original computer system (c) 1994-1996 A.S. Kaprelyants, A.A. Kaprelyants, A.N. Reylan .

New infiltration method permits use of freeze substitution for preparation of wood tissues for transmission electron microscopy

SummaryA new infiltration method was developed for preparation of wood tissues for transmission electron microscopy (TEM) using freeze substitution. The method included treatment of freeze‐substituted samples with sodium phosphate buffer before infiltration with epoxy resin. Samples from red osier (Cornus sericea L.) and flowering dogwood (C. florida L.) were studied to assess the quality of the infiltration method. The method produced well‐embedded samples with good trimming and cutting qualities. To our knowledge, this is the first report on preparation of wood tissues for TEM using freeze substitution.

A method for rapid fixation and dehydration of nematode tissue for transmission electron microscopy

SUMMARYA method is described using a microwave oven and acidified 2,2‐dimethoxypropane to fix and dehydrate nematode tissue rapidly in preparation for transmission electron microscopy. Nematode tissue prepared using this method is well preserved compared with tissue fixed by conventional methods. The technique also enabled fixation of unhatched nematodes in the egg, a process that is almost impossible to achieve using conventional methods of fixation. Using the method described, differences were found between the cuticles of different nematode stages.

Uranyl acetate staining under different conditions of preparation, storage and use

Uranyl acetate is routinely used in the staining of thin sections of biological tissue for transmission electron microscopy. Although many methods for its preparation and use have been described, there is seldom reference to the reasons for variations in concentration, solvent, storage time and staining time. Likewise, possible variations in the effects of staining under different conditions are largely ignored. In order to gain clarity on this issue an attempt has been made to test three variables (solvent, storage time and use in light or dark) under controlled experimental conditions.The tissues used for the experiment were the testis of a marine limpet, the gut epithelium of a fresh-water catfish, and the kidney of a rat; all of which were fixed and embedded by standard methods. The uranyl acetate solutions were prepared at the outset of the experiment and dispensed into small volumes and stored in the dark at 4°C until required.

A reliable epoxy resin mixture and its application in routine biological transmission electron microscopy

Although there are many papers in the literature on materials and procedures for the embedding of tissue for transmission electron microscopy (TEM), most recent publications have emphasized techniques for specialized applications. Although these may in many cases also be suitable for routine applications in addition to the specialized applications for which they were developed, this may not be clear from the literature. This paper describes the formulation and suggested procedures for the use of an epoxy resin mixture which has been routinely used by novice and experienced workers with success for a wide variety of biological TEM investigations in a multi-user multi-disciplinary EM facility. Results are given of the use of this embedding medium in the investigation of a variety of tissue types.

A rapid technique using epoxy resin Quetol 651 to prepare woody plant tissues for ultrastructural study

A new technique was evaluated for rapid preparation of woody plant tissues for transmission electron microscopy. A low-viscosity medium formulation was developed using a water-soluble epoxy resin, Quetol 651. No dehydrating solvents were needed during preparation. Samples from living trees and xylary tissues of sound and decayed wood were studied to assess the quality of the media and embedding techniques. Infiltration, preservation of ultrastructural detail, sectioning qualities, and staining of the wood samples were found to be very good. The technique provided a fast, reliable method for preparing wood samples for ultrastructural observation.

Polyethylene glycol embedding of plant tissues for transmission electron microscopy

SUMMARYThe use of high molecular weight polyethylene glycol as an embedding medium for the production of resin‐free sections of plant cells is described. Specimens can be observed at a range of accelerating voltages, are high in contrast and require no heavy metal staining. All the major organelles appear intact and a three‐dimensional network of filaments in the cytoplasm and nucleoplasm is reported.

Tissue Preparation and Fine Structure of the Radicle Apex from Cotton Seeds

A comparative methodological study was made of the fine structure of apical cortical cells in excised radicles from cotton (Gossypium hirsutum L. var M-8) seeds. Radicles from dry seed had 12% moisture content and were prepared for electron microscopy using several different techniques. These included different methods of chemical fixation or freeze-fracture and etching of unfixed tissue for transmission electron microscopy (TEM) and cryofracturing of fixed and dehydrated radicles for scanning electron microscopy (SEM). Cortical cells had a similar appearance regardless of the method used in tissue preparation. Cell walls had a pronounced waviness which was particularly evident in SEM images of cells lining the elongated intercellular air spaces. The plasma membrane (PM) delimited the cytoplasm of each cell as an intact unit membrane. Single layers of tightly-packed lipid bodies (LB) were apposed to the PM and protein bodies (PB). Distension of cells, membranous organelles and LB was observed in radicles fixed by immersion in aqueous solutions, suggesting that a certain amount of hydration occurred during fixation. This interpretation was supported by the compact appearance of cells and organelles in tissue prepared by freeze-etch or vapor fixation. We conclude that freeze-fracture and etching of unfixed tissue provided the best information for cell morphology and structure of membranes and organelles in dry tissue. Complementary data on the fine details of nuclei and cytoplasmic organelles were best observed with TEM of fixed tissue. These data when viewed collectively indicate the advantage of using several techniques to obtain analogous and complementary information essential for establishing a baseline level of information on the fine structure of cells in dry tissue.

TISSUE PREPARATION AND FINE STRUCTURE OF THE RADICLE APEX FROM COTTON SEEDS

A comparative methodological study was made of the fine structure of apical cortical cells in excised radicles from cotton (Gossypium hirsutum L. var M‐8) seeds. Radicles from dry seed had 12% moisture content and were prepared for electron microscopy using several different techniques. These included different methods of chemical fixation or freeze‐fracture and etching of unfixed tissue for transmission electron microscopy (TEM) and cryofracturing of fixed and dehydrated radicles for scanning electron microscopy (SEM). Cortical cells had a similar appearance regardless of the method used in tissue preparation. Cell walls had a pronounced waviness which was particularly evident in SEM images of cells lining the elongated intercellular air spaces. The plasma membrane (PM) delimited the cytoplasm of each cell as an intact unit membrane. Single layers of tightly‐packed lipid bodies (LB) were apposed to the PM and protein bodies (PB). Distension of cells, membranous organelles and LB was observed in radicles fixed by immersion in aqueous solutions, suggesting that a certain amount of hydration occurred during fixation. This interpretation was supported by the compact appearance of cells and organelles in tissue prepared by freeze‐etch or vapor fixation. We conclude that freeze‐fracture and etching of unfixed tissue provided the best information for cell morphology and structure of membranes and organelles in dry tissue. Complementary data on the fine details of nuclei and cytoplasmic organelles were best observed with TEM of fixed tissue. These data when viewed collectively indicate the advantage of using several techniques to obtain analogous and complementary information essential for establishing a baseline level of information on the fine structure of cells in dry tissue.