Cryofixation Method Research Articles

A cryo-fixation protocol to study the structure of the synaptonemal complex.

Genetic variability in sexually reproducing organisms results from an exchange of genetic material between homologous chromosomes. The genetic exchange mechanism is dependent on the synaptonemal complex (SC), a protein structure localized between the homologous chromosomes. The current structural models of the mammalian SC are based on electron microscopy, superresolution, and expansion microscopy studies using chemical fixatives and sample dehydration of gonads, which are methodologies known to produce structural artifacts. To further analyze the structure of the SC, without chemical fixation, we have adapted a cryo-fixation method for electron microscopy where pachytene cells are isolated from mouse testis by FACS, followed by cryo-fixation, cryo-substitution, and electron tomography. In parallel, we performed conventional chemical fixation and electron tomography on mouse seminiferous tubules to compare the SC structure obtained with the two fixation methods. We found several differences in the structure and organization of the SC in cryo-fixed samples when compared to chemically preserved samples. We found the central region of the SC to be wider and the transverse filaments to be more densely packed in the central region of the SC.

A Compartmentalized Neuronal Cell-Culture Platform Compatible With Cryo-Fixation by High-Pressure Freezing for Ultrastructural Imaging.

The human brain contains a wide array of billions of neurons and interconnections, which are often simplified for analysis in vitro using compartmentalized microfluidic devices for neuronal cell culturing, to better understand neuronal development and disease. However, such devices are traditionally incompatible for high-pressure freezing and high-resolution nanoscale imaging and analysis of their sub-cellular processes by methods including electron microscopy. Here we develop a novel compartmentalized neuronal co-culture platform allowing reconstruction of neuronal networks with high variable spatial control, which is uniquely compatible for high-pressure freezing. This cryo-fixation method is well-established to enable high-fidelity preservation of the reconstructed neuronal networks and their sub-cellular processes in a near-native vitreous state without requiring chemical fixatives. To direct the outgrowth of neurites originating from two distinct groups of neurons growing in the two different compartments, polymer microstructures akin to microchannels are fabricated atop of sapphire disks. Two populations of neurons expressing either enhanced green fluorescent protein (EGFP) or mCherry were grown in either compartment, facilitating the analysis of the specific interactions between the two separate groups of cells. Neuronally differentiated PC12 cells, murine hippocampal and striatal neurons were successfully used in this context. The design of this device permits direct observation of entire neuritic processes within microchannels by optical microscopy with high spatial and temporal resolution, prior to processing for high-pressure freezing and electron microscopy. Following freeze substitution, we demonstrate that it is possible to process the neuronal networks for ultrastructural imaging by electron microscopy. Several key features of the embedded neuronal networks, including mitochondria, synaptic vesicles, axonal terminals, microtubules, with well-preserved ultrastructures were observed at high resolution using focused ion beam – scanning electron microscopy (FIB-SEM) and serial sectioning – transmission electron microscopy (TEM). These results demonstrate the compatibility of the platform with optical microscopy, high-pressure freezing and electron microscopy. The platform can be extended to neuronal models of brain disease or development in future studies, enabling the investigation of subcellular processes at the nanoscale within two distinct groups of neurons in a functional neuronal pathway, as well as pharmacological testing and drug screening.

In situ Microfluidic Cryofixation for Cryo Focused Ion Beam Milling and Cryo Electron Tomography

We present a microfluidic platform for studying structure-function relationships at the cellular level by connecting video rate live cell imaging with in situ microfluidic cryofixation and cryo-electron tomography of near natively preserved, unstained specimens. Correlative light and electron microscopy (CLEM) has been limited by the time required to transfer live cells from the light microscope to dedicated cryofixation instruments, such as a plunge freezer or high-pressure freezer. We recently demonstrated a microfluidic based approach that enables sample cryofixation directly in the light microscope with millisecond time resolution, a speed improvement of up to three orders of magnitude. Here we show that this cryofixation method can be combined with cryo-electron tomography (cryo-ET) by using Focused Ion Beam milling at cryogenic temperatures (cryo-FIB) to prepare frozen hydrated electron transparent sections. To make cryo-FIB sectioning of rapidly frozen microfluidic channels achievable, we developed a sacrificial layer technique to fabricate microfluidic devices with a PDMS bottom wall <5 µm thick. We demonstrate the complete workflow by rapidly cryo-freezing Caenorhabditis elegans roundworms L1 larvae during live imaging in the light microscope, followed by cryo-FIB milling and lift out to produce thin, electron transparent sections for cryo-ET imaging. Cryo-ET analysis of initial results show that the structural preservation of the cryofixed C. elegans was suitable for high resolution cryo-ET work. The combination of cryofixation during live imaging enabled by microfluidic cryofixation with the molecular resolution capabilities of cryo-ET offers an exciting avenue to further advance space-time correlative light and electron microscopy (st-CLEM) for investigation of biological processes at high resolution in four dimensions.

Self-pressurised rapid freezing, a time-efficient and affordable cryo-fixation method for ultrastructural observations on unhatched cyst nematodes

Summary Ultrastructural analysis of the early development of nematodes is hampered by the impermeability of the eggshell to most commonly used fixatives. High-pressure freezing (HPF), a physical cryo-fixation method, facilitates a fast rate of fixation, and by using this method the issue of the uneven delivery of fixative is circumvented. Although HPF results in a superior preservation of the fine structure, the equipment costs impede a wider application of this method. Self-pressurised rapid freezing (SPRF) is an alternative low-cost cryo-fixation method, and its usefulness was evaluated in an ultrastructural study of the eggshell and the cuticle of unhatched second-stage juveniles (J2) of Globodera rostochiensis and Heterodera schachtii. A comparison with conventional (chemical) fixation demonstrates that SPRF fixation results in a remarkably well-preserved ultrastructure of the entire egg including both the eggshell and the cellular details of the unhatched J2. Therefore, SPRF fixation is proposed as an affordable, relatively easy-to-use and time-efficient technique to study the ultrastructure of unhatched J2 and eggs of nematodes.

Thermal stability of red algal galactans: Effect of molecular structure and counterions

Thermal degradation of κ-, ι-, λ-carrageenans, furcellaran, funoran and agarose samples in dry and sol states was investigated. The polysaccharides subjected to heat treatment were characterized by 1H NMR, 13C NMR and FTIR spectroscopy, size exclusion chromatography and static rheometry methods. The microstructure of galactan gels was studied using a cryofixation method in combination with freeze-drying and SEM techniques. Thermal stability at high temperatures decreases in the order of agarose>furcellaran>funoran>κ-carrageenan>λ-carrageenan>ι-carrageenan for dry preparations. The respective sequence for sol state is ι-carrageenan>λ-carrageenan>κ-carrageenan>furcellaran>funoran>agarose. The presence of methoxy groups stabilizes algal polysaccharides whereas divalent cations as counterions increase the susceptibility towards thermal degradation. In dry state the thermal treatment leads to significant desulfation of the galactan before the complete depolymerization occurs. Depending on the sulfation degree and the presence of 3,6-anhydrogalactose residues in the galactan, a notable amount of 5-hydroxymethylfurfural (yield 0.7–21.8%) is formed during the decomposition in sol state.

Self-Pressurised Rapid Freezing (SPRF): an easy-to-use and low-cost alternative cryo-fixation method for nematodes

Self-Pressurised Rapid Freezing (SPRF), an easy-to-use and low-cost alternative cryo-fixation method, was evaluated based on a comparative analysis of the ultrastructure of spermatozoa of the nematodesAcrobeles complexusandCaenorhabditis elegans. Sealed copper tubes, packed with active nematodes in water, were plunged into nitrogen slush, a semi-solid form of nitrogen. The water inside the capillary copper tube expands upon cooling due to the formation of hexagonal ice, thereby generating high pressure intrinsically for cryo-fixation of the sample. For sperm cells cryo-fixed by SPRF, the preservation of the ultrastructure was comparable to that achieved with high pressure freezing. This was evidenced by the clear details in mitochondria, membranous organelles and cytoskeleton in the pseudopod. It was demonstrated that SPRF fixation did not destroy antigenicity, based on the results of the immunolocalisation of the major sperm protein in both species. In conclusion, SPRF is a low-cost alternative cryo-fixation method for nematodes.

Technical Advances in Intracellular Detection Using Immuno-Gold Particles: Simple Cryofixation with Metal Contact Quick Freezing.

The preparation of biological specimens using cryofixation techniques ensures excellent visibility of intracellular structures and preserves the antigenic sites of subcellular molecules. Hence, cryofixation is an effective method of preparing samples for analyses using antibodies conjugated to gold nanoparticles that are designed to detect the localization of specific target molecules within cells. However, cryofixation cannot be utilized easily because it requires expensive equipment and skilled technologists, resulting in a high level of expense for researchers. Here, we describe a simple technical approach to cryofixation that uses metal contact quick freezing followed by a modified freeze substitution technique and immuno-gold labeling electron microscopy. Micrograph images of cells prepared using this modified cryofixation method demonstrated its superiority over chemical fixation for high contrast visualization of the morphologies of cellular components and preservation of antigenicity for immuno-gold labeling. This report provides valuable technical information related to the advancement of metal contact quick freezing techniques, which can be used to visualize biomedical events of interest in an easy, simple, and rapid manner.

Freeze-Fracture Electron Microscopy on Nano- and Micro-Delivery Vehicles for Biological Active Compounds

The potency of nano- and micro-vehicles, loaded with therapeutics or/and diagnostics (theranostics) is strongly depending upon their morphology adopted at their site of action. Freeze-fracture transmission electron microscopy (ff-TEM) as a cryo-fixation method is a powerful technique to monitor the morphology of nano- as well as micro-particles in biological relevant environments. At the resolution limit of 2nm we are able to characterize nano-particles such as quantum dots (coupled to drug-loaded immunoliposomes), gold nano-particles, superparamagnetic iron oxide nano-particles loaded in polymeric immunomicelles, micelles (spherical-, disc-, and worm-type micelles), single-wall carbon nano-tubes embedded in hydrated gels, and small unilamellar liposome (targeted and non-targeted). Furthermore, ff-TEM includes the production of beam-damage-resistant replica from much larger, micrometer-size objects such as multilamellar liposome, niosomes, cationic liposome/DNA complexes, integrin-targeted lipopolyplexes, polymer-, lipid- or surfactant-stabilized gas bubbles, cochleate cylinder, and depofoam particles. Ff-TEM enables us not only to monitor self-assembling of lipid-, polymer-, as well as protein/peptide-based carriers encapsulating drug-, gene-, vaccine, antimicrobial- and imaging molecules but is the method of choice as well to study their fate related to their pay load, application milieu, and during cell interaction. Furthermore, we investigated structural modifications within mono- and bilayers such as domain-formation but also transformations to non-bilayer structures such as hexagonal and cubic phases. Currently we are focused on cochleate cylinder as macrocarrier for antibiotics combating bacterial multidrug resistance.

Cryo-fixation and associated developments in transmission electron microscopy: a cool future for nematology

At present, the importance of sample preparation equipment for electron microscopy represents the driving force behind major breakthroughs in microscopy and cell biology. In this paper we present an introduction to the most commonly used cryo-fixation techniques, with special attention paid towards high-pressure freezing followed by freeze substitution. Techniques associated with cryo-fixation, such as immunolocalisation, cryo-sectioning, and correlative light and electron microscopy, are also highlighted. For studies that do not require high resolution, high quality results, or the immediate arrest of certain processes, conventional methods will provide answers to many questions. For some applications, such as immunocytochemistry, three-dimensional reconstruction of serial sections or electron tomography, improved preservation of the ultrastructure is required. This review of nematode cryo-fixation highlights that cryo-fixation not only results in a superior preservation of fine structural details, but also underlines the fact that some observations based on results solely obtained through conventional fixation approaches were either incorrect, or otherwise had severe limitations. Although the use of cryo-fixation has hitherto been largely restricted to model organisms, the advantages of cryo-fixation are sufficiently self-evident that we must conclude that the cryo-fixation method is highly likely to become the standard for nematode fixation in the near future.

Vaccinia virus mutations in the L4R gene encoding a virion structural protein produce abnormal mature particles lacking a nucleocapsid.

Electron micrographs from the 1960s revealed the presence of an S-shaped tubular structure in the center of the vaccinia virion core. Recently, we showed that packaging of virus transcription enzymes is necessary for the formation of the tubular structure, suggesting that the structure is equivalent to a nucleocapsid. Based on this study and on what is known about nucleocapsids of other viruses, we hypothesized that in addition to transcription enzymes, the tubular structure also contains the viral DNA and a structural protein as a scaffold. The vaccinia virion structural protein L4 stands out as the best candidate for the role of a nucleocapsid structural protein because it is abundant, it is localized in the center of the virion core, and it binds DNA. In order to gain more insight into the structure and relevance of the nucleocapsid, we analyzed thermosensitive and inducible mutants in the L4R gene. Using a cryo-fixation method for electron microscopy (high-pressure freezing followed by freeze-substitution) to preserve labile structures like the nucleocapsid, we were able to demonstrate that in the absence of functional L4, mature particles with defective internal structures are produced under nonpermissive conditions. These particles do not contain a nucleocapsid. In addition, the core wall of these virions is abnormal. This suggests that the nucleocapsid interacts with the core wall and that the nucleocapsid structure might be more complex than originally assumed. The vaccinia virus nucleocapsid has been neglected since the 1960s due to a lack of electron microscopy techniques to preserve this labile structure. With the advent of cryo-fixation techniques, like high-pressure freezing/freeze-substitution, we are now able to consistently preserve and visualize the nucleocapsid. Because vaccinia virus early transcription is coupled to the viral core structure, detailing the structure of the nucleocapsid is indispensable for determining the mechanisms of vaccinia virus core-directed transcription. The present study represents our second attempt to understand the structure and biological significance of the nucleocapsid. We demonstrate the importance of the protein L4 for the formation of the nucleocapsid and reveal in addition that the nucleocapsid and the core wall may be associated, suggesting a higher level of complexity of the nucleocapsid than predicted. In addition, we prove the utility of high-pressure freezing in preserving the vaccinia virus nucleocapsid.

Cryofixation rapidly preserves cytoskeletal arrays of leaf epidermal cells revealing microtubule co‐alignments between neighbouring cells and adjacent actin and microtubule bundles in the cortex

Accurate preservation of microtubule and actin microfilament arrays is crucial for investigating their roles in plant cell development. Aldehyde fixatives such as paraformaldehyde or glutaraldehyde preserve cortical microtubule arrays but, unless actin microfilaments are stabilized with drugs such as m-maleimidobenzoyl N-hydroxysuccinimide ester (MBS), ethylene glycol bis[sulfosuccinimidylsuccinate] (sulfo-EGS) or phalloidin, their arrays are often poorly preserved. Cryofixation, used primarily for electron microscopy, preserves actin microfilaments well but is used rarely to fix plant cells for optical microscopy. We developed a novel whole-mount cryofixation method to preserve microtubule and microfilament arrays within Tradescantia virginiana leaf epidermal cells for investigation using confocal microscopy. Cortical microtubule arrays were often oriented in different directions on the internal and external faces of the epidermal cells. A number of arrays were aligned in several directions, parallel to microtubules of neighbouring cells. Actin microfilaments were particularly well preserved possibly due to the speed with which they were immobilized. No transverse cortical microfilament arrays were observed. On occasion, we observed co-aligned microfilament and microtubule bundles lying adjacent to the plasma membrane and positioned side by side suggesting a potential direct interaction between the cytoskeletal filaments at these locations. Cryofixation is therefore a valuable tool to investigate the interactions between cytoskeletal arrays in plant cells using confocal microscopy.

Self‐pressurized rapid freezing (SPRF): a novel cryofixation method for specimen preparation in electron microscopy

A method is described for the cryofixation of biological specimens for ultrastructural analysis and immunocytochemical detection studies. The method employs plunge freezing of specimens in a sealed capillary tube into a cryogen such as liquid propane or liquid nitrogen. Using this method a number of single-cell test specimens were well preserved. Also multicellular organisms, such as Caenorhabditis elegans, could be frozen adequately in low ionic strength media or even in water. The preservation of these unprotected specimens is comparable to that achieved with high-pressure freezing in the presence of cryoprotectant. The results are explained by the fact that cooling of water in a confined space below the melting point gives rise to pressure build-up, which may originate from the conversion of a fraction of the water content into low-density hexagonal ice and/or expansion of water during supercooling. Calculations indicate the pressure may be similar in magnitude to that applied in high-pressure freezing. Because the specimens are plunge cooled, suitable cryogens are not limited to liquid nitrogen. It is shown that a range of cryogens and cryogen temperatures can be used successfully. Because the pressure is generated inside the specimen holders as a result of the cooling rather than applied from an external source as in high-pressure freezing, the technique has been referred to as self-pressurized rapid freezing.

The extraction, structure, and gelling properties of hybrid galactan from the red alga Furcellaria lumbricalis (Baltic Sea, Estonia)

The structure and composition of galactan from Furcellaria lumbricalis (furcellaran) were investigated in connection with rheological specificities, gel structure, and extraction conditions. The polysaccharide was characterized by 13C nuclear magnetic resonance (13C-NMR) and Fourier transform infrared spectroscopy, inductively coupled plasma-optical emission spectrometry, electrothermal atomization atomic absorption spectrometry, and gel permeation chromatography methods. The microstructure of polymer gels was studied using a cryofixation method in combination with freeze-drying and scanning electron microscopy (SEM) techniques. The undersulfated furcellaran backbone consists mainly of 3,6-anhydro-d-galactose (28.5–30.1%) and galactose residues, the latter being partly sulfated in positions 4 and 6, which give rise to some specific properties of the gel. Also, residues of 6-O-methyl-d-galactose as a minor component are found to be present. The water-extracted furcellaran with the average molecular weight about 290 kDa is rich in nitrogen, calcium, magnesium, and potassium, while the sodium content is rather low. The low sulfur content (5.3%) and 13C-NMR spectra refer to an undersulfated nature of this galactan. The extraction of seaweeds in low concentration alkaline solutions (instead of water) leads to a significant increase of the minimum size of the galactan particles and the value of gel strength (more than 12 times for Rb-containing gels). The properties of the gel are dependent on specific tentacle-like structure units present in furcellaran gels established by a high-resolution SEM.

Gel-forming structures and stages of red algal galactans of different sulfation levels

Sol-gel transition processes of algal galactans were studied using cryofixation method in combination with freeze-drying and scanning electron microscopy (SEM) techniques. The structures formed in successive stages of gelling process upon cooling were rapidly frozen at defined temperature points and viewed by SEM. It was established that in the case of both types of gelling galactans investigated, a fine honeycomb-like network exists for a wide range of solution temperatures. The formation and structure of this network depends on the structural type, gelling stage, and concentration of the galactan in solution. The honeycomb suprastructures exist also in carrageenan and agarose sols (at temperatures considerably exceeding the gelling temperatures). An additional helical network formed showed different behaviour in the case of carrageenan and agar-type polysaccharides. In the gel-formation process, tightening of the network takes place in both types of galactan gels; the honeycomb structures persist in carrageenan (furcellaran) but not in agarose gels.

Application of in vivo cryotechnique to the examination of cells and tissues in living animal organs.

When all biological materials in cells and tissues of living animal organs are quickly and promptly frozen, immunolocalization of their components and structural features in situ is necessary to understand their in vivo functioning states. However, these direct morphological analyses were difficult to achieve by conventional chemical fixation methods during the last century. A new cryofixation method, named the "in vivo cryotechnique", in which the normal blood circulation in living animals is always retained at the moment of freezing, has become a powerful tool to visualize the real native morphology of cells and tissues with functional meaning. The "in vivo cryotechnique" can usually be combined with a wide range of subsequent preparation techniques, and can thereby enable us to perform various direct analyses on biological samples, reflecting the physiological functions of living animal organs.

Immunocytochemical and stereological studies of the rat vestibular nucleus: optimal research methods using glucocorticoid receptors as an example

Many anatomists agree that stereological procedures for counting neurons are superior to simple density measurements because the latter are biased by the way that sections are sampled and the likelihood of selectively sampling neurons of particular sizes and orientations. Despite this, few stereological studies of the vestibular nucleus complex (VNC) have been conducted and all of the published immunocytochemical studies have used densitometry to quantify receptor numbers. One possible reason for this is the difficulty of combining stereological methods with immunocytochemical techniques required to label specific types of receptors. In this paper we detail the optimal methods for combining stereological and immunocytochemical techniques for the unbiased quantification of neuronal number, using glucocorticoid receptors as an example. We report that the cryofixation method is more effective than perfusion with paraformaldehyde, in order to obtain sections to estimate neuronal number using the Cavalieri and physical disector methods. Thionin proved to be the optimal counterstain for identifying both neurons and the boundaries of the VNC. The combination of stereological and immunocytochemical techniques offers an effective means to ensure accurate estimates of the number of neurons expressing a receptor.

Influence of aldehyde fixation on the morphology of endosomes and lysosomes: quantitative analysis and electron tomography.

Cryoimmobilization is regarded as the most reliable method to preserve cellular ultrastructure for electron microscopic analysis, because it is both fast (milliseconds) and avoids the use of harmful chemicals on living cells. For immunolabelling studies samples have to be dehydrated by freeze-substitution and embedded in a resin. Strangely, although most of the lipids are maintained, intracellular membranes such as endoplasmic reticulum, Golgi and mitochondrial membranes are often poorly contrasted and hardly visible. By contrast, Tokuyasu cryosectioning, based on chemical fixation with aldehydes is the best established and generally most efficient method for localization of proteins by immunogold labelling. Despite the invasive character of the aldehyde fixation, the Tokuyasu method yields a reasonably good ultrastructural preservation in combination with excellent membrane contrast. In some cases, however, dramatic differences in cellular ultrastructure, especially of membranous structures, could be revealed by comparison of the chemical with the cryofixation method. To make use of the advantages of the two different approaches a more general and quantitative knowledge of the influence of aldehyde fixation on ultrastructure is needed. Therefore, we have measured the size and shape of endosomes and lysosomes in high-pressure frozen and aldehyde-fixed cells and found that aldehyde fixation causes a significant deformation and reduction of endosomal volume without affecting the membrane length. There was no considerable influence on the lysosomes. Ultrastructural changes caused by aldehyde fixation are most dramatic for endosomes with tubular extensions, as could be visualized with electron tomography. The implications for the interpretation of immunogold localization studies on chemically fixed cells are discussed.

A New Concept and Machine for High Pressure Freezing

Abstract To date, high pressure freezing is the only reliable cryofixation method for vitrifying bulk biological samples up to 150μm in thickness (1). This proves the enormous potential of the method. Nevertheless, the procedure is not well accepted by the scientific community. It seems that people not completely convinced by the method have reservations to use high pressure freezing machines. The main reasons seem to be that the commercially available machines are noisy, large, very heavy, expensive and consume a lot of liquid nitrogen. Last but not least, a relatively large volume of cryogen is pressurized to 2000bar representing a potential risk of accident. Considering high pressure freezing to be a very potent method, it was tried to establish a new machine avoiding the above mentioned disadvantages. The first functional high pressure freezing apparatus was freezing fine metal rods with a boring of 0.2mm in diameter (2). This boring was filled with suspensions, the rod was connected to a pressure generator thus pressurizing the suspension, and simultaneously the metal rod was frozen from the outside by a low pressure jet of liquid nitrogen. The big disadvantage of the method was that only freeze-fracture experiments could be performed at the time because the sample could not be removed from the boring. As a consequence, this approach was never commercialised. In the mean time sample preparation has improved. Cellulose capillaries can be used to freeze suspensions and tissue samples (3,4). To freeze these capillaries in the new machine, they are introduced in a sample holder which consists of a copper tube with an inner diameter of 0.3mm and a length of 16mm. This tube fits into a concentrical stainless steel tube which stabilises the copper rod and helps to fix the sample in the cryofixation device.

Electron Tomography of Rat-Liver Mitochondria: Structure of Cristae and Interactions With Endoplasmic Reticulum

Abstract We are using electron tomography to investigate the internal organization of mitochondria and the interactions of these organelles with other cellular components. Two-axis tilt-series are recorded from 0.2-1.0-μm sections using either an AEI EM7 high-voltage or JEOL 4000 intermediate-voltage electron microscope. Reconstructions are computed using programs in SPIDER and visualized with the Sterecon system. Three-dimensional reconstructions of conventionally fixed and plastic-embedded rat-liver mitochondria (isolated and in situ) indicate that the cristae, the infoldings of the inner membrane, are pleiomorphic, defining compartments that are connected to each other and to the surface of the inner membrane by narrow (30-40-nm diameter) tubular regions (Fig. 1). This is contrary to the “baffle” model commonly shown in textbooks and has important implications for the diffusion of ions, metabolites, and macromolecules inside the organelle. The same basic organization is also observed in reconstructions of mitochondria in rat-liver sections prepared by the cryo-fixation method of Tokuyasu(Fig. 2).

A newly developed cryofixation appartus for physiological experiments and its application to squid photoreceptor cells

By cryofixation method for electron microscopy, structural change in squid photoreceptor microvilli accompanied by long period of light illumination has been studied by Tsukita et al., resulting that there are two states of the microvillar cytoskeletal structure under dark- and light-adapted conditions. In this paper we report the structural changes as a function of time after the light pulse application and their physiological significance.For this experiment, we have developed a new cryofixation device which can combine rapid freezing with physiological experiment2 (Fig. 1). The device cryofixes biological specimen at precisely specified time after the light illumination by driving a small silver block cooled down to 10°K with a computer-controlled actuator; that is, for cryofixation, the cooled silver block is pushed onto the specimen put on an experimental bench. Structural change in the microvilli is observed to take place for the specimens cryofixed at the time period over 50 msec after the light pulse is irradiated, but is not observed for the 20-30 msec light-illuminated photoreceptors (Fig. 2).