Agitation Modules: Flexible Means to Accelerate Automated Freeze Substitution.

For ultrafast fixation of biological samples to avoid artifacts, high-pressure freezing (HPF) followed by freeze substitution (FS) is preferred over chemical fixation at room temperature. After HPF, samples are maintained at low temperature during dehydration and fixation, while avoiding damaging recrystallization. This is a notoriously slow process. McDonald and Webb demonstrated, in 2011, that sample agitation during FS dramatically reduces the necessary time. Then, in 2015, we (H.G. and S.R.) introduced an agitation module into the cryochamber of an automated FS unit and demonstrated that the preparation of algae could be shortened from days to a couple of hours. We argued that variability in the processing, reproducibility, and safety issues are better addressed using automated FS units. For dissemination, we started low-cost manufacturing of agitation modules for two of the most widely used FS units, the Automatic Freeze Substitution Systems, AFS(1) and AFS2, from Leica Microsystems, using three dimensional (3D)-printing of the major components. To test them, several labs independently used the modules on a wide variety of specimens that had previously been processed by manual agitation, or without agitation. We demonstrate that automated processing with sample agitation saves time, increases flexibility with respect to sample requirements and protocols, and produces data of at least as good quality as other approaches.

Three-dimensional reconstruction methods for Caenorhabditis elegans ultrastructure.

The roundworm Caenorhabditis elegans is one of the major model organisms in modern cell and developmental biology. Here, we present methods for the three-dimensional (3D) reconstruction of the worm ultrastructure. We describe the use of (1) serial-section analysis, (2) electron tomography, and (3) serial block face imaging by scanning electron microscopy (SEM). Sample preparation for high-pressure freezing/freeze substitution (HPF/FS) has been extensively covered in a previous volume of this "Methods in Cell Biology" series and will only be described briefly. We will discuss these 3D methods in light of recent research activities related to worm and early embryo biology.

A new microbiopsy system enables rapid preparation of tissue for high-pressure freezing.

A microbiopsy system was developed to overcome long sampling times for tissues before they are cryo-fixed by high-pressure freezing. A commercially available biopsy gun was adapted to the needs of small-organ excisions, and biopsy needles were modified to allow small samples (0.6 mm x 1.2 mm x 0.3 mm) to be taken. Specimen platelets with a central slot of the same dimensions as the biopsy are used. A self-made transfer device (in the meantime optimized by Leica-Microsystems [Vienna, Austria]) coordinates the transfer of the excised sample from the biopsy needle into the platelet slot and the subsequent loading in a specimen holder, which is then introduced into a high-pressure freezer (Leica EM PACT; Leica Microsystems, Vienna, Austria). Thirty seconds preparation time is needed from excision until high-pressure freezing. Brain, liver, kidney and muscle excisions of anesthetised rats are shown to be well frozen.

Immunohistochemical analyses on albumin and immunoglobulin in acute hypertensive mouse kidneys by "in vivo cryotechnique".

The purpose of this study is to visualize topographical changes of serum proteins, albumin and immunoglobulin, passing through mouse glomerular capillary loops and their reabsorption in renal proximal tubules by immunohistochemistry in combination with our "in vivo cryotechnique". The "in vivo cryotechnique" was performed on left mouse kidneys under normotensive, experimentally acute hypertensive and heart-arrest conditions. The cryofixed tissues by the technique were routinely processed for freeze-substitution. Serial deparaffinized sections were stained with hematoxylin-eosine and immunostained with anti-mouse albumin, immunoglobulin G (IgG), kappa or lambda light chain and IgG1 heavy chain antibodies. Under the normotensive and heart-arrest conditions, albumin and IgG were clearly immunolocalized in blood vessels and slightly in apical cytoplasmic parts of some proximal tubules. Under the acute hypertensive condition, the albumin and kappa or lambda light chains, but not IgG1 heavy chain, were strongly immunolocalized in the apical cytoplasm of almost all proximal tubules. This study is the first in vivo visualization for glomerular passage of serum proteins and their transtubular absorption. Thus, the "in vivo cryotechnique" with freeze-substitution can be used for clarifying not only the functional morphology of living animal cells, but also in situ immunohistochemical localization of their components.

Retention of fluorescent probes during aldehyde-free anhydrous freeze-substitution.

Fluorescent probes are widely used for microscopy of live-cell processes, but few such probes can also be used with classically fixed or otherwise immobilized material, and none has been used without aldehyde fixation, which can introduce artefacts of structure and probe localization. Here we show that the fluorescence patterns in fungal hyphae loaded with chloromethyl aminocoumarin (CMAC), and then anhydrously freeze-substituted, without any aldehyde fixation, are similar to those seen in living hyphae. Probe loss into the mounting medium (Spurr's resin) with CMAC and five other probes tested indicated that some unwanted solubilization of probe occurred during embedding, but nevertheless vacuoles could be imaged by their retention of probe.

A new approach for cryofixation by high-pressure freezing.

A newly designed high-pressure freezing machine for cryofixation was established and tested (Leica EMPACT), based on ideas originally proposed by Moor & Riehle in 1968. The new machine, essentially an improved version of our prototype, pressurizes the sample to 2000 bar in a small container (using methylcyclohexane as hydraulic fluid) and at the same time cools the outer surface of the container with a jet of liquid nitrogen. The advantage of this approach is that the machine uses little liquid nitrogen and can be built small and light. The machine is able to vitrify and freeze well a variety of specimens, for example, plant leaves, yeast cells, liver or nerve tissue (more samples are shown at: http://www.ana.unibe.ch/empact). Cooling efficiency is the same as in the traditional machines that use liquid nitrogen to pressurize and simultaneously cool the sample.

Freeze-substitution: origins and applications.

Freeze-substitution is a physicochemical process in which biological specimens are immobilized and stabilized for microscopy. Water frozen within cells is replaced by organic solvents at subzero temperatures. Freeze-substitution is widely used for ultrastructural and immunocytochemical analyses of cells by transmission and scanning electron microscopy. Less well recognized is its superiority over conventional chemical fixation in preserving labile and rare tissue antigens for immunocytochemistry by light microscopy. In the postgenome era, the focus of molecular genetics will shift from analyzing DNA sequence structure to elucidating the function of gene networks, the intercellular effects of polygenetic diseases, and the conformational rearrangements of proteins in situ. Novel strategies will be needed to integrate knowledge of chemical structures of normal and abnormal macromolecules with the physiology and developmental biology of cells and tissues from whole organisms. This review summarizes the progress and future prospects of freeze-substitution for such explorations.

Cryofixation, cryosubstitution, cryoembedding for ultrastructural, immunocytochemical and microanalytical studies.

Cryofixation, cryosubstitution and cryoembedding are a set of low-temperature methods for immunocytochemical and microanalytical ultrastructural studies. This review covers the theoretical and practical aspects of these cryomethods, simple, low-cost, safe devices that provide reproducible results and a summary of recent results. Sections prepared by these three cryomethods can be used to determine elemental composition, molecular composition, functions and 3-D ultrastructure. The information obtained can be treated by multivariate statistical methods. Thus, each cellular compartment can be identified by its morphology, molecular and elemental composition and function and changes in these data during physiological and pathological processes can be monitored.

Programmable freeze-substitution and cryo-embedding device.

The construction and performance of a modular and fully controllable freeze-substitution device are described. The core of the device consists of a heavy brass block providing a large, stable thermal mass. The block is composed of two perforated plates and a base plate forming nine deep wells, which enable the concomitant substitution of several samples in various substitution fluids, and in large volumes. The wells are surrounded by an isometric network of tunnels through which either liquid nitrogen or hot air can flow. The isometric network enables heat transfer across short uniform distances throughout the entire block's volume, thus minimizing temperature gradients and differences. The temperature of the substitution fluid, rather than that of the metal block, is monitored by a programmable controller, enabling the presetting of any freeze-substitution regime.

A stimulus timing device for capturing fast physiologic events by quick-freezing.

A timing device was designed that, in conjunction with an impact type of quick-freezing apparatus and an externally-triggerable stimulus generator, allows the application of an electrical stimulus to a muscle preparation at a selected time interval before quick-freezing and the measurement of the interval with submillisecond precision. This is needed for stopping fast physiological events in calcium release and excitation-contraction coupling and allows studying the morphological parameters (by freeze-fracture and freeze-substitution) and elemental distributions (by x-ray microanalysis) as a function of time after stimulation. The device should be adaptable for use with most equipment designed for quick-freezing electrically excitable tissue by impact on a cold solid surface.