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

Summary

Obtaining high-quality transmission electron microscopy images is challenging, especially in the case of plant cells, which have abundant large water-filled vacuoles and aerated spaces. Tandem high-pressure freezing and quick freeze substitution greatly reduce preparation time of plant samples for TEM while producing samples with excellent ultrastructural preservation.

Abstract

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.

Introduction

Our knowledge of cell ultrastructure comes mainly from electron microscopy, which can resolve details in the range of a few nanometers ¹. Despite being so powerful in resolution TEM is not considered user-friendly, as sample preparation requires time-consuming and laborious protocols, and demands some expertise from the practitioner. Traditional fixation of samples has combined the use of aldehydes and osmium tetroxide before further processing that includes dehydration, embedding in resin and then sectioning to produce ultra-thin sections that are then stained with heavy metals. However, it is known that chemical fixation can produce artifacts including pr....

Protocol

NOTE: The QFS procedure requires extreme care and caution by the user and we highlight these safety precautions here as Cautions and Notes where applicable.

1.Preparation for HPF Run

1. Before beginning sample preparation, turn on the high-pressure freezer following manufacturer’s instructions.
   NOTE: The HPF unit used in this protocol is a Wohlwend Compact 02 unit (Figure 1A), and it takes approximately one to one-and-a-half hours of start-up procedure before HPF runs can commence.
   1. Switching on the Wohlwend Compact 02 High Pressure Freezer.
      1. Switch on the instrument by turning ....

Results

Results presented below have been obtained using a Wohlwend Compact 02 for HPF (Figure 1A). One major advantage of this instrument is the ease of use of the specimen carriers and its holders. When using other instruments, McDonald recommends that two users should carry out the sample preparation and HPF, one preparing the samples while the other does the freezing and transfer to the FS cryovials ⁹. However, the Wohlwend specimen carriers and holder are easy enough for a single user to manipula.......

Discussion

The success of the protocol presented here depends heavily on the user. First, advanced preparation is required to ensure that all necessary materials are readily available and in sufficient quantity to complete an entire HPF-QFS run. Second, the user must work quickly, moving from step-to-step in an efficient manner that minimizes sample handling, thus minimizing changes to the native state of the tissue. Once samples are frozen and before they are dehydrated it is imperative that they be kept cold, so care must be take.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Wohlwend HPF Compact 02 High Pressure Freezing Machine	Technotrade International, Inc	HPF02	With integrated oscilloscope to display freezing and pressure curves; PC (not included) is required for display  of freezing parameters
Holder for DN 3 x 0.5 mm aluminum apecimen carriers	Technotrade International, Inc	290
Specimen carriers, P=1,000, DN 3 x 0.5 aluminum, type A	Technotrade International, Inc	241-200
Specimen carriers, P=1,000, DN 3 x 0.5 aluminum, type B	Technotrade International, Inc	242-200
Storage Dewar 20.5 L, MVE Millennium 2000 XC20	Chart
Baker's yeast			The older the better, to avoid excessive gas (CO2) production
Tooth picks
Thermocouple data logger EL-USB-TC	OMEGA Engineering Inc.	OM-EL-USB-TC	Replacement battery purchased separately
Temperature probe	Electron Microscopy Sciences	34505
Heater block 12/13 mm
Rotary shaker	Fisher Scientific	11-402-10
Leaf punch - Harris Uni-core 2.00	Ted-Pella Inc.	15076
Pink dental wax	Electron Microscopy Sciences	72660
Cryogenic vials 2 ml	Electron Microscopy Sciences	61802-02
Methanol
Blow dryer
Dry ice
Liquid nitrogen
Acetone
Forceps			Several pairs