How to increase freeze-substitution and electron microscopy embedding reliability

1 Introduction

Freeze substitution [1]–[3], a technique historically associated with High Pressure Freezing (HPF) protocols, plays a crucial role in replacing the water content of vitrified biological specimens with resin. This process prepares the sample for subsequent embedding, facilitating room temperature electron microscopy observation or, in correlative microscopy projects, in-resin fluorescence microscopy.

Despite its extensive history of use, maintaining sample integrity during freeze substitution remains a persistent challenge. Risks such as sample pellet resuspension, damage to fragile materials, and sample aspiration during the numerous washing steps involved in freeze substitution and embedding have prompted the development of various flow-through chambers [4]. However, existing solutions fail to accommodate all HPF carriers and do not effectively prevent resuspension issues.

We addressed these challenges, devising a novel consumable equipment piece aimed at optimizing the reliability of freeze substitution steps and ensuring a high success rate in all HPF-FS experiments. This innovative tool, referenced as ‘the FS-basket’, is designed to securely trap the carrier, containing the sample, between two fine meshes, thereby preventing unwanted sample movements during fluid exchange steps. Comprising two separate parts that tightly interlock, with each supporting one mesh, the dimensions of the tool are tailored to accommodate 3 mm or 6 mm carriers, as well as CryoCapsules^® [5], and are compatible with widely available Nunc^® CryoTubes^®.

In this manuscript, we introduce the FS basket, detailing its usage and exploring its potential applications within the broader HPF-FS workflow. Moreover, we note its suitability for fragile sample handling in room temperature dehydration and embedding protocols, thereby demonstrating its versatility and utility in various microscopy methodologies.

2 Results

Freeze-substitution is widely recognized for its ability to preserve biological specimen ultrastructure better than conventional chemical fixation and room temperature embedding, which is crucial for electron microscopy. However, the delicate nature of samples often leads to fragmentation and loss during washing and embedding steps. As a result, researchers frequently resort to replicating samples to enhance the chances of successful observation under the electron microscope. This practice significantly increases both time and cost, contributing to the perception of a low success rate in High Pressure Freezing (HPF) workflows.

Manufacturers have attempted to automate this process, focusing on gentle fluid exchanges through robots and delicate flow-through chambers (e.g., Leica AFS-2 + FSP or microscopy innovations mPrep ASP). Nevertheless, handling the most fragile samples typically remains a manual task entrusted to experts who closely monitor the sample and strive to salvage it when detaching from the HPF carrier.

In response to these challenges, we developed a tool, the FS basket (CryoCapCell, #C-006c FS basket) with the following objectives:

1. Maintain sample stability throughout fixation, washing, dehydration, and embedding steps

2. Facilitate unhindered fluid exchange

3. Be compatible with commonly available cryo-tubes

4. Have minimal chemical reactivity with freeze-substitution cocktails

5. Enable manual handling, even in narrow and deep freeze-substitution chambers

6. Withstand the extreme cold temperatures of liquid nitrogen

We opted for a passive mesh that does not react with the chemicals commonly used in freeze-substitution. The support structure to which the mesh is affixed is resistant to solvents. To prevent sample resuspension and ensure optimal sample retention, we designed the A carrier to be enclosed between a meshed bottom element and a top element also affixed with a mesh, effectively sandwiching the sample between two meshes. This configuration ensures that the carrier remains centred within the assembly, securely held above and below by meshes of the specified pore size (Figure 1C-1, C-2).

Video 1: transferring the carrier to the FS basket at the unloading station of the HPM, and storage to a cryo-tube: https://www.youtube.com/shorts/AjeiiwuDuRM

Video 2: manipulating the cryotubes containing the FS basket, during freeze substitution and embedding: https://youtube.com/shorts/KGEGNgie90c

On the day of the experiment, we anticipate the number of samples to be vitrified and prepare FS basket assemblies accordingly. To ensure proper fitting in the liquid nitrogen environment, we pre-mount the assembly pair and store them adjacent to the unloading station (Figure 2B-1, B-2). Following an HPF shot, the assembly is opened, and the bottom/support piece is placed onto the liquid nitrogen cold unloading tray of the station (refer to Video 1). The sample is collected from the HPM clamp, and at this stage, identifying the A and B carrier is straightforward as no ice contamination has obscured the planchettes. The A carrier, with the sample facing upwards, is placed directly into the bottom element and enclosed with the pre-cooled top element (refer to Video 1). The assembly, now containing the sample, is transferred into a Nunc^® CryoTube^® of 1.8 mL or more (manufacturer/tradename: Nunc363401) preloaded with frozen FS solution. Warning: Nunc^® CryoTubes^® of 1 mL will not accommodate the height of the FS basket. Alternatively, a perforated Nunc^® CryoTube^® may be used for long-term LN2 storage of the sample.

After approximately 30 min in the freeze substitution apparatus, the FS cocktail is typically thawed. We recommend pushing the FS basket deep inside the Nunc^® CryoTube^® to ensure complete submersion of the sample. Following the ramp-up to rinsing steps, emptying the cryotubes can be achieved by gently tilting the tube over a waste recipient. The subsequent solution is immediately pipetted onto the tube to prevent sample dehydration (see Video 2). These steps can be repeated until the final embedding stage.

During these operations, the experimenter should pay attention to avoid the FS basket to slide out. The FS basket fits tightly to the Nunc^® CryoTubes^® and in our hands, tilt slightly sideways and do not fall off. In case of a slightly larger cryotube, and if the FS basket falls off, the sample remains protected by the meshes and a rapid transfer back into the cryotube should preserve the sample.

Depending on the sectioning strategy, the embedded sample may be extracted from the FS basket and transferred to the desired mold (e.g., silicon, beam capsule, flat embedding), or polymerized together with the FS basket inside the cryotube.

We use the FS basket as a consumable. Composed of polymers only, recycling is challenging as chemical components are hard to clear without mechanical action. We do not recommend recycling the product to avoid variability in sample preparation.

We have successfully tested embedding with various resins, including EPON Embed812 (EMS #14120 kit), LRWhite (EMS #14380 kit), and R221 (CryoCapCell # R221mono-step) [6], [7], using flat embedding or en-bloc polymerization strategies directly within the cryotube (Supplementary Figure 1). Our observations report no alteration of the contrast nor the embedding quality using the FS basket. The detailed sample preparation protocols are provided in F° Low [8] format as Supplementary Datasheet S2–24.

Beta tester customer also reported testing successfully EPON Embed812 (EMS #14120 kit) and LRWhite (EMS #14380 kit), but experienced issues using Agar-Low-viscosity-resin-kit (Agarscientific #AGR1078 - Spurr like). They reported that this resin caused denaturation of the FS basket, resulting in softening deformation after overnight exposure. As of the time of writing the manuscript, we have not identified the chemical compound that specifically caused this denaturation, therefore we do not recommend using the FS basket in its current form with Spurr-like resin.

3 Discussion

In 2021, CryoCapCell established a collaboration agreement with the INSERM Unit, U1195, aiming to develop high-standard procedures for electron microscopy. Our initial focus was on establishing live-HPF-CLEM and HPF workflows [6] (HPM Live µ, CryoCapCell) and implementing freeze substitution processes (FS 8500, RMC) to advance our electron microscopy capabilities. However, we encountered a significant challenge when needing to embed samples vitrified in CryoCapsules^® or 6 mm carriers, as existing tools were primarily designed for 3 mm carriers, rendering them incompatible with our requirements. This limitation was particularly problematic when training a large group of next-generation electron microscopy users unfamiliar with cryo-methods.

Although we attempted to explore homemade solutions, their lack of reproducibility resulted in sample losses equivalent to those experienced with previously available tools. Consequently, we adopted an industrial approach to develop a standardized piece of equipment, applying production strategies commonly used in industry. The concept, illustrated in Figure 1, entails depositing a mesh with pore sizes smaller than the sample chunks below the carrier to maintain a tight seal while facilitating fluid exchange from below.

Figure 1:

3D representation of the FS basket. A-1: side view of both FS basket elements. A-2: top view of both FS basket elements. B-1: sectioned side view of both elements of the FS basket. A 6 mm carrier containing a figurative fish is represented to give a sense of scale. B-2: sectioned top view of both elements of the FS basket. C-1: sectioned side view of the assembled FS basket, holding a 6 mm carrier containing a figurative fish. C-2: sectioned top view of the assembled FS basket.

During the introduction of our novel tool to the market, we discovered that other scientists had independently arrived at similar conclusions and proposed handmade materials with a similar concept. However, our tool offers distinct advantages, primarily in its ability to tightly maintain the sample within a framed area while allowing for easy fluid exchange. The absence of carrier movement is particularly beneficial for large samples, facilitating recurrent orientation in multimodal imaging strategies such as live CLEM or biopsy orientation. Furthermore, this lack of movement is crucial in Quick Freeze Substitution protocols [9], as it mitigates the risk of the carrier acting as a grinder to the sample if detached during agitation. Figure 2 presents the produced material and its tight adjustment to the CryoCapsule^® (Figure 2C-2), or 3 and 6mm carriers (Figure 2C-3). A closer view of the fine mesh is also visible (Figure 2C-1).

Figure 2:

Real view of the FS basket. A-1 side view of both FS basket elements. A-2 top view of both FS basket elements. B-1 side view of the assembled FS basket. B-2 top view of the assembled basket. B-3 bottom view of the assembled basket. Arrow is pointing to a CryoCapsule held by the mesh. C-1 top view of the bottom element of the FS basket. The mesh is clearly visible. C-2 top view of the bottom element, and presence of a CryoCapsule to illustrate dimensions of the entire system. C-3 top view of the bottom element with a 6 and a 3 mm carrier. Scale bars 5 mm.

4 Conclusion

Although modest and simple, we hope that this novel device will minimize sample loss during freeze substitution and optimize associated costs and time expenses on electron microscopy studies.