In Situ Nanoindentation at Elevated Humidities

Nanoindentation is one of the most widespread methods to measure the mechanical performance of complex materials systems. As it allows for local characterization of composite architectures with sub-micron spatial features and a large range of properties, nanoindentation is commonly used to measure the properties of biological materials. In situ nanoindentation, a further development of the approach, is a powerful tool for the analysis of plastic deformation and failure of materials. Here, samples can be mechanically manipulated using the indenter, while their behavior is monitored with the resolution of a scanning electron microscope (SEM). Indeed, numerous studies demonstrate the potential of this approach for studying the most fundamental material characteristics. However, so far, these measurements are performed in high-vacuum conditions inherent to the conventional electron microscopy method, which are irrelevant when studying biological structures that evolved to perform in hydrated conditions. In this work, the ability to conduct nanoindentation experiments under controlled humidity and temperature inside an environmental SEM is developed. This technique has the potential to become crucial for materials design and characterization in many domains where humidity has a signiﬁcant impact on performance. These include organic/polymer systems, microelectronic and optoelectronic devices, materials for catalysis, batteries, and many more.


Introduction
[3][4] These include, for example, the highly mineralized shells of mollusks, corals, endoskeletons, and teeth of numerous organisms, the cell walls of a number of unicellular algae species, as well as purely organic architectures, such as cuticles in arthropods, plant and wood materials, and many more.[7] Ultimately, these composite architectures, grown under strict biological regulation in a so-called bottom-up manner and comprising mechanically modest building units, exhibit overall performance that is in many ways superior to engineered composite structures. [8,9]ignificant efforts are being made to understand the principles of the mechanical functionality of biological materials.These include multi-scale mechanical characterization of the materials from the level of a single component to the level of the performance of the entire tissue. [7,10]13][14] Nanoindentation is one of the most commonly used techniques to study the mechanical functionality of biological materials. [15]It allows for local mechanical characterization of complex composite structures with sub-micron spatial features and a large range of properties-ideal for gaining a comprehensive understanding of the performance of biological materials.In biomineralized tissues, for example, the elastic modulus of the mineral building units can reach a value of up to 100 GPa, while the modulus of the thin organic membranes that hold the structure together is two orders of magnitude smaller. [16,17]owever, biological materials are the result of billions of years of evolution under a number of driving forces and constraints, one of which is the immediate environment in which the material was formed.Therefore, when attempting to understand the mechanical behavior of a biocomposite, it is crucial to consider the habitat in which it evolved to perform.Intuitively, the properties of a dry wood material are significantly different from those of a fresh one.Indeed, in order to understand the origin of the mechanical behavior of wood, one should measure its properties hydrated.The same argument applies to absolutely all biocomposites.Furthermore, probing their properties under various hydration levels has the potential to provide a mechanistic understanding of the actual role of water in their behavior. [16,18]21] Recently, our group developed a platform in which all nanoindentation-based methods can be performed in a controlled environment, including the regulation of temperature and humidity. [22]This capacity already provided a number of insights into moisture-induced mechanical performance of a number of biological systems. [7,16]Furthermore, it allowed to perform nanoindentation measurements of various classical materials systems under well-regulated ambient conditions, which is of high importance in many technological applications and standardization practices.However, so far, in situ nanoindentation, namely, nanoindentation experiments performed inside a scanning electron microscope (SEM), could only be performed in high vacuum.
In situ nanoindentation is a powerful tool for the analysis of plastic deformation and failure mechanisms of materials. [23]ere, samples can be mechanically manipulated using the diamond indenter tip, while its behavior can be monitored with the resolution of an SEM.[26] However, inherently to the conventional electron microscopy method, so far, all these stud-ies were performed in high vacuum conditions, [27][28][29] which as pointed out above, are irrelevant when studying biological structures that definitely did not evolve to perform in space.
In this work, we develop an instrumental ability to conduct nanoindentation experiments in controlled humidity from absolutely dry to an elevated humidity in the range of 0-100% of relative humidities (RH) and up to full immersion in water in situ, i.e., inside a scanning electron microscope.The capacity of this method to measure the mechanical properties of biological tissues is demonstrated on three model systems: wood material taken from the spruce Picea abies, the diatom cells Craspedostauros australis, and silver fir needles from Abies alba.

Humidity Stage Assembly
Observation of hydrated samples using environmental scanning electron microscope (ESEM) is a standard practice in a variety of domains where the studied materials must remain hydrated.Using this technique, hydration of the samples is achieved by allowing water molecules that are introduced into the microscope chamber to condense on the sample stage that is being cooled using a Peltier element.Here, relative humidity around the sample is accurately maintained by controlling the temperature of the stage and the water pressure inside the microscope.In this work, a similar approach was used to construct a sample stage for the in situ nanoindenter system (Figure 1A,B).
To avoid designing an independent software that is capable of controlling stage temperature and microscope chamber pressure by interfacing it with the control software of the instrument, our experimental set-up was based on redesigning the cooling stage provided by the manufacturer of the microscope, in this case, Thermo Fisher Scientific (Figure 1C).Three major redesign considerations had to be taken into account.
First, the heat sink that houses the Peltier element provided by the manufacturer is too heavy for the nanoindenter stage.Its main goal is to remove the thermal energy that is generated by the thermoelectric Peltier element, which is being cooled by transferring heat from its one side to another when a voltage is applied.Basically, when the sample stage side of the element is cooled, the other side becomes hot.However, the piezo-controlled motors that are designed to move the sample on the nanoindenter cannot handle the weight of the device.As the nanoindentation experiments are not planned to be performed in negative temperatures, the large heat sink was not necessary for our experiments.Therefore, the Peltier element, extracted from the original stage (Figure 1C) was mounted on a much smaller custom copper heat sink (Figure 1A,B).The entire water cooling infrastructure remained the same and was used to stream cold water through the copper base instead of the bulky metal heat sink provided by the manufacturer.
Second, the reduction of the stage size was necessary in order to be able to bring the sample as close as possible to the detector that is mounted on the end of the electron gun (Figure 1B).Imaging resolution in ESEM degrades very significantly by increasing the sample-detector distance in the presence of water molecules in the chamber. [30]The higher the necessary relative humidity is, the higher is the chamber pressure that is required to maintain it.
Finally, the sample stage mounted on the cold side of the Peltier element was encapsulated by a flat Teflon plate having the same lateral dimensions as the copper heat sink.The role of the Teflon is to provide mechanical stability to the assembly while preventing temperature exchange with other parts of the device, thus, optimizing temperature control over the sample stage.In all standard in situ nanoindenters, the sample is placed with its studied surface parallel to the electron beam, i.e., perpendicular to the horizontally mounted indenter tip (Figure 1B).Therefore, in order to be able to image the sample during experiments, the indenter stage is usually tilted by 10-15 degrees, allowing the electron beam to raster the surface of the sample.To prevent the Teflon part from crashing into the detector, the upper part of the plate was cropped at 15 degrees with respect to the upper part of the stage (Figure 1B).
The final humidity stage/nanoindenter assembly mounted on the stage of the microscope is depicted in Figure 1D,E.Altogether, at a stage tilt of 10 degrees, we were able to generate a significant secondary electron contrast from all the studied specimens at a variety of relative humidifies, sample-detector distance of ≈7 mm, and a stage temperature of 4°C.More detailed information on stage design can be obtained by contacting the authors directly.

Results
Wood material is an ideal model system to demonstrate our ability to control the relative humidity around the sample and consequently, its properties.It was also previously used to develop the platform for lab nanoindentation in controlled humidity and temperature. [22]Wood consists of elongated cells having a number of layers: the primary outer cell wall and three secondary cell wall layers, of which the second layer, called S2, is by far the thickest one (Figure 2A,B).Whereas these layers contain lignin reinforced by cellulous fibrils, the different cells are bonded together by the middle lamella that contains lignin primarily.In this work, wood material was used to demonstrate our ability to image the sample at high relative humidity.
A free standing and polished sample of spruce wood was mounted on the developed humidity stage and indented at various atmospheres, at 50%, 70%, and 90% RH (Figure 2B,D, respectively).Sample cutting and polishing was performed perpendicular to the long axis of the cells (Figure 2A).Indentation of the S2 layer at different atmospheres using a maximal load of 500 μN demonstrated a trend expected from systematically increasing the relative humidity.Similar measurement can easily be performed in a platform nanoindentation system. [22]owever, these experiments were used to show the influence of humidity on indentation imaging inside the ESEM.Comparing 90% RH (Figure 2D) to 50% RH (Figure 2B), the reduction in image quality is clearly evident.However, due to the design of the stage, we were able to maintain a distance of 7 mm from the sample's surface and to resolve the middle lamella even at a relatively high water molecules pressure.Corresponding videos of the indentation process taken at 50%, 70%, and 90% RH are presented in Videos S1-S3 (Supporting Information), respectively.
Further experiments were performed to demonstrate that in situ indentation can also be performed while the sample remains to be fully immersed in water.For this purpose, relatively large diatom C. australis cells were placed on a thin glass slide together with the aqueous solution in which they were dispersed (Figure 3A).Diatoms are unicellular algae that form an intricate cell wall made of amorphous silica.Whereas, recently, a number of studies were performed to measure the properties of the cell wall using nanoindentation, [28,31,32] none of these experiments were carried out under hydrated conditions.
During the experiment, an atmosphere of 98-100% RH humidity was maintained.Due to the high humidity conditions, the cells remained immersed in water (Figure 3B).The cells could still be easily located inside the microscope and indented with a maximal force of ≈18 μN.As can be seen in a representative loaddisplacement curve (Figure 3E), despite the low applied force and the high humidity, the measurement remained extremely stable.
Moreover, as it is evident from the SEM images taken during indentation, water also condenses on the diamond tip but did not interfere with the measurement (Figure 3B).A corresponding video of the indentation process is presented in Video S4 (Supporting Information).
Finally, silver fir needle was used to demonstrate the capacity to perform mechanical manipulation of a fully hydrated organic tissue while observing the deformation process in high resolution using the SEM (Figure 3C).Here, the needle was sliced perpendicular to its long axis and mounted on the stage with its exposed surface toward the electron beam.Indentation was performed at 98% RH.This way the material remained hydrated without water condensing on the tip and the sample.As the imaging was performed perpendicular to the indented surface, the deformation process during indentation could be observed without any interference from the diamond tip.
Initially, low force nanoindentation with a maximal load of 500 μN was performed to probe the surface of the silver fir needle and to test our ability to image the mechanical deformation process while indenting at an elevated humidity (Figure 3F; Video S5, Supporting Information).Then, a larger force of 4000 μN was applied while the deforming tissue was imaged at higher magnification (Figure 3G; Video S6, Supporting Information).In both cases stable load-displacement data was successfully recorded while the deformation was recorded using the SEM.

Conclusion
The developed instrumentation described in this work provides a unique capacity to probe the mechanical performance, or to manipulate materials at controlled environmental conditions in situ, i.e., inside an environmental scanning electron microscope.The experiments can be carried out while recording the load displacement curves and imaging the deformation process with high resolution provided by the electron microscopy method.
Whereas, in this work, as a proof of concept, only biological materials were used, this technique has the potential to become crucial for materials design and characterization in many other domains where humidity has a significant impact on performance.These include synthetic organic systems, polymer systems, microelectronic and optoelectronic devices, materials for catalysis, batteries, and many more.

Experimental Section
All experiments were performed using PI85 SEM Piconindenter (Hysitron/Bruker) installed on a Scanning Electron Microscope Quanta 650 FEG (Thermo Fisher Scientific).

Figure 1 .
Figure 1.Humidity stage for in situ nanoindentation.A,B) Schematic representation of the stage design; C) Humidity sample holder for an ESEM provided by Thermo Fisher Scientific; D,E) In situ humidity stage mounted on an indenter inside the ESEM.

Figure 2 .
Figure 2. In situ nanoindentation of wood material.A) Secondary electron image of the polished surface of a sample taken from a spruce wood and a cubic diamond indenter tip above it; B-D) Secondary electron images of single cell nanoindentation performed at 50%, 70%, and 90% RH, respectively.Corresponding indentation videos are presented in Videos S1-S3 (Supporting Information), respectively.

Figure 3 .
Figure 3.In situ nanoindentation of diatom cells and silver fir needles.A) Light microscopy image of diatoms C. australis cells; B) Secondary electron image of nanoindentation performed on a single C. australis diatom cell covered with water; C) An image of a fresh silver fir branch taken from Abies alba; D) Secondary electron microscopy image of nanoindentation performed on the needle taken from the silver fir branch in (C); E) Representative load-displacement curve recorded during in situ nanoindentation of a single diatom cell at RH of 98%.A corresponding indentation videos is presented in Videos S4 (Supporting Information); and F,G) Representative load-displacement curves recorded during in situ nanoindentation of a silver fir needle at RH of 98% with a maximal load of 500 and 4000 μN, respectively.Corresponding indentation videos are presented in Videos S5 and S6 (Supporting Information), respectively.