# Development and validation of an interface for automated image acquisition during high-temperature environmental scanning electron microscopy experiments

J. Lautru, R. Podor

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J. Lautru, R. Podor. Development and validation of an interface for automated image acquisition during high-temperature environmental scanning electron microscopy experiments. Micron, 2026, 202, pp.103980. 10.1016/j.micron.2025.103980. hal-05422624

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Distributed under a Creative Commons Attribution 4.0 International License
Development and validation of an interface for automated image acquisition
during high-temperature environmental scanning electron microscopy
experiments.

J. Lautru and R. Podor*

ICSM, CEA, CNRS, ENSCM, Univ Montpellier, Marcoule, France

*Corresponding author: renaud.podor@cea.fr

**Abstract**

An interface that enables automatic image acquisition during high-temperature experiments in an environmental SEM is developed. It is optimized to work on multiple regions of interest at multiple magnifications, performing image focusing (focus and astigmatism) and automatic re-centering of regions of interest. Its operation has been validated by monitoring two regions of interest of a nickel-based superalloy undergoing oxidation at 950°C at different magnifications. Recording series of images at different magnifications on different regions of interest makes it possible to qualify the behavior of different areas of the sample in a single operation and/or to validate the reproducibility of the observations.

**Introduction**

High-temperature experiments using scanning electron microscopy (SEM) have been used since
the 1970s to study the behavior of materials under extreme conditions. [1,2]. One of the major
limitations of conventional SEMs is the need to work under high vacuum. Therefore, in most of the cases studied, the experiments were carried out under vacuum. The localized supply of gas via injectors positioned close to the sample surface made it possible to overcome this intrinsic limitation in SEM operation [3,4,5]. However, the issue of precise control of gas pressure in the sample environment has always remained a significant instrumental limitation [6,7]. The development of environmental or variable pressure scanning electron microscopes (VP-SEM or ESEM) at the end of the 1970s opened up new possibilities [8,9]. The ESEM is designed to operate between 10 and 2,400 Pa of gas in the chamber. It is equipped with a specific detector for secondary electrons known as 'environmental' electrons (Gaseous Secondary Electron Detector, or GSED), which enables electronic images to be recorded in the presence of gas. Combining ESEM with a furnace makes it possible to conduct high-temperature experiments under controlled gas pressure (any type of gas can be used) directly in the ESEM chamber. This technique has been widely used over the past two decades to study the numerous physical and chemical transformations associated with heating a sample [10].

The principle of these experiments is to continuously record images of the same area of the sample in order to visualize (and then analyze) local morphological changes in a comprehensible/intelligible manner. When conducting these experiments, ESEM operators have two options. The work can be carried out manually with regular adjustment of operating conditions (focus, contrast/brightness, others). In this case, magnification values can be modified according to the operator's needs. This operating mode is limited by the operator's ability to work continuously, which is usually a few hours at most. Another mode, known as 'automatic', is also available on the latest generations of SEMs. The parameters set by the operator are then fixed, and the images are automatically recorded by the SEM control computer. This option is interesting because it allows images to be recorded without an operator, but it has many limitations that make its use unpredictable in the long term and at high magnification. The focus, astigmatism and contrast/brightness settings are not changed during the experiment. They may change over time and lead to a gradual deterioration of the recorded images. In addition, sample drift may occur over time due to sample reaction (contraction related to a sintering process, expansion due to a chemical reaction, change in the position of the region of interest (ROI) due to prolonged exposure of this area to the electron beam compared to the rest of the sample), which then results in loss or degradation of the observation region (it is no longer in the field of automatically recorded images). These processes are all the more pronounced when the magnification at which the images are recorded is high. Finally, this solution proposed by the manufacturer only allows images to be recorded in a single area of interest, which limits the possibilities for observation and does not allow the reproducibility of the processes observed on different areas of the sample surface to be verified. It
should be noted here that, even in 'manual' mode (where the operator records images continuously), observation of different areas of the sample surface is rarely carried out because it requires numerous additional adjustments that are tedious to perform over the 'long term' and can lead to the loss of areas of interest, or even key moments in the transformations if these are rapid.

To overcome all these limitations, we propose developing an image recording automation interface that addresses all of the issues listed above. This interface will be used to validate its functionality and the value of its features in relation to experiments conducted under normal conditions, by studying the oxidation of a nickel-based superalloy.

## Description of the interface developed

The objective of this interface is to continuously capture SEM images at different magnifications, with automated centering of the region of interest, automatic refocusing and astigmatism correction, automatic adjustment of contrast and brightness values, and the ability to work on different regions of interest. The interface is written in Python. The 10 libraries required for the interface to run are os, tifffile, autoscript, numpy, matplotlib, time, datetime, tkinter, threading, and re. The code comprises 983 lines, corresponding to 39 kb of memory space. It has not been optimized for speed, as priority has been given to stability and safe handling of the microscope. The code will be made available upon request to the authors. The ESEM (ThermoFisher Quattro S) is controlled using AutoScript interface software (ThermoFisher). A library of functions for controlling the microscope is provided by the ESEM manufacturer (ThermoFisher).

The various parameters taken into account and operations carried out by the interface are as follows.

### Image recording conditions

The image recording conditions are defined prior to the user launching the image acquisition interface, via a dialogue interface. One to five ROIs can be defined for each experiment. The number of regions of interest has been limited to 5 so that there is not too much 'blind' time between two consecutive series of images recorded on the same ROI. Each time the position of a new ROI is acquired, a series of images at different magnifications (maxMAG/1, maxMAG/2, maxMAG/4, maxMAG/8, where maxMAG is the maximum magnification used in the image series) is recorded to allow for image drift correction during the experiment. Two types of drift correction are used: the first one uses a shift of the stage position (above 10 µm cumulated drift) and the second one uses an
electron beam shift (below 10 µm cumulated drift). The offset image correction is obtained using the
default cross-correlation template matching based on OpenCV library.

The number of image recording cycles during which the reference images used to recenter the
position of each ROI is defined by the user. For example, it may be decided to use the same reference
image for 10 cycles before acquiring a new one.

The magnification change cycle that will be used when acquiring images for each ROI is defined
(see the following paragraph for details). During the image acquisition cycle, the drift in the position
of the region of interest will be corrected using a stage and / or beam shift. The position of the stage
will be updated, and then the image will be recorded for each magnification defined for the current
image cycle.

The scan speed and the number of images accumulated to record an average image are also
defined through this user interface. These parameters will be used to record all images in the image
series.

The choice to automatically adjust the beam parameters (focus and astigmatism) and the time
delay between two consecutive images are defined in this communication interface.

It should be noted here that it is possible to modify these parameters at any time during the
acquisition of the image series by stopping and then restarting the interface via the user dialog
interface.

## Details of the protocol for defining image recording frequency

The magnifications at which the images will be recorded are defined by a divisor factor *d*. For
example, if the image at the highest magnification is (as an example) X16000, and the interface starts
with this initial magnification, the associated factor *d* is 1 in the interface. A factor of 2 leads to a
magnification of X8000, a factor of 4 to X4000, etc. The image series are defined by the series of
factors *d*, where *d* can take any positive value. For example, the series {1, 2, 4, 8, 2, 16, 32, 64} allows
(if the first image is recorded at X16000) a series of images to be recorded at different magnifications
(X16000, X8000, X4000, X2000, X8000, X1000, X500, X250). It is not necessary to start with a factor *d*
equal to 1, as the first magnification read by the interface when communicating with the microscope
is associated with a factor *d* equal to 1. Repeating a factor *d* within the same series means recording
more images at a given magnification relative to the total set of images. The focus and astigmatism
adjustment of the images, as well as the ROI drift correction, are performed when *d* = 1, if this option
is enabled.
For very long recording series (several days or weeks) and/or slow transformations, a recording delay between two images can be set in advance by the operator to limit the number of images recorded. This helps to limit any degradation of the ROI due to the electron beam effect and prevents the image storage space from becoming saturated.

## Defining multiple regions of interest (ROI)

When running the interface, the operator is asked to define different ROIs to which the previously defined image recording sequences will be applied. It should be noted that during this time, images on other ROIs are not recorded. Therefore, it may be counterproductive to increase the number of ROIs, as morphological transformations may occur without being recorded.

## Adjustment of electron beam conditions

Focus and astigmatism are adjusted automatically each time the operation described in the following paragraph on ROI drift correction is performed. Autofocus method is based on the classical approach based on maximization of high frequencies in image using Fast Fourier Transform. It is possible to disable this operation in the user dialog interface, as it is quite time-consuming and may limit image capture in conditions where rapid image capture is required.

Contrast/brightness adjustment is not performed automatically. In environmental mode and at high temperatures, this adjustment is performed using three parameters: contrast, brightness and bias. The functions that allow these three parameters to be managed automatically and independently are not available in the function library provided by the SEM manufacturer. The decision not to automatically adjust the contrast/brightness settings on images was also made in order to preserve contrast variations that can contain information. In fact, a backscattered electron detector operating at high temperature can be coupled with the ESEM's high-temperature mode [11]. In this case, contrast variations reflect changes in the composition of certain areas of the sample, and can only be exploited if they are not corrected.

## Recognition and centering of the region of interest

When the interface is launched, reference images of each ROI are recorded at four magnifications corresponding to factors equal to 1, 2, 4 and 8. They are stored in a dedicated folder. These reference images will be used $x$ times (the number $x$ is defined by the user at the start of the
experiment). The reference images are refreshed after x complete image capture cycles (i.e. when all images at all selected magnifications have been recorded).

When the interface is started, if there is a change in ROI, ROI recognition and centering is performed by comparing the stored reference images with the new ROI images stored with factors d equal to 4, 2 and then 1. This initial centering is performed by repositioning the stage for factors d equal to 4 and 2. Repositioning is then performed with a beam shift when the factor is equal to 1.

Once the ROI has been realigned, the centering is then performed only on the images recorded at the highest magnification (factor d equal to 1). If, in the series of defined factors d, it does not start with one, then this operation will only be performed when the interface is on d = 1. In this case, the displacement is corrected by a beam shift and, if this is too far off, the position of the stage is adjusted to compensate. The details of the ROI reference image are compared with the new recorded image using a function provided in the library supplied by the SEM manufacturer.

The conditions for recording reference images are the same as those determined for all images in the series. After a complete cycle of image recording on the same ROI (all d values are scanned), the stage is moved to the next position. The 'new' ROI is recognized, centered, and the position on the stage is updated. New reference images are recorded if the number x of image recording cycles is reached.

## Image storage

After each acquisition, the images are automatically saved. A folder is created for each ROI. Within this folder, a subfolder is created to store the reference images. Other subfolders associated with each magnification value defined by the d factor are created to store the images saved at each magnification. The images are automatically incremented by steps of 1. This numbering will be resumed if the interface is stopped by the user.

## Interface stop/restart

The operator can decide to stop the interface at any time to modify the image recording conditions. The interface supports two stop modes.

* By default, the interface stops after completing a full loop of image recording on all marked ROIs.
* If necessary, the operator can stop the interface after saving the image being acquired. The last images in the current series will not be saved.

The image recording conditions can be modified directly in the dialogue window and via the dialogue interface with the microscope (high voltage, beam current, scanning speed). When the interface is restarted, the user must check that the sample is correctly positioned on the first ROI. After validation, the interface resumes recording images with the new parameters on the first ROI, continuing the image numbering and saving them in the folders already created. The reference images are updated after a user-defined number of zoom cycles has been completed.

These various operations are carried out according to the workflow reported in Figure 1. The interface window is shown in Figure 2. Comments explain the purpose of the different fields and associated functions. The successive operations to be performed by the user are described from 1 to 10.

In practice, the preparation time for the experiment is approximately one to a few minutes (locating the ROIs if necessary). Next, the recording time for a series of images on a particular ROI depends on the image recording conditions selected. It can vary from a few seconds to several minutes. If the 'auto-focus / auto-stigmator' option is selected, this operation takes at least 40 seconds. During the experiment, the temperature curve is plotted and recorded automatically.
<figure><img src="image_2.png" /><figcaption>Figure 1. Workflow of the interface dedicated to controlling the ESEM and automated high-temperature image recording</figcaption></figure>
<figure><img src="image_3.png" /><figcaption>Figure 2. Interface window for controlling the ESEM at high temperatures and recording image series at different magnifications and in different areas. The comments explain the purpose of the different fields and buttons.</figcaption></figure>

## Interface functionality validation

A specific experiment that involves observing the first hours of oxidation of an experimental Ni-based superalloy (called Tropea [12]) and the formation of the protective oxide layer was carried out in the high-temperature ESEM. The surface of the sample (1 mm x 3 mm x 0.5 mm thick) was polished with colloidal silica. The oxidation heat treatment was carried out at 960°C for 15 hours, under 20 Pa of air. A high-temperature backscattered electron detector called Karmen (provided by the Crytur Company) was used to form the images [11].

The images were recorded as soon as the isothermal plateau was reached under the following conditions: images of 1536 x 1024 pixels, integration of 4 images recorded at 1 µs, magnifications of 8000, 2000 and 500X. Two distinct regions of interest were observed. One corresponds to the raw alloy (position 1), the other to an area where manufacturing defects were detected after oxidation began (position 2). During oxidation, these two areas will likely exhibit different behaviors, which can be studied in parallel using the interface developed and presented in this paper. The images corresponding to the two areas, recorded at different magnifications, are shown in Figure 2.
<figure><img src="image_4.png" /><figcaption>Figure 3. Selection of images from the series of images automatically recorded with the dedicated interface, on the 2 ROIs, at three different magnifications (8000X, 2000X and 500X).</figcaption></figure>

A set of images showing the oxidation behavior of the two ROIs observed was recorded, thus validating the correct functioning of the automated image recording interface over a period of 22 hours. The experiment was not extended beyond this duration because it did not provide any additional relevant information to validate the functioning of the interface. The series of images were edited into movies that are included in Supplementary Information SI1a, b and c (for the healthy area) and SI2a, b and c for the defect-containing area.

The recorded movies and selected images shown in Figure 3 demonstrate that the two areas exhibit different oxidation behaviors. In the healthy alloy area, a protective layer of NiO is established
as soon as the plateau temperature at 950°C is reached. It is formed of oxide grains whose diameter
increases steadily as the sample is kept at temperature. The main information concerning this ROI
(position 1) are obtained from the images recorded at the highest magnification. On this ROI, images
recorded at lowest magnifications show that the oxidation process is homogeneous on the whole
observed area. A layer of nickel oxide develops as soon as the isothermal plateau is reached and
gradually develops as the sample is kept at temperature, until it completely covers the surface of the
sample. In the defect-containing area (position 2), a complex microstructure consisting of various
oxides of Ta, Mo, W, Cr and Ni develops and covers an area of several tens of µm² (observable at the
highest magnification images – white box). The images recorded at lower magnification allow the
extent of the defect area and its evolution over time to be visualized, in comparison with the healthy
area of the alloy. Finally, on the images recorded at the lowest magnification, an area known as a
burn area (which corresponds to the onset of eutectic melting during alloy production and results in
a localized defect measuring a few µm to a few tens of µm [13]) can be observed. It appears white in
the images (because it contains heavy elements such as Mo and W). An enlarged insert of this area is
shown in the images of position 2 recorded at 500x magnification. The oxidation behavior of the alloy
in this zone is very different from that observed on the position corresponding to the healthy alloy.
As the purpose of this work is not to describe in detail the different stages of oxidation of the Tropea
alloy, this part will not be discussed further in this document. More information can be found in
several studies [14,15]. Dedicated articles are currently under preparation [16,17].

Discussion

The main information provided by the recording of these series of images is as follows.

Automated control of the ESEM during experiments conducted at high temperatures enables
quasi-simultaneous imaging of different ROIs on the sample surface. This enables additional
information on these areas to be acquired in a single experiment. The behavior of any
heterogeneities present in the material can be characterized, and the reproducibility of observations
made on a given area can be validated.

During this experiment, the location of the two ROIs was maintained. The maximum offset
recorded between two consecutive images taken from the same ROI was 900 nm over the course of
the twenty-four-hour validation experiment. This value corresponds to a loss of information of
approximatively 6% on the image series recorded at magnification X8000.
The sequence of images recorded at high speed without adjusting the beam conditions between two images (focus and astigmatism) allows each of the ROIs identified to be observed in approximately 10 seconds. As a result, the time during which the user is unaware of the behavior of one of the two ROIs remains limited and acceptable. There is approximately 10 seconds between two images recorded under the same conditions. The temporal resolution of the image series is high, and rapid changes (on the order of minutes) can be observed. The quality of these images is entirely acceptable and allows for the correct visualization of details on the order of 50 nm (directly measured on the smallest details observed on the images).

When the image recording time was increased and the focus and astigmatism were automatically corrected, the quality of the recorded images improved significantly, and more details could be observed in the images. However, this corresponds to an increase in the time between two images recorded on the same ROI at the same magnification, and therefore to a decrease in the temporal resolution of the image series. These conditions must be associated with the observation of slow transformations.

## Conclusions

The interface developed here allows an ESEM to operate in high-temperature mode in order to automatically record series of images at different magnifications across several areas of interest. To our knowledge, this interface is the first to perform these functions [18]. The interface's functionalities were validated by studying the oxidation of a nickel-based superalloy (Tropea). This specific study demonstrates the advantages of working at different magnifications. This operating mode allows information to be collected that provides insights into the local behavior of the material. At the same time, it provides access to more general information that allows specific localized behaviors to be visualized and described that could not have been observed without the use of this interface. The multi-zone study makes it possible to verify that the same behavior is observed (or not) over most of the sample surface.

The automated control of the ESEM over long periods of time opens up new possibilities of research for which morphological changes on the surface of materials are slow. In addition, this interface can potentially be used for other applications such as maintaining a sample in controlled humidity, performing long-term mechanical tests, or any other in situ experiment in the ESEM chamber (temperature-dependent or otherwise).
Acknowledgements

Part of the investigation was funded by the French National Agency for Research supporting the
ALUPLAT project (Grant ANR-21-CE08-0013).

References

[1] R.M. Fulrath, Scanning Electron Microscopy to 1600 °C. SEM (1972), pp. 17-24, Chicago (Illinois).
https://www.osti.gov/biblio/4627284-scanning-electron-microscopy-sup

[2] J.W. Brouillette, W.E. Leyshon, Hot sub-stage for a scanning electron microscope. US patent n°3919558 (1975). https://patents.google.com/patent/US3919558

[3] J.E. Castle, M.R. Hunt, In-situ oxidation of iron and steel in the scanning electron microscope. Corros. Sci., 16 (1976), pp. 137-142, 10.1016/0010-938X(76)90054-8

[4] S.K. Verma, G.M. Raynaud, R.A. Rapp, Hot-stage scanning electron microscope for high-temperature in-situ oxidation studies. Oxid. Met., 15 (1981), pp. 471-483. https://link.springer.com/article/10.1007/

[5] E. Charyshkin, N.N. Kinaev, M. Waterworth, D.R. Cousen, N. Calos, T. Bostrom, A. Ilyushechkin, In-situ electron microscopy studies of hot filament chemical vapour deposition diamond thin film growth in an environmental SEM. Phys. Status Solidi A, 154 (1996), pp. 43-54, 10.1002/pssa.2211540106

[6] A.M. Brown, P.L. Surman, Oxidation of iron within the scanning electron microscope: oxide crystallite growth kinetics during the oxidation of iron in CO2. Surf. Sci., 52 (1975), pp. 85-102, 10.1016/0039-6028(75)90010-2

[7] A.M. Brown, M.P. Hill, A hot stage SEM for gas-solid reaction studies. J. Microsc., 153 (1989), pp. 51-62.
https://onlinelibrary.wiley.com/doi/pdf/10.1111/j.1365-2818.1989.tb01466.x

[8] G.D. Danilatos, V.N.E. Robinson, Principles of scanning electron microscopy at high specimen pressures. Scanning, 2 (1979), pp. 72-82, 10.1002/sca.4950020202

[9] D.J. Stokes, Principles and Practice of Variable Pressure/Environmental Scanning Electron Microscopy (VP/ESEM). John Wiley & Sons Ltd, The Atrium, Southern Gate, Chichester, West Sussex, UK (2008)

[10] R. Podor, G.I. Nkou Bouala, J. Ravaux, J. Lautru, N. Clavier, Working with the ESEM at high temperature. Materials Characterization (2019) 151, 15-26. https://doi.org/10.1016/j.matchar.2019.02.036

[11] R. Podor, J. Mendonça, J. Lautru, H. P. Brau, D. Nogues, A. Candeias, P. Horodysky, A. Kolouch, M. Barreau, X. Carrier, N. Ramenatte, S. Mathieu, M. Vilasi, Evaluation and application of a new scintillator-based heat-resistant back-scattered electron detector during heat treatment in the scanning electron microscope. J. Microscopy (2021) 282 [1] 45-59. https://doi.org/10.1111/jmi.12979

[12] J. Rame, S. Utada, L. M. Bortoluci Ormastroni, L. Mataveli-Suave, E. Menou, L. Després, P. Kontis, J. Cormier, Platinum-Containing New Generation Nickel-Based Superalloy for Single Crystalline Applications. In: Tin, S., et al. Superalloys 2020. The Minerals, Metals & Materials Series. Springer, Cham. https://doi.org/10.1007/978-3-030-51834-9_7

[13] N. Warnken, D. Ma, M. Mathes, I. Steinbach, Investigation of eutectic island formation in SX superalloys. Materials Science and Engineering A 413-414 (2005) 267-271. https://doi.org/10.1016/j.msea.2005.09.067

[14] L. Hunault, S. Mathieu, J. Cormier, R. Podor, F. Pedraza, Oxidation Behavior of Platinum-Containing Model γ and γ' and TROPEA Superalloy. In: Cormier, J., et al. Superalloys 2024. ISS 2024. The Minerals, Metals & Materials Series. Springer, Cham. https://doi.org/10.1007/978-3-031-63937-1_62
[15] S. Mathieu, R. Podor, M. Emo, L. Hunault, M. Vilasi, J. Cormier, F. Pedraza, Short-Term Oxidation in HT-SEM of the Pt-Containing TROPEA Single Crystal Ni-Based Superalloy from 680 to 1000 °C. High Temperature Corrosion of matter. (2024) 101, 1211–1223. https://doi.org/10.1007/s11085-024-10272-9

[16] R. Podor, T. Duprat, F. Pedraza, J. Cormier, L. Aranda, K. El Bouassi Dos Santos, M. Odorico, J. Lautru, S. Mathieu, Ni-based single crystal superalloy oxidation studied by High Temperature Environmental Scanning Electron Microscopy. Part 1. Unveiling the first moments of oxidation. Unpublished data.

[17] R. Podor, J. Lautru, L. Hunault, F. Pedraza, J. Cormier, L. Aranda, M. Vilasi, S. Mathieu, Ni-based single crystal superalloy oxidation studied by High Temperature Environmental Scanning Electron Microscopy. Part 2. Long-term formation and stabilization of the oxide layer. Unpublished data.

[18] R. Podor, J. Lautru, T. Duprat, S. Mathieu, M. Vilasi, J. Cormier, L. Hunault, F. Pedraza NiO whiskers growth driven by the presence of Pt nanoparticles during Ni-base superalloy oxidation. Materials Characterization (2025) 230, 115754. https://doi.org/10.1016/j.matchar.2025.115754