Improving light microscopy training routines with evidence‐based education

The low reproducibility of scientific data published in articles has recently become a cause of concern in many scientific fields. Data involving light microscopy is no exception. The low awareness of researchers of the technologies they use in their research has been identified as one of the main causes of the problem. Potential solutions have hinted at the need to improve technological and methodological education within research.


INTRODUCTION
Light microscopy is a varied, complex and powerful technique used in many disciplines of research as well as in biotechnologies, engineering, education from school to university, and within public health at hospitals.Education, rigour and reproducibility in light microscopy are therefore important to many aspects of society.
2][3][4][5] A major source of irreproducibility of published microscopy data is the under-reporting of methods. 6,7The top suggestions to address this issue include the need for a better understanding of methods, better supervision, better teaching, more proficiency testing and longer mentoring time.These suggestions clearly point to the need to improve the quality of the education provided to researchers during their research career.
The microscopy community has led initiatives to improve the reproducibility of light microscopy data in published studies. 10,6,8,9The goal is to establish guidelines on various aspects of microscopy, addressing equipment calibration and reliability, as well as ensuring the accuracy and completeness of microscopy methods in research articles.Besides reading these guidelines, researchers can also improve their microscopy knowledge through a plethora of freely available online material, [11][12][13][14][15][16][17][18][19] excellent books, 20 articles 21,22 and courses. 16,23,24Unfortunately, having access to a wealth of educational materials is not equivalent to learning.Therefore, researchers who seek information often fail to understand how they can apply it in their research.
Most universities around the world have made a noticeable effort to modernise their pedagogical methods. 25owever, the implementation is slow to reach technological training at postgraduate level.When train-ing researchers to use technologies, it is widely agreed that what is being taught should be based on up-todate, evidence-based research, but surprisingly, it is less accepted that how the education is designed should follow the same principle.As a result, the teaching strategies that have been proven to vastly improve the students' learning process have not been applied to design technological training routines for researchers.This is also true in the field of light microscopy.
Over the past 20 years, core facilities have been increasingly recognised as central to ensuring the technological education of researchers.7][28][29] In the literature, several factors influencing the quality of microscopy training routines have been mentioned, including the training session/staff ratio, 30 the need for FS continuous education, 31 suggestions of a typical training content and workflow, 28,31,32 the need to customise training routines to the project, 28 and when to discuss image analysis. 32,33Nevertheless, besides a single mention that the educational aspect of training routines could be improved, 4 the possible impact of effective pedagogical methods on the quality of microscopy education is mostly unexplored.
Here, we first argue that effective pedagogical methods have a strong potential for improving the quality of training routines and the knowledge that researchers have of microscopy.This is likely, in turn, to increase the reliability and reproducibility of the data they publish.We also argue that improving the education of researchers requires that even FS themselves get access to quality education, not only in microscopy principles, but also in pedagogy.
Second, we discuss why teaching microscopy is challenging and we present the principles of Constructive Alignment, an evidence-based pedagogical method suitable in the context of microscopy training.We then give an example of how to gradually introduce Constructive Alignment into existing training routines in order to improve the researchers' learning outcomes.We also discuss how applying the Constructive Alignment method may clarify the respective responsibilities of FS and researchers in the acquisition of reliable data.
Finally, we suggest the creation by the microscopy community of quality standards relating to microscopy education, with the ambition of helping FS benchmark and improve their own training routines.We also advocate that the policies of funding agencies and universities be adapted in order to give time and incentive for FS to upgrade their technological and pedagogical knowledge, and to introduce pedagogy to the curricula at 'Train-thetrainer' courses.
With this article we hope to provide the imaging community with tools to improve the outcomes of training routines at light microscopy facilities.

CONSTRUCTIVE ALIGNMENT FOR EFFECTIVE TEACHING AND LEARNING
Over the past 50 years, in response to the evolution of both the university psychosocial environment and research on how students learn, 34,35 major reforms have resulted in the implementation of new pedagogical methods in higher education around the world. 25

Pedagogical trends at universities
In the past, access to universities was restricted to a handful of highly motivated students.It was thought that teaching was about delivering content and was the teachers' responsibility, while learning meant absorbing content and was the students' responsibility.The consensus was that some students could learn well while others could not, and examinations were designed as a mechanism to sort 'good' students from 'bad' ones. 25,34ccessing higher education gradually became easier.The diversity of students broadened considerably and, with it, the source and strength of their motivation to learn. 34n the modern concept of quality teaching, continuously developed since the late '70s, teaching and learning are intimately connected and require methods that activate students' motivation.As a result, education has moved from a teacher-centred model, where the most important aspect for the teacher is to master the subject, to a studentcentred model, where teachers focus less on what they say or do, and more on if and how well students can apply the new knowledge.
These major changes in education at universities parallel the typical evolution of teachers during their career. 36 the first stage of their development, teachers tend to focus on themselves as a performer, what they look and sound like, if they are nervous or not.Further in their development, teachers concentrate their efforts on their subject matter, so they try to improve the accuracy of their teaching material and how they formulate its content.As they evolve further, their focus becomes student-centred: first they try to understand how students learn, then how students can use the new knowledge and finally, the most advanced teachers tend to teach by guiding students to independently design their own learning.
Like other teachers, FS involved in training researchers evolve through the steps described above.Indeed, there is no difference between teaching and training.In both cases, the goal is to educate.Lectures and quizzes can be used to train small groups.Practical demonstrations and labs can be used to teach hundreds of students.Therefore, FS who train researchers are teachers, and their trainees are students.Even if few researchers are usually involved in training sessions, all responsibilities and pedagogical skills applying to teachers and students apply to trainers and trainees.One responsibility that is expected of all teachers in the education system is that they have received an education in the principles of teaching but FS usually do not fulfil this requirement.
Training researchers at microscopy facilities shows strong similarities with the process of life-long learning for medical staff in a healthcare context.There as well, a limited amount of time is available to improve the skills of working adults and their ability to make complex, educated decisions.8][39] Similarly, pedagogy should improve the learning outcomes when teaching microscopy to researchers.
Unfortunately, FS rarely use pedagogy to design their training routines and consequently, it is difficult for them to grab the attention of busy researchers.The risk is then that FS feel that researchers are not learning because they lack motivation and interest. 28,31On the other hand, researchers might get frustrated if despite spending time for training, they experience a poor learning outcome and remain insecure about how to conduct their microscopy experiments.In this situation, the trust relationship between FS and researchers is likely to be affected.

2.2
Effective pedagogical methods

Learning by building concepts
The Once we succeed in connecting several pieces of information together as a concept, our working memory considers this concept as a single element.This in turn allows us not only to remember, but also to understand and learn at a much higher level of complexity.Where a novice needs to deal with 100 individual elements, an expert sees only 2 of them, each consisting of 50 elements coherently assembled.Connecting elements into concepts takes time but it is a necessary step to remember and learn complex information. 40One key is thus that time to practise, discuss and reflect is needed to build concepts and ultimately, to learn.

Learning strategies
Educational research 34,41,42 also shows that the learning environment and the subsequent learning strategy adopted by students are key factors that can either enhance or hinder the learning process. 43tudents have a wide range of expectations about what they can gain from an education programme.Some consider that the new knowledge may help them develop and is likely to have a positive influence on their future in a deep and permanent way.Others believe that education is merely a process that one must go through to progress towards a practical goal, for example get a degree or gain access to a microscope. 34,42artly due to these expectations, students tend to adopt either a deep-learning strategy (not to be confused with the deep-learning methods used in computer science), or a surface-learning strategy. 44Students who adopt a deeplearning approach focus on understanding the overarching meaning of the subject matter, with the belief that it will be useful to them in the future.On the other hand, students who adopt a surface-learning approach set their focus on guessing what is required of them in the short term to reach the next step towards their goal, so they look for ways to minimise their efforts and tend to use short-term memory instead of trying to extract the overall meaning.As a result, surface-learning students often fail to reach a high level of understanding, and are more likely to forget. 45Evidently, it is harder for them to learn.

Learning environment
The students' learning strategy is often shaped by their past experiences of learning.When joining a new teaching session, students try to understand what is expected of them and tend to adapt their learning strategy accordingly. 45t is thus important to use pedagogical tools throughout the teaching session to signal to the students that they are expected to be engaged and adopt a deep-learning approach. 46The classification of 'good' and 'bad' students can consequently be reformulated as students who independently adopt a deep-learning approach versus students who need, to varying degrees, to be guided to do so.One of the roles of the teacher is thus to create a learning environment that signals to students that deep-learning is required.
The factors that channel students towards adopting a deep-learning approach (summarised in Figure 1) have been studied for decades. 34,41First, the teacher must explicitly convey the goal of the tasks being taught and show their importance and why they are worth doing.The assessment criteria the students are expected to fulfil to demonstrate success must also be clearly stated.Second, the teaching session must use activities involving different parts of the brain such as listening, watching, doing, writing and discussing. 34A range of levels of cognition must be involved, from the lowest like memorising, to the highest like summarising and extrapolating. 46Third, it must be clear from the start that the tasks will be challenging, and their outcome assessed, but that success can be expected.Stress, lack of time and tasks that are too difficult all contribute to creating a negative learning environment that channels students towards surface-learning.So do boredom and being overwhelmed with information without a chance to self-assess one's progress.
In summary, the role of a teacher is not only to master the topic of the teaching session and create good content, but, as importantly, to clearly communicate to the student what the goals of the teaching session are, what is required to reach them and to create a stress-free but challenging learning environment that channels students towards deep-learning, triggers their interest and grabs their attention from start to end. 47he exact same principles presented here apply when training researchers to design and conduct microscopy experiments.

Constructive Alignment
Constructive Alignment is a widely adopted, evidencebased educational strategy 48 to help teachers design and structure their teaching in a systematic way.The objective Failing to give feedback on how well a task has been performed, giving negaƟve feedback without explaining how to improve, or suggesƟng that it is unlikely that the student will succeed.
Failing to give feedback on how well a task has been performed, giving negaƟve feedback without explaining how to improve, or suggesƟng that it is unlikely that the student will succeed.
Assessing progress based on low-level cogniƟon like memorising.

Assessing progress based on low-level cogniƟon like memorising.
PresenƟng what is being taught has having a low value or as unlikely to be useful.Teaching or recommending one thing and doing the opposite.
PresenƟng what is being taught has having a low value or as unlikely to be useful.Teaching or recommending one thing and doing the opposite.
Causing students stress or anxiety, blaming them for the lack of success.
Causing students stress or anxiety, blaming them for the lack of success.
Failing to bring forward the goals and structure and show how elements are related to each other.
Failing to bring forward the goals and structure and show how elements are related to each other.
Teaching complex concepts before checking first that the basic level is acquired.
Teaching complex concepts before checking first that the basic level is acquired.
Teaching interacƟvely (asking a quesƟon before explaining a point) and with varied acƟviƟes (speaking, drawing, watching a video, doing, etc).
Teaching interacƟvely (asking a quesƟon before explaining a point) and with varied acƟviƟes (speaking, drawing, watching a video, doing, etc).
Explicitly announcing when the student has successfully fulfilled expectaƟons and requirements.
Explicitly announcing when the student has successfully fulfilled expectaƟons and requirements.
CorrecƟng misconcepƟons by asking the student to formulate important concepts in their own words then giving feedback.
CorrecƟng misconcepƟons by asking the student to formulate important concepts in their own words then giving feedback.
Explaining why what will be taught is important and connecƟng it to the researcher's project.
Explaining why what will be taught is important and connecƟng it to the researcher's project.
Assessing progress with challenging tasks that require the student's reflecƟon but where success is expected.
Assessing progress with challenging tasks that require the student's reflecƟon but where success is expected.
Being paƟent and kind when students make mistakes and explaining how they can progress further.
Being paƟent and kind when students make mistakes and explaining how they can progress further.

Foster surface learning Foster surface learning Foster deep learning Foster deep learning
CreaƟng a logical flow between the training secƟons.CreaƟng a logical flow between the training secƟons.Overwhelming students with informaƟon without relevant tasks and assessments.Giving too liƩle Ɵme to fulfil or pracƟse a task.
Overwhelming students with informaƟon without relevant tasks and assessments.Giving too liƩle Ɵme to fulfil or pracƟse a task.
Explicitly staƟng the goals and expectaƟons of the teaching and the requirements for success.
Explicitly staƟng the goals and expectaƟons of the teaching and the requirements for success.
F I G U R E 1 Aspects of teaching that encourage a surface or a deep approach to learning. 34 to engage student motivation in such a way that the student wants to learn and to create an environment that supports deep-learning.The strategy is said to be studentcentred and outcome-based because it focuses on the students' progress in what they can perform.
In educational sessions designed according to the Constructive Alignment principles, three aspects are aligned to coherently work together: (1) what students should be able to do at the end of the session and at what level (Intended Learning Outcomes, ILOs), ( 2) what needs to be done to support the students to reach these ILOs (Teaching and Learning Activities, TLAs), (3) what the students need to do to demonstrate how well they can perform the ILOs (Assessment Tasks, ATs).
The very first stage in implementing the Constructive Alignment strategy in a teaching session is to define the Intended Learning Outcomes.ILOs are student actions formulated as verbs. 49These actions are what the teacher expects the student to be able to perform after the teaching session. 46Writing ILOs is a challenge for inexperienced teachers.In the process, they may well realise that they only teach simple tasks while claiming that the students should be able to perform complex ones.Announced at the start of the teaching session, the ILOs ensure a match between teacher and student expectations.
The second stage of the design is to align the Teaching and Learning Activities (TLAs) to the ILOs: what needs to be done to enable the students to reach the ILOs?Which information must be provided?What do they need to practise?ILOs usually describe actions that require a high level of cognition, where students engage their reasoning. 46Performing these complex actions often requires the student to first be able to perform other lesser tasks.For example, an ILO might be for the student to explain why the objective they selected is best for their experiment.This entails that the student is already able to turn on the equipment, select the correct objective, calculate the optical resolution delivered by the objective and assess the resolution, field of view and working distance required to answer the research question.Each of these tasks requires explanation and practice activities described as TLAs.
The third and final stage is to create assignment tasks (ATs) for each TLA and ILO.Assessments are not examinations.They are a way for the teacher to gauge if and how well the student can perform the task.They also create an opportunity to give feedback and correct misunderstandings.As importantly, assessments are a way for students to engage their attention and assess their own progress.Assessments should be embedded all along the training session in a stress-free manner and should simulate situations that the student is likely to face in the future.They should involve different levels of cognition like memorising, calculating and summarising, but also reflecting or concluding. 46t is a common mistake for inexperienced teachers to assume that if someone has understood, they have also learnt.Understanding and learning are not the same.If something is reasonably well explained to us, we may understand what is being said or shown but it does not mean we can explain or perform it ourselves.This requires learning.The principle of using assessable action verbs to describe ILOs and TLAs excludes verbs like 'understand' or 'know'.This is because it is not possible to assess if or how well someone understands or knows.Instead, one can assess how well someone does something, explains and summarises it, or draws conclusions.If they can explain well, then it can be assumed that not only they understand, but that they have indeed learnt.
In a constructively aligned teaching session, students know very clearly, from the start, what is expected of them and what they need to do to succeed.This creates a positive and encouraging learning environment where students witness their own progress as a reward for their engagement.As importantly, Constructive Alignment is a tool for teachers to ensure that the goals, content and methods of their teaching sessions are not only clearly defined, but also are coherent.

Misconceptions about light microscopy
One issue with teaching microscopy is the widespread notion that it is easy and highly standardised.In reality, microscopy is a complex technique with many hidden potential pitfalls that unfortunately, imaging researchers and their group leaders are often unaware of. 2,50ne can find several explanations to this unfortunate situation.
First, our own teaching experience at undergraduate biology classes shows that light microscopy is commonly used as a tool without the support of theoretical teaching.Students participating in labs involving light microscopes are often shown little more than how to change filters and press the Acquire button.Teaching how to avoid typical microscopy pitfalls, like bleed-through or saturation is missing from the curriculum.In contrast, labs requiring the use of PCR machines usually include explanations about primer design and the theory behind each PCR cycle.Therefore, very early in their career, students are given the false impression that light microscopy is a simple technique without challenges.This mindset is unfortunately likely to follow them when they start their PhD.
Another reason why microscopy is deemed easy is that, unlike techniques which fail when basic principles are not applied, pressing the Acquire button always delivers an image.In turn, images always contain data that can be analysed, regardless of whether they are meaningful and reliable, or not.Unaware of this fact, researchers who have not been trained to critically assess their images are unable to detect hidden problems.Unfortunately, they are then at high risk of acquiring and publishing inaccurate data extracted from images, possibly with systematic biases, without even being aware of it.
One more difficulty with teaching microscopy is that everything is interconnected.Many elements of sample design and imaging parameter choices directly depend on both the research question and the way the images will later be analysed.In other words, all the intermediary steps of a full microscopy experiment are connected to the first and the last steps.Educated choices that consider the whole experiment must be made along the way, with a direct impact on the quality and reliability of the results.Correctly designing a microscopy experiment thus requires a reverse-thinking approach. 33Unfortunately, many researchers consider that sample preparation, image acquisition and image analysis should be dealt with separately and one after the other.They consider the first two trivial and requiring little discussion, and the latter difficult.Addressing image analysis is thus often conveniently postponed until all images are acquired, strategy that is very likely to fail.On the contrary, all moments of a microscopy experiment are intimately intertwined, and the difficulties encountered with analysing images often stem from poor sample and experimental design.
For the reasons listed above, the time, effort and funding that researchers and their group leaders are willing to allocate to a microscopy training session are often minimal.The pressure on FS to 'get it done' as fast as possible and avoid challenging concepts is high, with likely consequences on the training quality.

Need for flexibility in training design
Besides the false preconceptions that light microscopy is easy and requires minimal training, another challenge facing the design of microscopy education is that it requires a high degree of flexibility.
In today's highly international universities, researchers come to microscopy facilities not only with different learning cultures and language skills, but also with a wide range of research questions and sample types.Unfortunately, because of the lack of consensus across facilities about what researchers should learn to produce reliable microscopy data, their knowledge often also contains large and varied gaps.Additionally, depending on which parts of the microscopy experiment they will perform themselves, their learning requirements are quite diverse.Some need to be able to design and conduct the whole microscopy experiment on their own, adapting sample preparation, imaging settings and analysis pipelines to their project's ever-changing demands.Others want to image a few more samples for an article revision, using the exact settings established by a colleague, months earlier.For others, the best approach is that FS offer a full-service, 50 including preparing the sample and setting the imaging parameters, so that the researcher is left with choosing the area to image and the interpretation of the results.This is common at electron microscopy facilities but can also be the case at facilities that specialise in one type of technique such as light sheet or super resolution microscopy.
The training content and design need to be adapted to each project, but because of time pressure, the temptation is high for FS to reduce the time allocated to training to a mere common denominator needed for all, instead of customising it as required.

3.3
The problem with traditional microscopy training

Training in labs
In the past, it was common for individual labs involved in microscopy to get funding to buy their own complex and expensive systems, such as single-point confocal microscopes.The initial training session would be delivered by the application specialist of the company selling the microscope.Typically, a junior researcher would then take on the responsibility of training newcomers.After a few years, the trainer would leave the lab in pursuit of a career, and someone else would inherit the training role, despite never being trained by the application specialist.In this process, there is a high risk that knowledge and experience get lost from one generation of trainer to the next, leading to a decrease in the training quality.As time passes and many poorly trained researchers abuse the equipment, the need for maintenance rises.This situation can quickly snowball into the trainer losing interest in spending time on maintaining the equipment and improving the training quality.As the training routine offers less and less value, the trainer comes under pressure to reduce it further.
When asking the researchers at our facility and at our course what type of training they have received, we find that many of today's imaging researchers are trained in these poor conditions, receiving, in the worst case, sadly quite common, training mainly aimed at minimising potential damage to the equipment.Not surprisingly, it is difficult for researchers trained in this manner to be confident that the microscopy data they produce is reliable and does not contain any hidden flaws.

Education of FS in microscopy
Later in their career, researchers who have used microscopy extensively in their projects may join a core facility as staff and become responsible for training other researchers.By that time, they have usually worked in science for many years, 30 thus gaining experience.They might then realise that microscopy is not as simple as it might first appear.However, many take on their new role without having themselves received any education in the fundamental principles of light microscopy.Additionally, despite shouldering a teaching role in higher education, they rarely get the opportunity and incentive to learn about effective pedagogical methods.Consequently, it is natural for FS to train researchers following the methods used earlier by their own teachers.
As FS interact with many researchers, varied samples and challenging projects, their experience increases.To improve their knowledge, they have access to the many education resources mentioned earlier, as well as microscopy-related conferences, 51,52 articles specifically aimed at FS, 23,28,31,53 microscopy forums 54,55 or a few 'Train-the-trainer' courses. 23,56These give them access to invaluable peer support; provide them with best practice guidance on sample preparation, image acquisition and analysis; and instruct them in managing equipment, staff and money.However, the principles of pedagogy are surprisingly absent from these resources.
We have not found any published data about where and when during their career FS acquire their microscopy knowledge, but it is likely to be a mix of some of the abovementioned sources, assembled randomly like pieces of a giant puzzle, likely leaving many gaps.As their experience and knowledge increase, FS might identify some fundamental aspects of microscopy as important and feel the urge to communicate them during their training routines.
To our opinion, this is unfortunately where a knowledge gap might start forming between FS who know the importance of understanding microscopy fundamentals to acquire reliable data, and the overstressed researchers who believe, supported by their colleagues, that microscopy is easy, requires little training and certainly does not involve difficult concepts.
Without the support of pedagogy, FS are likely to struggle to teach important fundamental concepts in a way that grabs the researchers' attention.Frustration on one side and boredom on the other may then lead to a break in the trust relationship that should naturally form between someone with a high level of knowledge and the urge to transmit it, and someone in need of that knowledge.

Education of FS in training design
Only a few articles have been published to support FS in improving the design of microscopy training routines. 28,31,32,57The importance of starting with a project meeting prior to the training session, of customising the training content to the needs of the project and of allowing researchers to practise afterwards is always highlighted.It is also recommended to include some theoretical aspects of microscopy.Trained researchers are then encouraged to contact the FS if they have questions.These recommendations form excellent general guidelines for FS, but despite complaints in the literature about the lack of researcher's motivation during training, 28,31 upgrading FS' pedagogical knowledge is never mentioned.Unfortunately, without pedagogy, it is likely that FS will meet a lack of motivation among researchers, eventually leading to a degradation of their trust in FS and a reluctance to ask questions. 50ecommendations about the duration of training sessions are even more rare in the literature, despite time pressure being a major concern for FS. 2 One to two sessions of a few hours each have been recommended. 28,31s mentioned earlier, learning new information requires that one connects it with past knowledge to form coherent concepts.Unconnected elements, if not rehearsed, are forgotten within 20 seconds. 40,58Two sessions of a few hours is thus very little time for researchers to learn to use the equipment with confidence, independently recognise and avoid the pitfalls of microscopy, and make several educated decisions about which objective and pixel size to use or which fluorophores to combine.

Training of researchers at facilities
When FS consider that their task is to deliver information, this is what they focus on during the few hours of the training session.They say and show everything they think the researcher must or should know.In the Must category fall the actions that researchers must perform to be able to acquire images at all.These are low cognition-level tasks such as turning on the equipment, placing the sample correctly on the stage, focusing, setting the light path, setting the top and bottom of a z-stack, etc.If the researchers are unable to perform these tasks after the training session, they are not able to conduct their experiments, which clearly demonstrates the training failure.To avoid this situation, FS naturally assign these tasks enough time for practice and for assessment.
The tasks in the Should category enable researchers to confidently and independently acquire images in a reliable way.Examples are to be able to assess bleed-through or choose the most appropriate objective or pixel size for the experiment.These tasks require a higher level of cognition and thus need more time for conceptualisation.If insufficient time is assigned to the training session, the tasks in the Should category are often explained but not prac-tised and assessed.The learning of these tasks is unlikely to occur.
Because of the way students learn, 40 tasks that are shown but not assessed not only overwhelm researchers' memory but also send signals that they are not important, thereby diminishing the researcher's motivation to learn them.Consequently, very short training sessions that lack practice and assessment time might result in researchers knowing how to perform the simplest tasks but being confused about the mostly forgotten complex concepts.On the other hand, FS who have 'covered' the theory, feel reassured that the important message they wanted to convey has been 'delivered', without realising that it might not have been learnt.
Our facility runs an extensive microscopy doctoral course that to this date, 200 international students have taken.The preselection process ensures that all students have already received microscopy training (not at our facility) and have extensive and recent experience with working on a fluorescence microscope.Many have been trained at several universities, in different countries.The majority have only received three hours of microscopy training, sometimes less.Only a few of them remember ever hearing about numerical aperture, and even fewer know its roles.Almost no one remembers ever hearing about the Point Spread Function or the optical aberrations potentially created by poor sample design.All of them report facing major challenges with imaging but not knowing where the problem lies or where to ask for help.Researchers in this situation sometimes waste many precious resources and months of research without knowing how to move on.
After the training session, FS may consider that it is the researcher's responsibility to come back and ask questions.Unfortunately, without enough knowledge, it is difficult for researchers to identify problems and understand that they need to ask questions, let alone how to clearly formulate them.If no one asks for help, it is natural for FS to be comforted in that their job is done.On the other hand, the researchers might turn to colleagues for answers to their problems and myths such as 'the more magnification the better' or 'single-point confocal microscopes are always best' resurface.Besides, the root of the imaging issues they are facing is not addressed.

IMPLEMENTING CONSTRUCTIVE ALIGNMENT TO EXISTING LIGHT MICROSCOPY TRAINING ROUTINES
To improve researchers' attention levels and learning outcomes, effective teaching methods can be progressively implemented in existing microscopy training routines.As an added benefit, this implementation is likely to result in increased appreciation of the role of FS.

Reminder of Constructive Alignment
As mentioned before, using Constructive Alignment in training routines requires defining Intended Learning Outcomes (ILOs: what the teacher wants the students to be able to perform independently after the training session), Teaching and Learning Tasks (TLAs: what is required to enable students to perform the ILOs), and Assessment Tasks (ATs: how the level of progress is assessed).
ILOs must always be an action verb and be assessable.For example, 'not damaging the equipment' is not a valid ILO because the researcher cannot demonstrate this skill.Instead, to avoid instrument damage, the teacher needs to include several TLAs involving the main risks for instrument damage such as focusing on the sample or swapping objectives.The training session should include enough supervised practice to reassure the teacher that damage is unlikely to occur.
Additionally, because researchers have different needs, ILOs, TLAs and ATs need to be adapted to match the responsibilities the researcher is expected to take on once trained.Researchers who will perform all tasks independently require the most complete training sessions.On the other hand, in full-service facilities where sample preparation and image acquisition are performed by FS, researchers do not need to be trained to perform these tasks.

Duration and number of training sessions and sample choice
For effective learning to occur, the training routine must include enough time for repeated practice and assessment.A few long training sessions can be replaced with several shorter ones.Explanations and demonstrations by the teacher should be interrupted every 15-20 min with TLAs and ATs, lowering the risk of overwhelming, signalling to the researcher to stay alert and indicating that progress is made. 40Importantly, if several researchers are trained at the same time, the teacher must ensure enough time to assess progress for each of them individually.
Using the researchers' samples to conduct the training session is ideal from a pedagogical perspective, as it strongly connects what is being taught with the researcher's actual needs.To avoid researchers being distracted by the use of their sample and focusing on collecting data instead of on the training session, 28,31 the teacher needs to relate everything that is taught, including the TLAs, to the researcher's sample and research question.Doing so will encourage deep-learning and engage researchers' attention.It also creates opportunities to troubleshoot samples and experimental design, which is directly helpful to the project.Finally, it signals that the FS is knowledgeable, can help and therefore be trusted.

4.3
Where to start and an example Constructive Alignment ultimately prevents teachers from placing their focus on what they say and show.Instead, it leads them to spending a large part of the training session as assessors of progress.Speaking and acting gives teachers a feeling of control which is hard to give up.However, research shows that students learn when they do something, not when the teacher does it, 34 so the shift is key to improving the effectiveness of training.Implementing Constructive Alignment to existing training routines does not need to be a daunting task and can be done gradually.A good place to start is a part of the training routine that the teacher is thoroughly confident with.
For example, the teacher may identify that an important training objective is for researchers not to acquire saturated images.The ILOs can be formulated as follows: the researcher should be able to identify saturated pixels, explain why saturation is a problem and modify the appropriate settings to avoid this artefact.All these ILOs are action verbs that are assessable.The teacher should then decide on how the researcher's performance level will be assessed (ATs), then deduct what needs to be said and shown to researchers and what they need to practice (TLAs) in order to be able to reach the ILO at the expected level.
Next, the teacher should test the new training design with a researcher.The ILOs are first announced as what will be expected of the researcher, after the training session.The teacher explains saturation, shows how to detect it and which microscope parameters influence it.The researcher acquires a few images of their own sample, varying the settings that affect saturation.Simple image analysis tools to measure the brightness and size of a thresholded region are introduced to familiarise the researcher with extracting data from images.The teacher assesses the proficiency level of the researcher who uses the Lookup Tables and image analysis tools to assess the presence of saturation, draws conclusions about the consequences of saturation on object intensity, size and number and adapts the settings to acquire images without saturation.Finally, the teacher points out the success in achieving the ILOs.In this example, researchers clearly know what is expected of them and how to succeed.They are assessed when practicing situations that they are very likely to often encounter in their research.Everything that is being said, shown and practised is fully related to the ILOs.Another example and a workflow for FS to design their training routines following the Constructive Alignment method is given in Figure 2.
In our experience, implementing the example above requires no dramatic changes.It might somewhat lengthen the training session, but it clearly improves its effectiveness.The enhancement in researchers' level of attention and learning is noticeable.At first, formulating the ILOs in terms of assessable researcher actions and designing TLAs and ATs can be challenging, but with a little practice, it becomes easier and even very natural.

Clarifying responsibilities
Because science is highly collaborative, the responsibility for ensuring that acquired data are reliable is usually shared among several researchers who may design the project, collect and prepare the sample, acquire the images, and analyse or interpret them.Consequently, FS cannot be held responsible for ensuring that the data produced by researchers are reliable, since this is beyond their control.
On the other hand, should researchers be held responsible for the unreliability of the data they acquire if this reflects the low quality of the way they were trained?Should FS who possibly never received any structured education in the principles of microscopy and in pedagogy be held responsible for the poor content of their training routines and the ineffectiveness of the learning environment?It is the responsibility of FS to provide researchers with enough training to enable them to acquire reliable data.What does that mean and where does this responsibility end?Surprisingly, the crucial question of responsibility has found very little echo in the literature.Some 28 argue that 'whether or not a user actually fully understands what he or she is doing is the user's responsibility in the end'.Others 50 consider that the question of responsibility is central to the training quality at research facilities and that 'responsibility cannot be simply off-loaded'.It is challenging to define the limits of FS responsibilities because their role in research lies at the border between service, education, mentorship and scientific collaboration.This uncertainty leads to the impact of FS on the reliability of produced data often being undervalued.In turn, the relationship between FS and researchers may be undermined, leading to frustration and discouragement.
We believe that using Constructive Alignment can help FS clarify their responsibilities.In their role as teachers, they must provide training of high enough quality, with clearly defined ILOs, TLAs and ATs as well as an appropriate learning environment, to enable the researcher to fulfil the ILOs.Once it is assessed that what was taught during the training session has also been learnt, it becomes the responsibility of the researcher to use the new knowledge when performing their experiment.Any additional advice and guidance provided by knowledgeable FS about experimental design or sample preparation for example, must be considered as mentorship or scientific collaboration.As with any collaboration, the researcher's knowledge might increase in the process, but it is not the responsibility of the FS to ensure that it does.Clearly communicating to researchers and their group leaders the goals and limits of their teaching responsibilities allows researchers and FS to match their respective expectations and fosters a healthy collaborative mindset.
Importantly, in the same way that students cannot learn in a bad environment, FS cannot fulfil their responsibilities if they are not themselves provided with effective education both in microscopy and in pedagogy, or when they are overstressed.It is thus the responsibility of those who manage and fund facilities to ensure that the working environment of FS and the time allocated to training are appropriate.Only then can FS really and truly fulfil their mission of supporting researchers in producing reliable microscopy data.

CONCLUSIONS
Universities around the world have succeeded in implementing modern pedagogy in their teaching at undergraduate and graduate levels. 25At doctoral level, formal courses have also benefited from new pedagogical input.Unfortunately, these pedagogical improvements have yet to be applied to training of the technologies used to conduct research.This oversight needs to be addressed to break the cycle where poorly trained researchers become trainers for the next generation of researchers.
In this article, we present the positive impact that pedagogical strategies like Constructive Alignment could have on what and how well researchers learn during microscopy training sessions.We believe that the reproducibility of published microscopy data will increase over time if tested pedagogical methods are used during training sessions.
We explain how the communication and trust relationship between FS and researchers may also be improved.We urge FS to reflect upon the potential gap between what they are trying to teach and what researchers indeed learn.This reflection may reveal that much is said but little is learnt, that only the simplest tasks are assessed, and that very important aspects of their teaching is likely to be lost.
Teaching someone who does not pay attention or is clearly not learning is frustrating but realising that the researcher's attitude might stem from the poor quality of the teaching is a painful experience.It is nevertheless a necessary journey for anyone who wants to improve their skills as a teacher. 36To progress along this path, FS involved in training imaging researchers need to fully embrace their role as educators.With this title comes the joy of sharing knowledge and helping others achieve their full potential, but also the responsibility for checking that what has been taught has also been learnt.
It is urgent to investigate if FS involved in teaching ever received any solid education in the technology they support as well as their pedagogical skills.If the investigation shows that the majority have indeed learnt on the job without any structured and effective training, this needs to be addressed by all stakeholders, university and facility management, funding agencies, global and national microscopy organisations.The necessary time and funding resources need to be allocated to incite FS to upgrade their knowledge.'Train-the-trainer' courses and activities, such as job shadowing 59 or reciprocal Critical friends, 60 must be actively supported, and the creation of a network of FS in their role as teachers must be promoted.Many universities offer pedagogical support videos or courses to their teachers involved in graduate studies.The material they offer is easily accessible and can be invaluable for FS so they should be encouraged to use these resources.
The microscopy community is trying hard to introduce standards at different levels of microscopy experiments, and this is an essential effort.However, it is important to remember that perfectly calibrating microscopes and fully reporting metadata will not prevent poorly trained researchers from unknowingly acquiring and publishing flawed and unreliable data.We advocate the creation of community-based recommendations about training content, duration and pedagogical design, to serve as guidelines to improve the quality of researcher training routines at facilities.The consequences of training the trainers and systematically developing pedagogy at facilities are far-reaching and will lead to the new generations of researchers having not only better microscopy skills, but also a higher standard and better role models when they themselves become the next trainers.
Finally, it is likely that the recommendations in this article for microscopy can be applied to facilities that train researchers to use other technologies.Improving the quality of FS education in the technology they teach and in pedagogy at all core facilities involved in teaching has a strong potential for increasing the reproducibility of published research data.

A C K N O W L E D G E M E N T S
The Live Cell Imaging Core facility/Nikon Center of Excellence, at Karolinska Institutet, is supported by the KI infrastructure council.We are grateful to Rebecca Oliver, Per Palmgren and Claude Le Guyader for critically assessing the manuscript.

F I G U R E 2
Recommended workflow to clarify responsibilities and improve training routines at facilities.
40ndings of research on how we learn can greatly help FS understand what motivates researchers during training and what triggers their interest.A researcher to whom a new task is explained or shown without time to practise, discuss or reflect can only learn through memorising.As explained by Hattie et al in 'Visible learning and the science of how we learn',40knowledge builds on knowledge.The authors explain that when trying to remember something new, we can only deal with three elements at the same time.That is if these elements are not related to other elements that are already known.If more elements are presented, our working memory is overwhelmed and stops working.