This month we share some reflections from Grace Baruta, who is a PhD candidate in the Hirota Laboratory at the University of Calgary. I have known Grace since she was a summer student, and she has always taken the time to research options and map out an approach, from the sample preparation to image analysis. Grace also builds community and is the lead facilitator for our imaging platform’s Data Café, an informal opportunity for graduate students to share results and exchange advice about imaging.
My journey with microscopy started several years ago, as a young and hopeful scientist seeing beautiful images in scientific publications and presentations. I began my microscopy journey with a widefield microscope, generating wound healing images. Using phase contrast microscopy, I examined the effect of various nuclear receptor agonists on the rate of wound healing (See figure in publication). Although, in the end, my workflow was streamlined, it took months to optimize the various steps. In the beginning, image analysis was a daunting challenge, but over time and with guidance from others I found a way to individually track individual cells over time. Now looking back, I see that there were more automated ways to measure individual cell migration, but I learned so much from this experience of tracking individual cells over time using ImageJ. I enjoyed my imaging-centred project so much that I decided to pursue graduate school and it’s been quite the adventure. However, if I could impart some imaging wisdom to my younger biologist self, this is what I would say.
First, do your research. Before touching a microscope or examining a sample, take the time to understand what it is you are doing and why you are using imaging to do it. As an eager master’s student, I didn’t always take the time to assess the reasoning behind my initial experiments, costing me time and causing frustration. Generating a concrete hypothesis for what you expect to see and why can help to prevent this. Consider your experimental rationale and its foundation in preliminary data or published research. Only once you understand the fundamentals of your experiments can you embark on imaging. This knowledge will shape the controls that you use and will aid you in interpreting unexpected results.
Second, seek guidance from others but only after you’ve done your own research. No one can be successful in isolation and the benefits of working on experiments on your own are often limited. Instead, seek out your peers and experts in your research area; often, they will be willing and happy to help. The prerequisite to seeking out guidance is to put in the time to search for answers on your own first. This will give you insight on what to ask and will respect the advisor’s time. Beyond this, it will aid in retaining the advice you receive, since you will have a framework of knowledge based on your review of the literature.
Finally, be flexible. Experiments will not work, and imaging will often not go as planned. Your project and environment might change during graduate school, and you will certainly change from this experience. When I was young, my mother would tell me I needed an “open hand” to accept what life chooses to give me. I internalize this concept a lot more now as an adult working towards a PhD. I can only control what I put into a scientific experiment and must keep an “open hand” about the results. Put your all into the hypothesis and rationale behind your imaging experiment but be flexible about what the results may tell you or what changes may need to be implemented when processing data.
Figure 1: Host-pathogen interactions in a mouse intestinal monolayer with a heterogenous cell population
I think about the lessons I have learned often as I pursue my PhD. I’ve been fortunate to be able to hone my imaging and image analysis skills in the past few years, while exploring the host-pathogen interactions of a certain bacteria in mouse small intestinal organoids. I’ve had opportunities to be flexible while generating a novel model to study host-pathogen interactions in a mouse intestinal monolayer with a heterogenous cell population (See figure 1 above). I wouldn’t have been able to generate this model without a well rationed hypothesis and innumerable hours of research and failed investigations. For example, generating a model system to study the pathogen I was interested in took months of failed attempts before I stumbled on a protocol that actually generated a heterogenous monolayer. I also wouldn’t have been able to do this on my own; I’ve received mentorship from many individuals that have paved the way. Currently, my life quote is “traveler, there is no path, the path is made by walking” – Antonio Machado, Border of a Dream: Selected Poems. The imaging scientist designs the experiment to the best of their ability, but the path to successful imaging and analysis is “made by walking” in that imaging scientists must experiment and find their own way.
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