So far, we’ve touched on the mindset, planning, and decisions required when carrying out imaging experiments. To highlight this framework with a real-life example, I have invited Louise Neave to share some of her experiences as a researcher new to imaging. Louise is a graduate student in biomedical engineering in Dr. Martino’s Laboratory here at the University of Calgary. The Di Martino group investigates problems in tissue biomechanics, with a focus on aortic embolism mitigation.
Just a few months ago, I had very limited knowledge of microscopy. I had some idea of what I wanted to achieve by imaging my samples, but without knowing how to use a microscope and with no one in my lab currently imaging and able to train me, I was initially stuck on where to even begin. I was also intimidated by the idea of using a complex and expensive machine that I knew nothing about.
Luckily, since then, I have had the opportunity to learn from and collaborate with people who have much more experience in microscopy and experimental design for imaging. Learning about how a microscope works (at a basic level) was an important initial step for me, as it helped me to understand the relationship between the parameters I input on the computer screen and the output image that is generated. It also allows me to continuously examine and improve my imaging procedure if I’m not seeing the results that I want.
I have found that, surprisingly, the most challenging aspect of microscopy is not actually taking the images. It is the preparation that happens before.
One of my main imaging goals is to examine the architecture of collagen and elastin in aortic tissue and how it relates to the mechanical properties of the tissue. Most of the traditional sample preparation techniques I had read about required tissue fixation, which has been shown to alter the structure of the extracellular matrix. We wanted to avoid the use of fixation and slide preparation, as it was important for us to observe the natural, 3-dimensional orientation of structural components. This also introduced a challenge in that the tissue thickness (ranging from 0.6mm to 2mm) limited our options for staining, as it is difficult to confirm that stains would permeate to the depths we were imaging. In the process of discussing and defining these limitations, it became clear that second harmonic generation (SHG) using a multiphoton microscope would be the best method to use. On the multiphoton microscope, I can collect 3D image stacks of unfixed tissue, visualizing both collagen and elastin without the use of staining by taking advantage of collagen’s strong SHG signal, and elastin’s autofluorescence that is captured in a separate channel. Because of the minimal specimen preparation required, I can image my samples in a PBS bath immediately after mechanical testing, which keeps them as close to in vivo conditions as possible.
In summary, defining my imaging priorities was a critical step in approaching my experimental design. Learning the basic principles of the microscope allowed me to understand the limitations that emerge from using unfixed, unsectioned samples and work to find equipment and procedures that accommodate this. Most importantly, finding people who could answer my questions and discuss with me throughout the experimental design process has been invaluable in getting me on the microscope collecting images.
Interested in learning more about SHG and multiphoton microscopy? Recommended readings here.