Ask the Expert

Spectroscopic Imaging for Tissues

Dr. Nancy Pleshko discusses her work in applying spectroscopic techniques to better understand how connective tissues and other types develop, and to understand what happens in disease states and with therapies

Rachel Muenz

Dr. Nancy Pleshko is a professor in the Department of Bioengineering and director of the Tissue Imaging & Spectroscopy Laboratory at Temple University (Philadelphia, PA). Dr. Pleshko is recognized for her research on the assessment of tissues at the molecular, cellular, and structural level through application of state-of-the-art vibrational spectroscopy. She has substantial expertise in basic and translational research in connective tissue pathophysiology and orthopedics, including osteoporosis and other bone pathologies, and osteoarthritis and cartilage repair. She has over 100 publications, and has received more than $6 million in federal funding.

Q: I see that your lab works with a variety of different types of tissues. What is the lab’s main mission?

A: We’re interested in applying spectroscopic techniques to help us understand how connective tissues and other types develop, and to understand what happens in disease states and with therapies. That involves applying spectroscopy to look at tissue matrix and sometimes cellular activity as well. Our primary mission is applying and developing spectroscopic techniques to evaluate tissue development and pathology.

Q: How many people work in your lab?

A: Right now, our lab has about 15 people. I have two postdocs, four grad students, and many undergrads. That is possibly one of the unique things about my lab, the number of undergrads; but in our department, we are very focused on giving our undergrads research experiences, and we have a lot of very enthusiastic students. They really add a lot to everyone’s lab, either by helping out the grad students and the postdocs or by eventually having their own projects.

Q: What major types of spectroscopy do you use in your lab?

A: We work with infrared spectroscopy in the mid- and near-infrared [NIR] range and we use spectroscopic imaging and fiber optics. We probably use those [technologies] about the same amount in our lab, but for different applications, of course. For the fiber optics, one of our main projects is to use NIR to follow the development of engineered tissues in situ as they are growing in the lab. The spectroscopic imaging comes into play in that project to help us understand the fiber optic data, so we typically will be evaluating tissues in situ or as they’re growing and then we use spectroscopic imaging to get a more detailed understanding of how they’re developing. For example, [part of one project involves looking at] the distribution of proteins in the tissue as they’re growing over a two-month period. We can see that very nicely with the spectroscopic imaging data. We also use spectroscopic imaging to look at pathology. For example, in preclinical disease models, we use it to try to understand differences in the tissues at the micron level, between normal and diseased states. We’re interested in different bone diseases, so we use this [technology] a lot to look at what’s different in the bone and if it’s responding to a specific therapy and how the molecular structure of the bone is changing in response to therapy.

Q: What are some of the key challenges you run into in your work with spectroscopy?

A: I would say one of the key challenges is that in every project, the methodology is slightly different. So, for almost every project that we work on, we have to develop the method to optimize our data collection. That’s one of the key challenges—optimizing the method to make sure the data we’re collecting is robust. Then, the other main challenge is keeping the spectrometer up and running and happy. Our imaging spectrometer is a PerkinElmer Spotlight 400, and it’s a great instrument, but we definitely have to maintain it. If you’re collecting data and you have a whole day of data collection planned, and then all of a sudden you run into an error in the instrumentation, sometimes you just have to be patient and restart. But I think that’s very typical for any high-end instrumentation—it doesn’t always work perfectly.

Q: How do you deal with those challenges?

A: To deal with those challenges, it’s just a matter of being aware that you’re going to have those challenges and having a very good relationship with your field engineer, which we do.

Q: What are some of the changes or trends you’ve noticed recently in spectroscopic imaging?

A: The main change is the trend to go toward faster data collections, and data collection at higher-pixel resolution. With a lot of the new instruments, the sources are different; for example, now we have quantum cascade laser sources for spectroscopic imaging, so you can get better data because the power is much higher. Also, the pixel resolution is now better because nano-IR spectrometers [are available] as opposed to just a micron-level pixel imaging spectrometer. Things are just basically getting faster and the resolution is getting better.

Q: How have those changes affected your lab?

A: In my lab, when I first started, there was no imaging capability. That was in the early 90s, and the imaging capability was just starting, so it was really just an infrared microscope with pixel-by-pixel data collection, as opposed to imaging. Since the imaging capability has been available, we’ve taken advantage of that, and now the instrumentation has gotten much better and faster. We have not gone to the level of purchasing, for example, a nano-IR to increase the spatial resolution that we can get our data at, but we would like to.

Q: What advice do you have for lab professionals who are setting up a spectroscopic imaging lab or are getting into a similar line of work?

A: What’s most important is choosing an instrument that fits the needs of your lab and your research goals, because right now there are so many choices for spectroscopic imaging. Before you purchase an instrument, you really have to decide what’s most important; is it the high pixel resolution, is it faster data collection, is it the frequency range you’re going to be collecting your data in? The different spectrometers have all these options and there are different price points of course, so you really have to make sure you know that you’re getting the instrument that’s going to best suit what you’re going to be doing.

Q: How do you expect things to change farther into the future, when it comes to spectroscopic imaging?

A: Definitely, we’re going to be getting the capability to image smaller and smaller regions. So, right now, the best that we can do with the infrared technology is about 50–100 nanometers, that’s not with the instrument that [our lab has] but with other instruments we would like to have. I’m guessing that down the road, we’ll be getting down to the actual nanometer level and that is going to be incredibly helpful for looking at things like cellular processes and how cells are responding to therapeutics or changing in disease states. I think that will be very helpful in the healthcare industry and, presumably, in the polymer industry where there are complex materials that have heterogeneous structures at that level. That’s probably going to be the main advance, just getting really nice resolution.

Q: You mentioned earlier that your lab is looking into getting a nano-IR. Did you have any other plans for new technologies or projects for farther down the road?

A: One thing we would like, and I think a lot of researchers would be very excited to have, is the capability of imaging in a fiber optic modality, essentially infrared spectral imaging in a fiber optic. That seems to be a little bit slower in development. Right now, we are using fiber optics and they’re working, and using our imaging spectrometer is working, but we would definitely like the opportunity to combine the two.

Q: Is there anything else you wanted to add?

A: Just a further comment on the fiber optic imaging. The reason that a development in that area would be useful for infrared in particular is the ability to use that in vivo in, for example, evaluation of tissues and tissue margins, such as in definition and extraction of tumors. There are some other microscopic techniques that are being developed for that, such as fluorescence microscopy, where you can use that in vivo. The development of that [technology] for infrared spectral imaging would be a really great advance because then you would have the ability to look at molecular-level information in vivo during surgeries. Right now, people are working on that just with fiber optics, which is essentially collecting one spectra at a time, so it would be really nice to do that with imaging. Just overall, in terms of running the lab, any imaging spectrometer is a piece of equipment that requires maintenance and taking care of it every day and, given that, it will probably be very reliable for a very long time.