January 30th - February 3rd
This week, I had the chance to reflect on the papers I used to create my literature review and I was trained on how to use the bioluminescence imaging system in person. Here, I will provide a run down of what BLI is and what I have learned thus far.
What is Bioluminescence Imaging (BLI)?
BLI is an imaging modality with high sensitivity and resolution that can be used both in vivo and in vitro to image cells stably expressing luciferase. It can be used for longitudinal cell tracking studies, such as those tracking cancer metastasis in mice models. Since mammalian cells do not exhibit endogenous bioluminescent light, BLI images in vivo and in vitro typically have low background signal. However, a major limitation of BLI when used in vivo is that tissue depth can attenuate the signal.
The cells of interest are first transfected with luciferase (for which there are different types). Cells stably expressing luciferase are either imaged in vitro, or injected into an animal model of interest. In vitro, cells stably expressing luciferase are seeded in a well plate and the substrate luciferin is delivered. Specific luciferin substrates are used with each type of luciferase. The luciferase enzyme stably expressed by the cells of interest catalyzes the oxidation of the added luciferin substrate to produce oxyluciferin, which is responsible for emitting bioluminescent light. A sensitive charge-coupled device (CCD) camera then captures the light (Sadikot & Blackwell, 2005).
Analysis of papers and reflecting on my study
Many papers run in vitro BLI experiments to quantify the lowest cell number that can be detected with this imaging modality. In my experiment, I am seeking to determine whether there are ways to improve the sensitivity of BLI to quantify lower cell numbers.
Two commonly used luciferase systems are Firefly luciferase (Fluc) and Akaluc. One paper reports quantifying down to a single cell in vivo in the mouse lung microvasculature using BLI with Akaluc-positive cells (Iwano et al., 2018). Additionally, another paper performed in vitro BLI with Fluc-positive circulating tumour cells in in blood samples. After optimization of their experimental procedures, they were able to quantify down to two cells using this system. Optimization included using FACS (fluorescence activated cell sorting) to select for the 5% brightest cell population amongst the Fluc-positive circulating tumour cells. This was based on the brightness of GFP fluorescence. A red blood cell lysis step followed this to simplify the blood sample (Sasportas et al., 2014). FACS sorting is a commonly used optimization procedure for BLI. When transfecting cells with luciferase, a reporter gene, typically GFP, is also added with the luciferase gene. GFP is fluorescent, thereby allowing a researcher to confirm the successful integration of luciferase in the cell's genome via GFP expression on a fluorescent microscope. FACS sorting then allows a researcher to select for the brightest population of cells.
In my experiment, I will be imaging on both BLI and MPI systems, hence it is helpful to review research papers which quantify BLI signal with cells that are also labelled with iron. Miyami and colleagues used BLI to quantify 1500 Fluc positive peritoneal macrophages labelled with SPIO that were collected from L2G85 transgenic mice (Miyami et al., 2012).
On an unrelated administrative note, this week I completed my online lab safety training required to work in the lab, and toured the animal facility. These admin tasks are important for my success with my research this semester.