The Basics

Why use neural stem cells?

In addition to the importance of studying the effects of the e-cigarettes on the brain, neural stem cells (NSCs) have well-defined spherical mitochondria and fast replication rates in vitro, making it a perfect choice for studying the mitochondrial stress response. The ones used in this study were taken from mice.

What is fluorescence microscopy?

Fluorescence is the absorption and subsequent re-radiation of light; these two occur almost simultaneously (MicroscopyU, n.d.). British scientist Sir George G. Stokes was the first to describe fluorescence in 1852 and he noticed that the wavelength of the fluorescence emission was always longer than that of the excitation light. This technique has become invaluable in biology for its ability to visualize cells and cellular components with a high degree of specificity. Chemicals termed fluorochromes or fluorophores are stains that are often specific in their attachment and are excited by specific wavelengths and emit light at various intensities.

In this experiment, the authors inserted a variety of highly specific plasmids that label specific organelles and tagged these plasmids with fluorescent proteins. The plasmids that they used include:

• mRFP-GFP-tagged LC3 – labels autophagosomes and is tagged with both red and green fluorescent protein
• mApple-LAMP1-pHluorin-N-8 – labels autolysosomes and is used to determine if an increase in pH will lower the activity or proteolysis
• mCerulean3-Mito-7 – labels mitochondria and produces a blue hue
• pCMV-CEPIA2mt – mitochondrial-tagged calcium reporter and measures level of calcium
• GCaMP5 – a calcium reporter used to evaluate calcium signalling in nicotinic acetylcholine receptors

They also used a plasmid called MitoTimer, a special tool for measuring mitochondrial turnover. This cunning tool is actually a mutant of a red fluorescent protein and its fluorescence changes from red to green as the protein matures. In this study, the authors used the ratio of red to green fluorescence to measure protein oxidation and aging of the cell.

E-liquids vs Aerosols – What’s the difference?

This study makes a distinction between the two, with E-liquids being measured in terms of concentration and aerosols being measured in terms of total puff equivalents. They’re basically the same thing, except that aerosols have gone through the smoking machine and are collected as vapour.

In The Lab: Imaging

CellProfiler Imaging Software

CellProfiler is a free, open-source image analysis software that is capable of handling hundreds of thousands of images a day. CellProfiler has many features that range from simple assays such as cell count to more complex assays such as localization of proteins. It has many benefits over human observation, such as being able to process such a high volume of images in a short amount of time, being able to produce quantitative versus qualitative measures, and being able to identify features not easily detectable by human observers. In addition, the fact that its code is open-source means that people can understand its methodology and add to it, testing and developing new methods for image analysis.

For more information about CellProfiler, check out this article.

CL-Quant Imaging Software

CL-Quant is another imaging software that has many applications such as tracking cell motility, generating growth curves, and automatic cell identification. It is also particularly useful for imaging live cells and measuring intensity of fluorescence, as seen in the article.

For more information about CL-Quant and some videos of it in action, check out this site.