7 Things You Didn’t Know About Imaging Cytometry
It has been said that “a picture is worth a thousand words.” We are visual creatures, and we seek to capture and describe the world around us. Some of the earliest evidence for this comes from very old cave paintings found around the world, like this painting of a horse found in the caves in Lascaux, France.
With the development of reliable microscopes, such as those developed by the dutch draper Antonie van Leeuwenhoek, we were able to see what was previously invisible, probing the unseen and learning in great detail how organisms worked.
Over time, the field of cytometry (the analysis of biological processes at the whole-cell level) has expanded in many different directions. Flow cytometry can be thought of as a microscope with very poor resolution. The power of flow cytometry lies in its ability to analyze thousands of cells through many dimensions, providing an amazingly detailed understanding of the cell. However, due to the resolution, it is not possible to tell where these signals are located.Read More
The Truth About Flow Cytometry Measurement Compensation
The topic of compensation is a critical one for the cytometrist to understand. It requires adherence to some specific rules, an understanding of how the instrument works, and how fluorescence occurs. Poor or incorrect compensation can easily lead to incorrect conclusions, and decreases the reliability and robustness of the data generated.
It is critical to question the wisdom of the “Protocol’s Book” and understand that the “truths” in this book are not always correct anymore. The new user doesn’t necessarily know any differently, and for this reason there are suboptimal practices that permeate flow cytometry experiments to this day.
Understanding compensation, and being armed with the knowledge, allows the researcher to combat those fairytales that continue to make their rounds in science. It is time to put them to bed and move forward with a full understanding of the process.Read More
Reproducibility In Flow Cytometry Requires Correct Compensation
Understanding the 3 rules of compensation, and applying them to your everyday workflows, is an essential step in good, consistent, and reproducible flow cytometry data. Making sure the controls are bright, and treated the same way, is essential. Don’t bring unfixed controls when your samples are fixed, as the controls will not reflect the spectra from the fixed samples. Make sure not to rely on the “Universal Negative”, use a single sample to set background, and collect enough events to make sure an accurate measurement is made, as this will further improve the quality of your control and therefore the data.Read More
3 Requirements For Accurate Flow Cytometry Compensation
For those new to flow cytometry, compensation is confusing at best and terrifying at worst. Likewise, those who have been doing flow cytometry since the analog ages may be holding on to practices that, while suited to the analog instruments, should be left to the annals of history. As such, a lot of time is spent discussing compensation and the best practices for this critical process. There are 3 rules that guide proper compensation, and they’ve been written about extensively since they first appeared in the “Daily Dongle” in 2011. Here, we will review the classic rules and expand upon the tacit assumptions required to fulfill them.Read More
5 Best Practices For Accurate Flow Cytometry Results
Here, we cover 5 lessons from the trenches of flow cytometry looking at important aspects of how best practices have changed over time, which practices need to be adopted, and which are outdated. Put those old, coffee-stained protocols away and take advantage of the best practices for digital instruments to write new and improved ones (coffee stains optional). Your data will thank you.Read More