Flow Cytometry and Image-Based Cell Counting: Complementary Technologies to Achieve Accurate Cell Quantifications and Characterizations

Written by Ben Capozzoli, DeNovix Application Scientist

Understanding the number and characteristics of a given cell population is paramount to many cell/molecular biology studies. Many fields, like immunology, rely heavily upon the ability to quantify the number of cells in a population and the various characteristics those cells possess.

Hemocytometers: A Traditional Cell Counting Method

Over the years, a number of technologies have been developed to enable scientists to ask and answer these questions. One very old technique is the hemocytometer, which allows scientists to manually count cells in a specified volume. Simply, the different grids and squares on a hemocytometer are defined by precise surface areas. Surface area multiplied by the height of the cover slip above the grid provides a carefully specified volume.

Imprecision with hemocytometers is caused by a few factors that more modern technologies have improved upon. For example, relying on a human’s interpretation of the tiny objects on the grid introduces variability between users, which is further stratified by years of experience. Imperfect machining of the slide means that the chamber height is rarely a true 100 µm, which adds assumptions to the equation that can cause deviations from the true counted population. 

Modern Solutions for Cell Counting

Automated Cell Counters

Automated cell counters like the CellDrop overcome these challenges by utilizing an algorithm to count the exact same way each time. The CellDrop’s patented DirectPipette™ technology also utilizes high precision motors to set a precise chamber height of 100 µm that is nearly perfect every single time. Automated cell counters also benefit from rapid image acquisition, removing user bias and significantly improving consistency across experiments.

Flow Cytometers

Another modern solution for flow cytometry cell counting is flow cytometers. Flow cytometers analyze a large number of individual cells rapidly by intaking cells through a capillary, then passing each cell in a single file line past a series of lasers. These lasers detect fluorophores bound to the surfaces of the cells, in addition to forward scatter (cell diameter) and side scatter (cell density or volume). 

Flow cytometry allows high throughput analysis, rapidly characterizing thousands to millions of cells quickly, facilitating efficient data acquisition. Typically, analyses are set to run until a specified number of cells is reached, e.g., one million cells. Fluorescent antibodies that target unique surface proteins allow researchers to elucidate the percentage of cells in different subpopulations, identifying various cell types within the total sample analyzed.

Limitations of Flow Cytometers in Cell Counting

Like any technology, flow cytometers have limitations as well. While flow cytometers are excellent at knowing how many events have passed over the internal detectors, knowing the precise intake volume containing those events is less easily measured. For some flow cytometers, the internal fluidic pumps can be calibrated yearly to ensure the intake volume is precise enough to derive accurate cell counts. Often, the pumps are not easy to calibrate or not calibrated at all, which can lead to inaccurate cell/mL quantifications. Additionally, traditional flow cytometers typically lack the ability to provide spatial information or detailed single cell images.

Complementary Technologies: Flow Cytometry and Imaging

To overcome these limitations, experts often complement flow cytometry with imaging flow cytometry. Imaging flow cytometers combine high resolution image capture with traditional flow cytometry principles, allowing detailed flow cytometry image analysis. This provides valuable spatial information and quantification of fluorescence intensity across cell populations, significantly enhancing cell analysis capabilities. Imaging flow cytometry can also clearly differentiate red blood cells from nucleated cells, crucial for accurate cell counts.

Another practical solution used by many flow cytometry experts is counting cells with another method, such as an automated cell counter, before measurement on the flow cytometer. This combines the strengths of both technologies, providing precise quantifications and detailed characterization. 

Counting cells with fluorescent viability stains such as AO/PI produces clear fluorescent images, giving visual confirmation of cell viability status. For more details on how AO/PI staining provides reliable sample images for viability assessments, read this technical document. When the same cell population is subsequently analyzed by the flow cytometer, the percentage of cells belonging to each subpopulation becomes clear. This data can be combined with the live cell concentration to determine the number of cells in each category analyzed.

Summary

Every tool has a specific purpose where it excels. While many tools can be utilized to complete tasks they were not designed for, they often fail to perform as well as a tool that is purposely built for the job. 

This principle holds true for the different cell analysis methods in biology. No single tool is superior in all respects. Instead, each technology complements others, excelling in specific niches and providing optimal data. 

By combining powerful analysis tools such as image-based cell counting and flow cytometry, the most accurate cell population quantifications and detailed characterizations are achieved, significantly benefiting biomedical research.

Want to learn more about the CellDrop Automated Cell Counter? Speak to a DeNovix application scientist!