Neon C. Jung, Ph.D.
Over the last decades, manual cell counting using a hemocytometer and light microscope has been widely used to count cells. This technique, however, is time consuming, tedious and error-prone. Image-based cell counters utilize light microscopy technology, but offer increased accuracy, reproducibility, speed, and also lend themselves to automation. Small sample volumes are another perk of image-based cell counters. Many of these systems utilize disposable slides that typically have 100 um chamber depths, allowing only 10 uL sample volume containing a suspension of live cells. The slides are inserted into the automated cell counter and the image is captured by a camera attached to the microscope. The integrated software then analyzes the captured image to present accurate cell numbers and viability data.
Here are three major tips to consider when performing image-based cell counting.
Accurate focusing
Like other light microscopy, accurate focusing of the instrument is crucial for precise cell counting. During manual cell counting, a well-trained human brain can easily identify the cells. However, the camera inside an automated cell counter detects bright centers and dark edges of live cells and subsequently processes them into black and white pixel information. If a cell is not focused properly, this bright center will have a significant grey level and can be misinterpreted as a dead cell.
Most researchers will not find it difficult to focus on the cells as long as the image quality displayed on the monitor of the cell counter is similar to that of the conventional microscopes. And it’s worth taking the time to make sure you have a crisp image. Another point to be aware of is the resolution of the camera in your cell counter. If the automated cell counter displays a low frame rate, such as less than 15 frames/second, the displayed image cannot respond in real time to the tuning of the focusing knob. Make sure your counter is up to the task.
Control for slide-to-slide variability
Most image-based automated cell counters provide disposable, instrument-specific slides with a defined measuring area (field of view) for cell counting. This measuring area is set by the magnification of the objective lens and the size of the image sensor of the camera. These cell counters are designed to replace manual hemocytometer cell counting; they typically measure 0.4 - 0.5 uL sample volume which is equivalent to 4 - 5 large squares of the hemocytometer.
Unlike the glass hemocytometer, disposable slides are only for a single use. Therefore, differences between slides can significantly impact cell counting results. You can minimize these differences by using slides from the same lot number for a given set of experiments. Testing a few slides with a standard bead is also recommended (before changing lots, for example). Most cell counter manufacturers provide standard beads of known concentration. Using the same bead sample to test different slides (even from the same lot) will help to identify variations.
Evalute the declustering performance of the software
The core of the automated cell counter is the software that does the analysis, which is equivalent to the human brain of the manual cell counting. Most automated cell counters contain integrated software that offers decent counting accuracy for a well-isolated single cell population. However, cell counters can vary significantly when it comes to performance in counting clustered cells (Figure 1). Because samples obtained from the cultured cells do not always have well-defined single cell populations, and thus, sometimes have multiple cell clumps; the declustering performance of the automated cell counters must be carefully evaluated. To evaluate declustering, perform a test as described in Figure 1.
Conclusion
Automated cell counters are replacing tedious manual methods of cell counting. With attention to some of the basics, automated image-based cell counters will enable you to obtain optimal results with minimal stress.
Figure 1. De-clustering performance of automated cell counters. HL-60 cells were artificially killed by heating to 70°C for 20 minutes in order to obtain dead cell samples. The samples were then tested using two different automated cell counters. After the cell counting, circled images provided by each manufacturer’s own software were captured for comparison. Red circles: dead cells, Blue circles: live cells: Black circles: excluded objects.
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