Automated Fluorescence Cell Counting
LUNA-FL™ DUAL FLUORESCENCE CELL COUNTERdownload pdf
To determine cell viability, dye-exclusion methods have been widely used. Popular dyes used for cell viability are trypan blue and methylene blue. Because stained dead cells are easily discriminated using bright field imaging, this technique is successfully used in most automated cell counters including the LUNA™ automated cell counter (see “How to Count Cells: An Overview of Cell Counting Methods” for general review).
Although bright field image analysis has been successfully applied to most mammalian cell counting, a lot of cell or sample types remain challenging. For example, primary cells and peripheral blood mononuclear cells (PBMCs) are recognized as difficult-to-count cells with the bright field image analysis. Typically, these cells are mixed with a large population of red blood cells, which can be mistakenly counted as dead cells in bright field image analysis. Other challenging cell samples include: yeast cells due to their small size and sperm cell because of regular impurities.
To overcome the current limitations of bright field image based cell counting, more sensitive and accurate cell counting methods are necessary. The LUNA-FL™ fluorescence cell counter is a recently introduced fluorescence cell counter used to count cells without the limitations of cell types.
- Figure 1. A diagram showing the principle of fluorescence from Pierce homepage.
- Figure 2. Spectra of Acridine Orange and Propidium Iodide from Invitrogen homepage.
- Table 1. Classification of nucleic acid-binding FL dyes
- Important factors to be considered for fluorescence cell counting include 1) cell membrane permeability, 2) fluorescence spectrum and 3) fluorescence strength. Because cell membranes consist of lipid bilayers containing long stretches of hydrocarbon fatty acid tails, they act as hydrophobic barriers of cells and have selective permeability to various molecules. In general, small, non-polar FL dyes can readily cross the cell membrane and stain nucleic acids whereas bulky, polar, especially charged dyes cannot. Acridine Orange (AO) and Propidium Iodide (PI) are good examples of cell membrane permeable and impermeable FL dyes, respectively (Fig 3).
- Figure 3. Chemical structure of Acridine orange(upper) and Propidium iodide(lower)
- Figure 4. Counting human peripheral blood with Luna-FL and the AO/PI kit.
- Figure 5. Counting mouse peripheral blood mononuclear cells (PBMCs) with Luna-FL and the AO/PI kit.
Despite the advantages of flow cytometric cell counting, the instruments are relatively expensive with costs ranging from $40,000 to over $100,000. Because of the complicated optical, fluidic and electro-mechanical configuration, users need to take special training before using it or core facilities need to hire a professional operator. Moreover, continuous maintenance is required for the acquisition of reliable data, which includes de-clogging, optical alignment and laser adjustment. For these reasons, they are not widely used for general cell counting applications.
The concept of image-based cell counting was implemented by combining fluorescence microscopy and computer software for digital image analysis (7, 8 and 9). To better fit the need for automatic cell counting, dedicated cell counters have started to come into the market, which are equipped with a fluorescence microscope module and an image analyzing/counting algorithm. The current trend is that the instruments are designed in a small, all-in-one configuration so that they can be employed in space-limited environments, and the instrument/consumable price is affordable by most laboratories.
When compared to flow cytometers, image-based cell counters are not as versatile as their flow-based counterparts, but they have more capability than required for general purpose cell counting. They are more affordable and easier to learn than the flow-based system, and essentially maintenance-free. Another noteworthy advantage over flow cytometry is that cells being counted can be visually inspected so users can verify counting accuracy in real-time. The presence of any cellular debris and abnormal cell morphology can be checked as well.
Currently, a number of fluorescence cell counters are commercially available ranging from simple (a single fluorescence channel) to comprehensive models (up to seven fluorescence channels). They differ in their hardware (size, measuring volume, built-in monitor and counting principle; image- or flow-based), software (speed, de-clustering and image cytometry capability), list price and running cost (the price of consumables per count).
- Using the LUNA Reusable Slide for accurate cell counting with automated cell counters
- Fast automated yeast cell counting algorithm using bright-field and fluorescence microscopic images
Quick Start Guide2018-12-05
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