Automated Fluorescence Cell Counters for Cell Counting and Viability Analysis

From the late 1990s, a range of image-based automated cell counters have been launched that provide precise data on cell number and viability. These counters essentially use a combination of image analysis software and light microscopy. A digital camera captures magnified cell images and software algorithms are used to precisely detect cells and distinguish them from non-cellular objects such as cell debris.

Bright Field Imaging

LUNA-FL Dual Fluorescence Cell Counter

Figure 1. LUNA-FL Dual Fluorescence Cell Counter

In order to determine cell viability, dye-exclusion techniques have been extensively employed. Two examples of well-known dyes used for this purpose are methylene blue and trypan blue. Due to the ease of discriminating stained dead cells using bright field imaging, most automated cell counters use this technique, including the LUNA-FL automated cell counter (Figure 1) which provides dual fluorescence counting as well as bright field counting.

While bright field image analysis has been effectively applied to most mammalian cell counting, many sample or cell types are still difficult to count.

For instance, the technique is difficult to apply to peripheral blood mononuclear cells (PBMCs) and primary cells. These cells are usually mixed with large amounts of red blood cells, which may be counted as dead cells in bright field image analysis.

Other difficult-to-count cell samples include sperm cells due to regular impurities and yeast cells due to their small size.

To overcome these restrictions of bright field image-based cell counting, more accurate and sensitive cell counting techniques are required. The LUNA-FL dual fluorescence cell counter provides a new cell counting technique that is not limited by cell type.

Fluorescence Dyes for Cell Counting

The fluorescence dyes used in nucleic acid staining can be categorized according to their chemical, physical and fluorescent characteristics as summarized in the table below:

Table 1. Classification of nucleic acid-binding FL dyes

Chemical nature

Cyanine

TOTO, TO-PRO, SYTO family

Phenantbridine

Etbidium bromide (EtBr) Propidium Iodide (PI)

Acridine

Acridine Orange (AO)

Indole and Imidazole

Hoechst dyes, DAPI

DNA binding mode

Intercalating

EtBr, PI, AO

Minor-groove binding

Hoechst dyes, DAPI

fluorescence color

Blue

EtBr, Hoechst dyes, DAPI

Green

AO

Red

PI

Cell membrane

Permeable

AO, Hoechst dyes, DAPI, SYTO family

Impermeable

PI, EtBr, TOTO, TO-PRO family

In fluorescence cell counting, important factors that need to be considered include:

  • Cell membrane permeability
  • Fluorescence strength
  • Fluorescence spectrum

Cell membranes are made up of lipid bilayers comprising long stretches of hydrocarbon fatty acid tails, and they therefore serve as hydrophobic barriers that are selectively permeable to different molecules. Generally, small, non-polar fluorescence dyes can easily cross the cell membrane and stain nucleic acids, whilst large, polar, charged dyes cannot cross.

Examples of cell membrane permeable and impermeable fluorescence dyes are Acridine Orange (AO) and Propidium Iodide (PI), respectively (Figure 2).

Chemical structure of Acridine orange (upper) and Propidium iodide (lower).

Figure 2. Chemical structure of Acridine orange (upper) and Propidium iodide (lower).

Cell membrane permeable fluorescence dyes are capable of staining the nucleic acids inside both dead and live cells, meaning the entire cell population can be visualized. Cell membrane impermeable fluorescence dyes, on the other hand, can only penetrate and visualize dead cells that have compromised membrane integrity.

Combining AO/PI dual staining with an automated cell counting system can therefore distinguish dead cells from live cells as well as accurately determining their numbers.

Application of Fluorescence Automated Cell Counters

Counting human peripheral blood with Luna-FL and the AO/PI kit.

Figure 3. Counting human peripheral blood with Luna-FL and the AO/PI kit.

Fluorescence-based cell counting provides a useful alternative for those applications where bright field-based cell counting is not viable. Figure 3 shows an example of counting white blood cells in peripheral blood.

Not only are blood cells already of a small size for bright field-based cell counting, but the white blood cells are mixed among a vast amount of anucleated red blood cells and platelets.

Using automated cell counting with AO/PI dual staining, the red blood cells and platelets can be excluded from the total cell count. Since mature red blood cells and platelets lose their nuclei during cell differentiation, they do not stain with either dye.

Counting mouse peripheral blood mononuclear cells with Luna-FL and the AO/PI kit.

Figure 4. Counting mouse peripheral blood mononuclear cells with Luna-FL and the AO/PI kit.

Using the fluorescence-based automated cell counter, otherwise tedious, time-consuming and error-prone white blood cell counting can instead be carried out easily. Figure 4 shows an example of counting PBMCs in the presence of contaminating red blood cells and platelets.

Flow Cytometry versus Image-Based Cell Counting

At present, flow cytometry and image-based analysis are the two main techniques used to count fluorescently labelled cells. One of the most powerful tools for analysing cells through fluorescence detection is flow cytometry. Conventional flow cytometers cannot adequately determine cell numbers because they do not measure the volume of liquid passed while fluorescently labelled cells are being counted.

In contrast, volumetric flow cytometry can be used to count the absolute number of cells in unit volumes of liquid and give highly accurate cell counts that many researchers have shown to be comparable to the numbers achieved with hemocytometers.

Traditional flow cytometers can be used to determine the absolute cell number, provided fluorescence beads with a known number are combined with experimental samples with an unknown number. The cell numbers achieved from the direct volumetric flow cytometry and the bead-based estimation are not statistically different.

Although flow cytometric cell counting provides a number of benefits, the instruments are very expensive. Moreover, due to the complex fluidic, optical and electro-mechanical configuration of the devices, special training is required prior to using the instruments. The instruments also require continuous maintenance if the data obtained is going to be reliable. Therefore, flow cytometers are not commonly used for general cell counting applications.

The idea of image-based cell counting was executed by integrating computer software for digital image analysis and fluorescence microscopy. In order to accommodate the need for automatic cell counting, dedicated cell counters were introduced to the market that have both an image analyzing/counting algorithm and a fluorescence microscope module.

Although image-based cell counters are not as flexible as flow-based cell counters, they have more capability when it comes to general purpose cell counting. They are also cost-effective, easy to use, and basically maintenance-free. Another benefit over flow cytometry is that the counted cells can be checked visually so users can validate the precision of the counting in real-time. The presence of any abnormal cell morphology and cellular debris can also be checked.

Conclusion

The LUNA-FL dual fluorescence cell counter has been specifically designed to provide precise and sensitive live/dead cell counting results without being restricted by sample type. The instrument captures and inspects three different images: red fluorescence, green fluorescence and bright field. It is also cost-effective and based on precise and sensitive fluorescence staining techniques to deliver a reliable cell counting solution.

About Logos Biosystems, Inc.

Dedicated to the development and commercialization of innovative technologies to support life science research community. Founded at year 2008, Logos Bio is developing a series of lab automation systems and imaging instruments. The primary commercial target of Logos Biosystems is cell biology and molecular biology.


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Last updated: Mar 29, 2019 at 7:00 AM

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