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Counting unstained, confluent cells by modified bright-field microscopy
 
L. Louis Drey, Michael C. Graber, and Jan Bieschke
Department of Biomedical Engineering, Washington University in St. Louis, St. Louis, MO


L.L.D.'s current address is St. Louis, MO, USA
BioTechniques, Vol. 55, No. 1, July 2013, pp. 28–33
Full Text (PDF)
Abstract

We present a very simple procedure yielding high-contrast images of adherent, confluent cells such as human neuroblastoma (SH-EP) cells by ordinary bright-field microscopy. Cells are illuminated through a color filter and a pinhole aperture placed between the condenser and the cell culture surface. Refraction by each cell body generates a sharp, bright spot when the image is defocused. The technique allows robust, automatic cell counting from a single bright-field image in a wide range of focal positions using free, readily available image-analysis tools. Contrast may be enhanced by swelling cell bodies with a brief incubation in PBS. The procedure was benchmarked against manual and automated counting of fluorescently labeled cell nuclei. Counts from day-old and freshly seeded plates were compared in a range of densities, from sparse to densely overgrown. On average, bright-field images produced the same counts as fluorescence images, with less than 5% error. This method will allow routine cell counting using a plain bright-field microscope without cell-line modification or cell staining.

In cell culture experiments where the effect of one or more chemicals on cell number must be assessed, it would be ideal to know precisely how many cells have been plated into a well or a subregion of a well at the beginning of an experiment, how many cells were alive days later before addition of the chemical, and how many cells were present at the end of the experiment. This is especially true when subject chemicals affect cell proliferation, yet the experiment aims to quantitate a different parameter such as metabolic activity, which then needs to be normalized against cell number.

Counting cells is required when adherent mammalian cells are cultivated for numerous other experimental applications such as measuring protein overexpression or RNAi gene silencing. Each procedure may affect cellular growth, skewing measurement of the effects. When cells must be harvested and replated one or more times, traditional cell counting with a hemocytometer (1) can be time-consuming and error-prone.

Fluorescence microscopy is the most widely used method to visualize and quantitate cellular proteins. When cell proteins are quantitated, usually it is necessary to normalize the data as a ratio of cell number against total protein content. Cell counting from fluorescence images may readily be achieved (2). However, when common nuclear dyes such as Hoechst 33342 or DAPI are used, counts may be less than precise if the dyes themselves reduce cell viability or affect growth rates (2). Hence, such methods can impinge on data and thus skew results.

Alternately, cells may be engineered to express a nuclear protein, such as the histone protein H2B, that is fused to green fluorescent protein (GFP) in order to provide highly accurate cell counts (3). With this approach, creating new stable cell lines before every experiment may be required. Since GFP fluorescence microscopy uses one fluorescence channel, immunofluorescence analysis of other proteins may also be limited.

Such difficulties have prompted a search for alternative methods of cell counting using bright-field microscopy. Generally, bright-field microscopy of flat, adherent cells suffers because cultured cells are transparent. As a result, contrast is very poor, particularly when imaging is done in the growth plane itself. Several software-based algorithms have recently been developed to improve contrast in bright-field images (4-6). Counting cells is relatively easy for naturally round, individually growing cells such as yeast (7). However, when flat, adherent cells must be quantitated, digital holography (8), z-projection of multiple z-stacked images (9), or intensity derivation (10) may be required to improve contrast and allow cell counting. These methods require acquisition of multipleimages followed by application of an image analysis algorithm, making them best suited to automated, high content screening microscopy.

Method summary

Unstained adherent cells are illuminated with monochromatic light through a pinhole aperture. In defocused bright-field images, each cell creates a bright spot that allows easy automated cell counting from single images. Contrast and counting accuracy can be further enhanced by brief swelling of cells in PBS.

Here we present a simple procedure to generate high-contrast images of flat, adherent and possibly confluent cells. The resulting images can be analyzed with free, readily available software tools, such as the ITCN plugin for ImageJ (11) or CellC analysis software (12). Cells may be counted directly from single bright-field images. Our approach allows users to count cells using a standard microscope present in most cell culture laboratories. Furthermore, it does not necessitate tagging of cell lines with fluorescent dyes or any other method of nuclear staining.

Methods

Cell culture

Cell culture plates were pretreated with 0.1% poly-L-lysine solution for 15 min, washed 4 times, and stored at 4°C. Human neuroblastoma (SH-EP) cells expressing the GFP-tagged nuclear histone H2B protein (SHEP-GFP) were cultured in DMEM supplemented with 4.5 g/l D-glucose, 10% fetal bovine serum, 100 IU/mL penicillin, and 100 µg/mL streptomycin at 37°C/5% CO2. SHEP-GFP cells were plated at densities of 50,000–5 million per well in 12-well plates and grown overnight. Live cells were imaged directly in culture media. Alternatively, the culture media was suctioned and replaced by PBS for 15 min prior to imaging to induce swelling of cells.

Imaging

Images were acquired on an Olympus IX70 inverted fluorescence microscope (Olympus America Inc., Center Valley, NJ), using a 4×/0.13 NA Uplan air objective and a 2 megapixel Olympus MicroFire CCD camera. Parallel GFP fluorescence and pinhole illumination images were recorded for each growth region. Fluorescence images were recorded in-focus at exposure times of 300 ms, using the Fluorescein/ GFP filter cube (U-MNIB). For pinhole illumination, cells were illuminated by the tungsten halogen lamp at full power with the condenser fully open, without phase rings or filters in the illumination path.

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