It is possible to obtain non-contact images of cell surface topography using ion conductive microscopy (ICM). The technique has been recently used to image live cells in a culture medium. As cell volume control is the basis of the cellular homeostatic mechanism, cell volume is a significant factor for cell research. ICM allows to obtain precise volume measurement and quantitative dimension information of cells. Plus, cell volume changes caused by changes in the physiological environment or in the cell status can be determined over time.
In this article, Park ICM is used to successfully image the live HEK293T cell and monitor its volume changes.
A 293T cell from the human embryonic kidney (HEK) was cultured on a glass coverslip in dulbeco’s modified eagle medium (DMEM) having 10% fetal bovine serum (FBS), comprising 1% penicillin at 37 °C in a 5% CO2 humidified atmosphere from Multi Gas Cell Incubator, SANYO, JAPAN. The cultured cell coverslip was moved to the Live Cell Chamber present in the Park XE-Bio from Park Systems. During culture in the live cell chamber (37°C, 5% CO2 and above 70% humidity), a live images of the cell were obtained by ICM-ARS. The pipette used for the ICM was made from borosilicate glass (O.D., 1.0 mm, I.D., 0.58 mm, length 90 mm; Warner Instruments, US) with the help of a CO2-laser-based micropipette puller from P-2000, Sutter Instruments, Novato, US. The outer and inner diameters of the pipette were about 200 nm and 100 nm, respectively.
Figure 1. a) Sequential HEK 293T live cell images. It took approximately 0.6 hour to obtain each image and a total of 3.3 hours for 6 images. b) Volume change of HEK 293T: the initial cell volume was 5008 μm3; 2.6 hours later it shrank to 3645 μm3.
Cell Volume Analysis Method
The cell volume was determined from the ICM image data using Park XEI image processing software. After selection of the cell area, the volume was automatically calculated (Fig. 2).
Figure 2. Illustration of the process for the cell volume measurement. a) Park ICM image of HEK cell (0.6h) loaded at main frame of Park XEI software. b) Cell area selected as pink, marked as ‘1’. c) The volume of the selected cell area (pink, 1) calculated as 5395 μm3.
Figure 1 shows how well ICM measures the morphology of the cell in detail. The ICM images show the shape of the whole cell clearly including its delicate structures. The tiny projections around the cell or filopidia can also be seen clearly. They could not be distinguished precisely using conventional optical microscopy. Furthermore, the quantitative dimension information from the ICM allows accurate cell volume calculation as shown in figure 2. The cell volume measurement method by ICM offers more quantitative and precise results when compared to the method that controls reagents such as ions or fluorescence dyes with ion-sensitive microelectrodes or quantitative fluorescence microscopy.
In conclusion, the ICM method provides useful information to cell researches related to hypertrophy. The quantitative volume measurement offers a new method for analyzing and gauging the changes in the cell structure of cardiomyocyte which are frequently accompanied by changes in volume.
This information has been sourced, reviewed and adapted from materials provided by Park Systems Inc.
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