An Alternative Approach to Detecting Cancer Cells by Multi-Directional Fluorescence Detection System Using Cost-Effective LED and Photodiode

Abstract

The enumeration of cellular proliferation by covering from hemocytometer to flow cytometer is an important procedure in the study of cancer development. For example, hemocytometer has been popularly employed to perform manual cell counting. It is easily achieved at a low-cost, however, manual cell counting is labor-intensive and prone to error for a large number of cells. On the other hand, flow cytometer is a highly sophisticated instrument in biomedical and clinical research fields. It provides detailed physical parameters of fluorescently labeled single cells or micro-sized particles depending on the fluorescence characteristics of the target sample. Generally, optical setup to detect fluorescence uses a laser, dichroic filter, and photomultiplier tube as a light source, optical filter, and photodetector, respectively. These components are assembled to set up an instrument to measure the amount of scattering light from the target particle; however, these components are costly, bulky, and have limitations in selecting diverse fluorescence dyes. Moreover, they require multiple refined and expensive modules such as cooling or pumping systems. Thus, alternative cost-effective components have been intensively developed. In this study, a low-cost and miniaturized fluorescence detection system is proposed, i.e., costing less than 100 US dollars, which is customizable by a 3D printer and light source/filter/sensor operating at a specific wavelength using a light-emitting diode with a photodiode, which can be freely replaceable. The fluorescence detection system can quantify multi-directional scattering lights simultaneously from the fluorescently labeled cervical cancer cells. Linear regression was applied to the acquired fluorescence intensities, and excellent linear correlations (R² > 0.9) were observed. In addition, the enumeration of the cells using hemocytometer to determine its performance accuracy was analyzed by Student's t-test, and no statistically significant difference was found. Therefore, different cell concentrations are reversely calculated, and the system can provide a rapid and cost-effective alternative to commercial hemocytometer for live cell or microparticle counting.

Keywords: cancer cells; fluorescence detection system; light-emitting diode; photodiode.

Figure 1

Representative R6G treated images of HeLa cells under a microscope with 200 magnification (a) bright field image; (b) fluorescence image; (c) merged image of fluorescently labeled HeLa cells.

Figure 2

Proposed fluorescence detection system. Light-emitting diode (LED) with specific wavelength excites the HeLa cells sample through the excitation filter (ex filter). Emission light including side scatter (SSC) and forward scatter (FSC) is collected through the emission filter (em filter). Collected light is analyzed and displayed on the liquid crystal display (LCD) module.

Figure 3

Computer aided design (CAD) of the main module: (a) top 3D view including filter holder (gray region), LED & lens holder (purple region), cuvette holder (green region), and lens and PD holder (red region); (b) isometric view of cuvette holder, lens, and PD holder parts.

Figure 4

Schematic design of the fluorescence detection system.

Figure 5

Data acquisition procedure of fluorescence detection system for a single filter set and load resistor.

Figure 6

Comparison of the SSCs and FSCs of the fluorescence intensities of HeLa cells using one of the three different load resistors (100, 200, and 390 kΩ) with a specific filter set: (a) no excitation filter, but only emission filter (center wavelength (CWL) at 500 nm); (b) excitation filter of CWL at 480 nm with emission filter of CWL at 500 nm; (c) excitation filter of CWL at 470 nm with emission filter of CWL at 500 nm. RSD (%) was calculated for each concentration group of stained HeLa cells with one of the selected load resistors.

Figure 6

Comparison of the SSCs and FSCs of the fluorescence intensities of HeLa cells using one of the three different load resistors (100, 200, and 390 kΩ) with a specific filter set: (a) no excitation filter, but only emission filter (center wavelength (CWL) at 500 nm); (b) excitation filter of CWL at 480 nm with emission filter of CWL at 500 nm; (c) excitation filter of CWL at 470 nm with emission filter of CWL at 500 nm. RSD (%) was calculated for each concentration group of stained HeLa cells with one of the selected load resistors.

Figure 7

Representative R6G treated images of HeLa cells on the ruled hemocytometer chamber under a fluorescence microscope and its performance results: (a) merged image of bright field image and fluorescently labeled HeLa cells; (b) fluorescence intensity of HeLa cells using 390 kΩ load resistor with no excitation filter, but only emission filter (center wavelength (CWL) at 500 nm).