# Chip-Based Cytometry Illuminated by a Blade-Shape Continuous Light for Multispectral Detection
## 3 Results and Discussion
### Discrimination for Particles and Bio-Cells  
The ability of the developed cytometer to discriminate and count microspheres of different types and sizes was evaluated using a mixed sample comprised of green fluorescent-labeled particles (7 \(\upmu\)m), red fluorescent-labeled particles (15 \(\upmu\)m), crimson fluorescent-labeled particles (10 \(\upmu\)m), and non-labeled particles (12 \(\upmu\)m). Figure 6a presents the results obtained for the four sub-population clusters within the sample when classifying the particles in terms of the green, red, and crimson fluorescent indices. For clarity, Figure 6b-d present the 2D scatter plots obtained by plotting the three fluorescent indices against the side-scatter index. In general, the results confirm the ability of the proposed cytometer to simultaneously detect and count particles with different fluorescence-labels and sizes using a single white light source and no filter sets.  
Figure 6: Flow cytometer detection results for the mixed sample composed of green-, red-, crimson-labeled and non-labeled particles: (**a**) 3D scatter plot without side-scatter index; (**b–d**) 2D scatter plots with different fluorescent and side-scatter indices.  
The feasibility of the proposed cytometer for practical bio-detection applications was evaluated using a sample comprising a mixture of Trypan-blue and Erythrosin-bluish stained AGS cells and unstained AGS cells. Figure 7 presents the dark-field images and detected spectra of the different cells. The dark-field images confirm the ability of the proposed illumination system to provide an effective excitation source for cells stained with different dyes. For microsphere samples, scattering occurs only at the boundary. However, cells and bio-samples contain both water and cytoplasma with a refractive index close to that of the buffer solution. As a result, the scattering phenomenon occurs not only at the boundary of the cells or bio-samples, but also at various points on their surface. Consequently, the dark-field images shown in Figure 7 exhibit a homogenous scattered color expression. When the cells are illuminated by the white light excitation source, certain wavelengths are absorbed (depending on the absorbance properties of the particular dye), whilst others are reflected or scattered. The Erythrosin-bluish stained cells absorb blue-green light (400-550 nm), and therefore have a red color when illuminated by the excitation light (see insets of Figure 7a). Similarly, the Trypan-blue-blue stained cells absorb green-red light (550-700 nm), and therefore have a blue appearance. The non-labeled cell yields a peak intensity at a wavelength of around 510 nm due to the absorption of water at the near infrared range. As a result, it has a light green color.  
Figure 7b presents the 3D scatter plots of the mixed sample shown in Figure 7a. The results confirm that the information-rich spectra obtained using the proposed dark-field illumination configuration enable the multi-parameter analysis and characterization of bio-samples containing cells or particles with different dimensions, optical properties, and dye labels (i.e., fluorescence or absorbance). As a result, the system provides a simple and low-cost alternative to conventional multi-color cytometer systems based on multiple laser illumination sources and filter sets.