# Chip-Based Cytometry Illuminated by a Blade-Shape Continuous Light for Multispectral Detection
## 3 Results and Discussion  
### Spectral Analysis and Performance Measurement  
The discrimination capability of the micro-flow cytometer was evaluated using a mixed sample comprising green fluorescent-labeled beads (468/508 nm), red fluorescent-labeled beads (542/612 nm), crimson fluorescent-labeled beads (625/645 nm), and non-labeled beads (blank) with diameters of 5, 15, 10 and 12 \(\upmu\)m, respectively. Figure 4a presents a dark-field image of the particles captured through the eyepiece lens of the microscope using a digital camera (SP-350, Olympus, Center Valley, PA, USA) with an exposure time of 25 ms. The image clearly shows a scattering phenomenon with different color expression at the boundary of each polystyrene bead due to the difference in refractive indices of the particles (1.59 at 589 nm) and the sample solution (deionized water, 1.33 at 589 nm), respectively. It is noted that the scatter patterns correspond to the pattern of the light-stop film. Figure 4b presents the detected wavelength spectra of the labeled and non-labeled particles. The difference in the four spectra enables the different particles to be reliably identified. Specifically, the green, red, and crimson fluorescent-labeled particles result in a scattering of the green, red, and blue light, respectively. By contrast, the non-labeled particles simply reflect the white excitation light, i.e., the normalized detected spectrum is identical to the excitation spectrum. It is observed that the spectra of the green and red fluorescent-labeled particles exhibit slighter absorption at the shorter wavelengths than the emitted florescence corresponding to the excitation wavelength of each fluorescence label. However, in general the emission spectra of the green and red fluorescence labels dominate the overall color expression of the particles in the dark-field image. Therefore, the color expressions of the green- and red-labeled particles correspond to the detected spectra (i.e., green fluorescence at 502 nm and red fluorescence at 580 nm). By contrast, the spectrum of the crimson fluorescent-labeled particles exhibits strong and broad band absorption at wavelengths between 550 and 650 nm and has a fluorescence peak at 690 nm. Figure 4: (**a**) Images obtained under diascop illumination of green-labeled (5 \(\upmu\)m), red-labeled (15 \(\upmu\)m), crimson-labeled (10 \(\upmu\)m), and non-labeled (12 \(\upmu\)m) polystyrene beads. (**b**) Corresponding detected wavelength spectra. Note that the spectrum for the non-labeled particle in (**b**) was used as reference spectra and was used to calculate the fluorescent, absorbance, and side-scatter indices. Although the detected spectra provide the means to distinguish between the different fluorescent-labeled particles, real-world cytometry applications require a more quantitative analysis of the sample. Accordingly, the sampled spectrogram data acquired in the previous experiments were processed using a simple linear superposition scheme in order to extract the corresponding absorption, fluorescence, and side-scatter information. In general, the detected spectrum, \(I_{\text{d}}\) (\(\lambda\)), (i.e., the lines for non-labeled particle in Figure 4b) composed of absorption and fluorescence spectral signals can be expressed as  
\[I_{\text{d}}(\lambda)=I_{\text{abs}}(\lambda)+I_{\text{fluor}}(\lambda)+I_{ \text{ss}}(\lambda) \tag{1}\]  
where \(I_{\text{abs}}\) (\(\lambda\)) and \(I_{\text{fluor}}\) (\(\lambda\)) are the absorbance (negative sign) and fluorescence (positive sign) spectra of the specific fluorophore probe, respectively, \(I_{\text{ss}}\) (\(\lambda\)) is the side-scatter (positive sign) spectrum (i.e., the line for the labeled particle shown in Figure 4b which is identical to the spectrum of non-labeled particles for the reference of the linear superposition calculation), and \(\lambda\) is the wavelength.