# Efficient Multifocal Structured Illumination Microscopy Utilizing a Spatial Light Modulator
## 3 Results
### Resolution Improvement by Imaging Biological Samples  
To investigate the performance of this system in biological applications, we began with imaging the fixed sample of Melosira (unicellular organism, usually shown as chain-like clusters). Figure 6a-i is the nine raw multifocal images of this sample. Figure 7 illustrates the MSIM performance compared with the conventional WF image. In comparison with the wide-field image shown in Figure 7a, the sum result of the nine raw multifocal images shown in Figure 7b shows the suppression of the background noise. Furthermore, the MSIM images shown in Figure 7c could distinguish the two chain-like structures which are blurred in the former two images as depicted in Figure 7d,e. To verify the accuracy and reliability of this experiment, the same magnified area of this sample was imaged using an objective with a higher numerical aperture (0.65). The result confirmed that there was a gap between the two chain structures, as shown in Figure 7h and its inset.  
Figure 5: Image results of three separate microspheres by wide-field (WF) microscopy (**a**) and MSIM (**b**). (**c**) Cross-section through one of the microsphere images in (**a**,**b**), respectively. Scale bars: 4 μm.  
Figure 6: Nine raw multifocal images (**a–i**) of the fixed sample of Melosira. Scale bars: 40 μm.  
In addition, we imaged a fixed transverse section sample (for teaching purposes) of earthworm (Figure 8) and esophagus (Figure 9), respectively. Compared with the uniform illuminated WF microscopy, the finer structures marked by the colored areas in Figures 8c and 9c are resolved by MSIM. For a quantitative analysis, the image modulation contrast \(I_{contrast}\) can be introduced by  
\[I_{contrast}=\frac{I_{max}-I_{min}}{I_{max}+I_{min}}, \tag{6}\]  
where \(I_{max}\) and \(I_{min}\) are the maxima and minima of certain regions in the images, respectively. Compared with WF, the contrasts of MSIM in Figures 8c and 9c are enhanced by a factor of 1.1319 and 1.2083, respectively. Based on SIM, only the high-frequency components of the sample modulated by MSIM are demodulated out to enhance the resolution of the final images, while the random noise of the background cannot be modulated by the patterned illumination. Once the high-frequency components representing the details of the sample are enhanced, the overall amplitude of the fluorescent signal-to-noise ratio (SNR) will be improved. Therefore, our structured illumination modality and reconstruction approach could achieve great performance with high image contrast.  
Figure 8: Results of the fixed partial transverse section of earthworm (dehydration structure of body cavity fluid) by WF microscopy (**a**) and MSIM (**b**). (**c**) Intensity profiles along the colored dash lines in (**a**,**b**), respectively. Scale bars: 40 \(\upmu\)m.  
Figure 7: Comparison of WF (**a**) and MSIM imaging (**c**) of the fixed sample of Melosira. (**b**) The sum result of nine raw multifocal images. (**d–f**) The magnifications of the colored boxed regions in (**a–c**), respectively. (**g**) Normalized intensity profiles along the respectively colored lines in (**a–c**), (**h**) the inset is the same areas in (**d–f**) observed by an objective with a higher numerical aperture (0.65) and the plot is the normalized intensity profile along the dashed line in the inset. Scale bars in (**a–c**): 40 \(\upmu\)m, (**d–e**) and inset in (**h**): 4 \(\upmu\)m.