# Optical Diffraction Tomography Using Nearly In-Line Holography with a Broadband LED Source
## 4 Effect of Misalignment on the Reconstruction Quality  
In this section, we study the effect of optical misalignment on the quality of the 3D reconstruction. As described in the above sections, it is critical that the illumination and imaging _NA_ are identical (i.e., conical illumination with the center aligned with the optical axis). Any misalignment will result in the \"Principal\" and the \"Twin\" images overlapping, which will alter the contrast quality of the final 3D RI reconstruction. To study this, the same optical setup was used; however, the LED was slightly misaligned from the optical axis. Figure 7 shows an image for two LED illuminations. As clearly seen, when the \"Principal\" and the \"Twin\" image overlap (i.e., in Fourier transform) we see very low contrast in the intensity images since the \"Principal\" and the \"Twin\" image tend to cancel each other [28]. However, for the other case when they do not overlap, we see that the contrast is enhanced. As a result of this effect, the final 3D RI reconstruction will not reflect the true 3D refractive index distribution of the sample, since we cannot retrieve the complex scattered field \"alone\" from the intensity images because of the overlapping circles for certain projections. Figure 8 shows the retrieved 3D RI distribution as a result of optical misalignment. From the figure, it is observed how the bacteria highlighted in blue box takes an RI value less than the surrounding medium (i.e., water), which does not agree with literature [17; 31]; this is also in contradiction to the reconstruction shown in Figure 6 when the LED were perfectly aligned, and the illumination and imaging _NA_ were matched. This error in the 3D RI reconstruction might be attributed to the twin image and the principal image overlapping which resulted in the wrong calculation of the 3D reconstructions. In addition, we see that the refractive index contrast decreased due to the cancelation of the low frequency components from the overlapping of the principal and the twin image.  
Figure 6: 3D RI reconstruction at different depths. The red box is a magnified picture of the highlighted red region. It shows the higher refractive index of this bacterial structure as compared to the cytoplasm of the cell.  
Figure 8: 3D RI reconstruction for misaligned LED ring. As shown in the red box, the contrast is highly suppressed as a result of the cancelation of the low spatial frequency due to the overlap between the two circles in some of the projections. On the other hand, as shown in the dark blue box, we see an artifact where the contrast is inverted for the bacterial structure, which does not agree with the literature [17; 31], where the bacterial structures have higher refractive index than cytoplasm.  
Figure 7: Effect of misalignment on the captured intensity images under LED illumination. An illumination angle smaller than the maximum angle allowed by the _NA_ of the objective lens, results in overlapping spectra (circles), and we obtain low contrast images. This is not the case when the illumination angle is maximized, and the spectra do not overlap.