# Advanced Biological Imaging for Intracellular Micromanipulation: Methods and Applications
## 2 Image Sampling Methods
### Wide-Field Fluorescence Microscope (WFFM)  
With low purchase expenses and high accessibility [96], WFFM is popular in biological imaging. Figure 3a shows the WFFM observations of a burr-like porous spherical microrobot loaded with MC3T3-E1 fibroblasts, and the microrobot movement inside the yolk of a zebrafish embryo was measured for automation control [97]. Similar to CFM, WFFM suffer from a diffraction barrier. The resolution of WFFM can be expressed as \(\frac{\lambda}{2 NA}\) (lateral) and \(\frac{2\lambda}{NA^{2}}\) (axial) [98], where \(\lambda\) is the wavelength of the emission light, and NA is the numerical aperture of the objective lens. Even for a WFFM with a high-NA objective lens, its resolution hardly reaches 230 nm laterally and 800 nm axially because the visible light wavelength varies from 390 to 780 nm [99]. During WFFM operation, the whole specimens are illuminated with emission light, and sampled images contain in-plane features, out-of-focus blurs projected from adjoined depths, and noises, which undoubtedly deteriorate the imaging quality, especially for depths that are far from the focused plane [100]. Such optical behavior can be modeled as a convolution process between point spread functions (PSFs) and the measured specimen [101]. To restore the sampled images of WFFM, many methods have been proposed on the basis of deconvolution algorithms that numerically invert blur processes, such as the Landweber method [102], Tikhonov-Miller gradient descent method [103], and Richardson-Lucy maximum likelihood method [104; 105]. Figure 3b shows an unfocused sampled image of _Caenorhabditis elegans_ and deconvoluted images with different deconvolution methods [106]. The processed images exhibit sharper features with more details than the original one, providing more accurate information for intracellular micromanipulation tasks. Through focal plane movement, the whole specimen can be scanned, and the sampled images via a WFFM are optically sectioned and stored in a computer. The sampled images can also be reconstructed into a 3D model after the restoration processes of deconvolution, segmentation, and reconstruction [107]. Figure 3c presents the sampled image series of a mitochondrion in a THP-1 cell and its 3D reconstruction model.  
Due to advantages of great compatibility, controllability, and programmability, WFFM is widely used to visualize cells, intracellular organelles, microrobots, and other micro-objects. It should be noted that the size of measured specimens is usually larger than 1 \(\upmu\)m, because WFFM resolution is limited even if the recovery algorithm is implemented. On the other hand, due to its simple configuration and large operation space, robot-aided manipulation tools are easy to integrate with WFFM. Therefore, WFFM is frequently applied in intracellular micromanipulation.  
Figure 3: Intracellular imaging practices with WFFM. (**a**) WFFM observations of a burr-like porous spherical microrobot. (1) A microrobot loaded with MC3T3-E1 fibroblasts. (2) The microrobot moving inside the yolk of a zebrafish embryo. Adapted with permission from Reference [97]; (**b**) Blurred sampled image of _Caenorhabditis elegans_ and restored images with different deconvolution methods. Adapted with permission from Reference [106]; (**c**) 3D reconstruction method based on a WFFM. (1) The reconstruction process. (2) The fluorescence image series of the selected mitochondrion (orange). (3) The reconstructed 3D model of the mitochondrion. Adapted with permission from Reference [107].