# Advanced Biological Imaging for Intracellular Micromanipulation: Methods and Applications
## 3 Intracellular Micromanipulation with Computational Image Information
### Planar Localization
#### Tracking  
In intracellular micromanipulation, manipulation tools and targets are normally small and fragile, so their localization requires skilled operations and tedious time. The automated tracking of manipulation tools based on image information can update operation position, adjust the moving path simultaneously, and increase micromanipulation success rate. A tracking algorithm based on motion history image was proposed [136], as shown in Figure 7a. The subtraction of two neighboring frames records the motion history of tool tips. Once the pixel value was larger than a threshold, the algorithm initiated an active contour model and detected boundaries of the manipulation tools. However, this tracking method may probably fail when the targeted specimens suddenly appear in the region of interest (ROI), or manipulation tools come in contact with them. Thus, a reinitialization and recovery mechanism named as Delock and ReloCk has been proposed [137]. When a targeted cell occluded the ROI, the reinitialization process was triggered, and an instantaneous template was recorded for accurate tracking. When the micropipette touched and deformed the cell, the pipette tip was localized by a template updated from the last two frames, as shown in Figure 7b. Besides, a confidence-based hybrid method has been developed to enhance tracking robustness; in this method, a confidence vector was weighted on estimations from motion-cue feature detection and similarity score-based matching methods [138]. Another tracking demand in micromanipulations lies in investigations on targeted specimens. A modified joint probabilistic data association filter method has been applied to track sperms. Sperm shape information was derived from differential interference contrast images obtained with a WFFM [139]. Figure 7c presents a robust multiple sperm tracking and sperm immobilization for intracytoplasmic sperm injection [140]. In cell aspiration measurements, a cell is deformed under negative pressure, and the aspirated length should be tracked to characterize cellular mechanics. Gradient subtraction method [141], Sobel filtration method [142], or Shi-Tomasi corner detection method [143] was used to enhance cell edges and eliminate adjacent interferences. The light intensity of cell edges is higher than the surrounding area. Therefore, the edge profile should remain bright, and the adjacent interferences will disappear after the filtering operation. The contact position between the cell and the aspiration micropipette can be initially identified. Then, the sampled images were separated into two ROIs containing the aspirated part and the remaining part outside the micropipette. The aspiration length can be tracked in the ROI of the aspirated part, as depicted in Figure 7d. In comparison with the sampled images from WFFMs, fluorescence sampling from CFMs provides clearer localization, which does not require complicated image processing procedures. The aspirated length was easily tracked with CFM-sampled images [144], as displayed in Figure 7e. Therapies based on microrobots has shown great potential for disease treatment, in which microrobots loaded with therapeutic cells can navigate to desired locations in the body. PAM provides image guidance for transportation of microrobots in deep tissues and organs. Figure 7f illustrates the trajectory of the burr-like porous microrobot in the mouse inferior vena cava. The microrobot showed a clear light difference with the mouse vena cava at the imaging depth of 6 mm, and can be tracked with minor position errors [145]. Recently, some photon-activated molecules have been manipulated and tracked to study intracellular activity. Compared with the WFFM illumination that activates many cells at the same time, with the help of one photon excitation of TPFM, the manipulation of a single cell can be realized. Photosensitive optogenetic actuators were activated and tracked with TPFM to study the functional reactions of immune cells involved in calcium interaction [146].