# Advanced Biological Imaging for Intracellular Micromanipulation: Methods and Applications
## 1 Introduction  
Intracellular organelles and molecules governing the liveness [1; 2], dynamics [3; 4], organizations [5; 6], and functionalization [7; 8] at cellular, multicellular, and tissue levels have attracted considerable interests from the field of biomedical research. Intracellular manipulation, measurement, and other surgical operations were conducted to unveil underlying mysteries inside cells [9]. For example, intracellular physical and chemical properties, such as Young's modulus [10], viscosity [11], pressure [12], pH [13], and temperature [14], were measured to study biological processes and biomedical reactions in living cells. Some tracking macromolecules were delivered inside mammalian cells for the exploration of intracellular mechanisms, such as the entry of cytoplasmic molecules into primary cilia [15] and the release of spliced mRNAs from nuclear speckle domains [16]. Furthermore, genome-editing tasks have attracted research interest, particularly those that involve the transfer of DNA [17; 18] and RNA molecules [19; 20] to different cell types for cell transfection. Although many intracellular manipulation techniques have been proposed, traditional clinical operations still depend on well-trained operators to fulfill various tasks [21]. In practice, operators laterally align manipulation tools with targeted specimens, manually adjust the relative height positions between manipulation tools and targeted specimens, and then initiate a given operation. Fatigue- and experience-induced failures always occur and result in low operation success rates and large damage, thus degrading the performance of intracellular manipulation [22; 23]. Generally, intracellular objects are fragile [24; 25] and usually have irregular shapes [26; 27], distribution [28; 29], and small sizes [30; 31]. For example, mitochondria in mammalian animals are usually less than 2 \(\upmu\)m and randomly distribute among the cytoplasm [32]. An inappropriate operation position not only leads to operation failure butalso largely damages the targeted cells and even kills them. Therefore, the acquired position should be precisely manipulated through computational imaging during intracellular operations. The complexity of intracellular environments and culture media inevitably causes difficulties in localizing operation positions [33; 34; 35]. The identification of targeted biological specimens is easily disturbed by unavoidable surroundings, such as counterpart cells and auxiliary manipulation tools, in an imaging field. Importantly, the morphological characteristics of most organelles are similar to those of their intracellular neighbors; as such, they become undistinguishable under optical observation [36]. Optical observation should be carried out several times to ensure that the imaging plane contains the target organelles because of the random distribution [37]. Meanwhile, most organelles have an irregular shape, so their appropriate operation position should be selected through comprehensive considerations [38]. Moreover, imaging blurs and noises during an optical observation deteriorate the imaging performance and make the organelles boundary unclear [39]. Apart from the automation demand of existing intracellular micromanipulation systems, two main challenges are encountered in current studies: how to obtain the sampled images of targeted intracellular specimens with high resolution and contrast and how to precisely extract operation information from the sampled images for a successful intracellular micromanipulation. The first challenge can be overcome with emerging advanced imaging technologies. With the help of some start-of-the-art imaging instruments and post-computation procedures, high-quality, real-time images during manipulation can be obtained. For the second challenge, many intelligent image computational processing methods have been proposed to restore the sampled images, analyze the targeted specimens, and provide precise visual feedback.