# Holographic Optical Tweezers: Techniques and Biomedical Applications
## 3 HOT Technique for Bio-Application
### Assembly and Bio-Structure Fabrication  
HOT can be used to program the assembly of nano- and microsized particles [45; 46] into crystal growths [47] or microstructures [103]. Several research groups have verified the feasibility of HOT-driven in 3D space. [PERSON] et al. [45; 48] lined up several silica spheres and then moved them into a 3D structure. They also constructed 3D crystal templates for photonic bandgap materials [50] by using microspheres ranging from 0.8 to 3 \(\upmu\)m. Other studies [104; 105] have reported the assembly of microsized particles into crystalline photonic heterostructures.  
On the other hand, [PERSON] et al. [46] adopted real-time feature recognition to automate the assembly and sorting of colloidal silica by size. Subsequently, [PERSON] et al. [106] improved the throughput of automation via efficient path planning and particle identification. The assembly technique can be extended to crystal growth and fabrication [47] by combining it with a fixing process, such as polymerization [49; 107].  
The assembly function of HOT is also suitable for microfluidic cytometry and lab-on-a-chip devices [55; 108; 109]. [PERSON] et al. [109] were the first to assemble living bacteria into the designing 2D and 3D arrangements in hydrogels without losing viability. Later, [PERSON] et al. [110] arranged the live embryonic stem cells of mice along straight lines, curves, and circles and studied the interaction between multiple bio-samples. Using a suitable gelatin medium, [PERSON] et al. [111] and [PERSON] et al. [112] created permanent 3D arrangements of isolated _E. coli_. The fabricated structures remained intact, and the _E. coli_ survived for several days even after removing the laser beam. However, the aforementioned bio-sample matrix array is insufficient to study the bio-system. The surrounding microenvironment also affects system operation. Even in this context, HOT can be used to assemble biological structures and manufacture complex cellular structures accurately [113; 114].  
Additionally, HOT has been applied to the fields of micro-robotics and micro-tools. [PERSON] et al. [115] employed HOT to fabricate living micro-robots comprising a rod-shaped prokaryotic bacterium and a single elongated zeolite L crystal. As depicted in Figure 7, HOT controls the orientation of zeolite L crystal and the position of bacterium and then assembles them together. After the trapping laser is turned off, the bacterium-zeolite L living machine can swim itself by using the bacterium's flagella. Unlike the aforementioned studies that used mobile bacteria for transport, [PERSON] et al. focused on automatically transporting [54; 56; 57; 60] by moving laser spots. They fabricated a micro  
Figure 6: Bio-applications of HOT.  
tool to transport biological cells indirectly. Figure 8 depicts the design of the micro-tool and the transportation results. Three trapping spots generated via HOT are used to manipulate the position and orientation of the micro-tool and transport the biological cell. Three-spots tweezers trap the micro-tool and indirectly move the cell along the designed path (dash line) to the desired position. The images during transportation are shown in Figure 8a-d.