# Holographic Optical Tweezers: Techniques and Biomedical Applications
## 3 HOT Technique for Bio-Application
### Parallel Measurement of the Bio-Samples in a Fluid  
HOT can be used to sustain multiple sample positions in a fluid, enabling parallel measurement and study of a single cell [116]. For instance, fluorescence microscopy can be used to acquire image stacks to extract quantitative data with high spatial and temporal protein distributions. [PERSON] et al. [117] combined 3D CGH, HOT, and image analysis to optimize the axial position of trapped cells in an array. As depicted in Figure 9a,c, via 2D optical trapping, all trapping points are aligned on the same plane. To obtain better images of all nuclei, their automated system displaced the nuclei onto a single imaging plane (Figure 9b,d), and a greater number of images could be obtained via time-lapse experiments before photobleaching. HOT can also be used to increase the parallel processing capability of digital holographic microscopy. [PERSON] et al. [118] aligned several bacterial cells precisely on the surface of living host cells by using HOT and simultaneously monitored them via self-interference digital holographic microscopy. Their system enables the 3D modeling and imaging of infection scenarios on the single-cell level. Figure 8: The indirect transportation of biological cells with HOT [60]. **(a)** Three-spots HOTs trap and move the microtool. The microtool push the cell. **(b)** The cell is carried to the waypoint to avoid the obstacle. **(c)** The moving direction of the microtool is redirected to the desired position. **(d)** Cell is transported to the desired location. Figure 7: Optical assembly of bio-hybrid micro-robot [115]. **(a)** HOTs trap bacterium and zeolite L crystal. **(b)** The orientation of zeolite L crystal is rotated by a two-spots HOT. **(c)** HOT assembles them together. **(d)** Trapping laser is turn off, then bacterium–zeolite L living machine utilizes the bacterium’s flagella to swim on its own. **(e–h)** Top views of the experiment during the assembling process **(a–d)**. The incorporation of HOT also enhances the performance of Raman spectroscopy [119, 120]. Raman spectroscopy [121] can be used to probe the biochemical composition of microorganisms without preprocessing. However, the floating motion of motile microbial samples influences the temporal and spatial resolution of the Raman signal. By integrating it with optical trapping, Raman tweezers [13, 15] enable the spectroscopic study of the optically immobilized single microorganism under animate conditions [14]. [PERSON] et al. [122] sorted and transported yeast cells and polystyrenes by using Raman tweezers. Initially, HOT was used to trap eight particles, and the Raman spectra of the particles were obtained via spectroscopy. From the spectral results, polystyrene particles and yeast cells were identified. The HOT then transported the polystyrene particles away. OT can also be applied in photonic force microscopy (PFM) [19] to measure forces in the pico-Newton range by detecting the Brownian motion of single trapped samples. This enables the study of the viscoelasticity of the bio-sample. The use of CGH enhances the dynamic control ability and lets HOT manipulate the movement of beads in vivo. For instance, [PERSON] et al. [123] injected fluorescent beads into zebrafish embryos and then applied HOT to control the movement of the beads in vivo to investigate the viscoelastic properties of the embryos, enabling the study of embryonic development without invasive intervention. Moreover, the incorporation of CGH enables the simultaneous study of multiple cells or molecules by using PFM. [PERSON] et al. [124] measured the mechanical coupling between an _Aplysia_ growth cone and a bead functionalized with the neuronal cell adhesion molecule, apCAM. As depicted in Figure 10, HOT was used to position several beads near the leading edge of the growth cone--subsequently, the cell pulled the beads, which escaped from the optical force. Cell adhesion and related motility were then measured. HOT can also be applied to the measurement of the viscoelasticity of proteins and membranes [125].