# Holographic Optical Tweezers: Techniques and Biomedical Applications
## 3 HOT Technique for Bio-Application
### Digital Hologram  
Digital holography microscopy (DHM) [62, 128, 129, 130] is a label-free optical microscopic imaging system which is suitable for the study of biological samples study [64, 131]. The computer program can numerically reconstruct the quantitative refractive index (RI) distribution inside bio-samples by digitally recording the interference pattern as a hologram. OT can rotate the orientation of the sample to a specified angle to gather part of the sample's information. [PERSON] et al. [132] measured the RI distribution for RBCs (red blood cells) from two perspective angles--when the long axis lies on the slide and when it is rotated by the OT to perpendicular to the slide. This application is extended to real 3D imaging in holographic tomography [66, 133, 134, 135, 136], which employs beam-rotation [137, 138] or sample-rotation schemes [63, 67, 139, 136, 139] to scan entire samples under the different angles of illumination and calculate the integral phase. The beam-rotation method can be implemented by using a galvano-mirror, LCD, or digital micro-mirror device (DMD), which keeps the sample and the observation system static. The beam-rotation method exhibits a limited measuring angle, leading to an anisotropic resolution with incomplete spatial frequency coverage [141, 142]. However, the sample-rotation method retains an illumination system with better reconstruction features.  
Among the multiple techniques used for sample rotation, HOT offers an all-optical setup [67, 139, 140, 143, 144] with stable control and exhaustive measuring angles. [PERSON] et al. [140] applied the HOT technique to rotate samples during recording processing in tomography-phase microscopy. They utilized a two-spot trap to orient a yeast cell on the y-z plane over 180\({}^{\circ}\) in angular steps of 5\({}^{\circ}\) and captured the off-axis interferogram at each stage. Our team [67, 139] also proposed a two-spot trap to implement full-angle rotation for living yeast cells and _Candida rugosa_ in a fluid and acquired an entire symmetric spectrum for tomography reconstruction. Figure 13 depicts the HT-HOT joint system. Green laser light with 2D-GSA CGH information was used to illuminate the sample area to hold the sample and rotate its orientation. The HT system was built up by using the red laser, and the 3D-RI distribution of the sample was recorded.  
To extend the application of HOT and DHM to arbitrarily shaped samples, [PERSON] et al. proposed the tomographic holds for optical trapping (TOMOTRAP) [143, 144]. Initially, their system measured the 3D-RI distributions of the samples. Then, based on them, they applied a 3D-GSA to generate a continuous light field distribution for the optical trapping.  
Figure 13: HT-HOT joint system [67]. (**a**) Optical system. (**b**) The schematic diagram. (**c**) The measuring index distributions of yeast cell.  
This 3D reconstructed beam intensity distribution is identical to the sample volume and can be used to manipulate the sample. They demonstrated orientation control, folding, and assembly of RBCs and eukaryotic cells [143]. They recently developed isotropically resolved label-free tomographic imaging based on tomographic models for optical trapping [144]. As illustrated in Figure 14, their proposed method yields an isotropic resolution of 230 nm and captures the structural details of live RBCs.