# Advanced Biological Imaging for Intracellular Micromanipulation: Methods and Applications
## 2 Image Sampling Methods
### Photoacoustic Microscope (PAM)  
Photoacoustic imaging is inspired by the photoacoustic effect converting optical energy to measurable pressure waves [84]. This effect is initiated when specimens are irradiated with a modulated laser source, resulting in thermoelastic expansion and photoacoustic wave formation corresponding to the geometric characters of specimens [85]. Figure 2a shows the two main configurations of a photoacoustic microscope (PAM) in terms of imaging modes. One is the optical resolution (OR) mode in which the focused laser light is illuminated on specimens. The optical region of an OR-PAM is restricted, but its lateral resolution is improved. The other one is the acoustic resolution (AR) mode that adopts uniform illumination. The unfocused light enables deeper penetration but yields lower resolution [86]. Photoacoustic imaging technology is suitable for deep depth imaging, and because acoustic waves can transmit across soft tissues and skins, high-resolution imaging can be achieved at depths far exceeding the optical diffusion limit. In fact, photoacoustic imaging technology has been widely adopted for navigating microrobots in vivo. The migration of capsule micromotors was controlled to move to the targeted region in the intestines under the guidance of photoacoustic imaging [87], as shown in Figure 2b. PAM also has the advantages of label-free imaging and out-of-focus blur suppression, and has become an ideal technology for non-toxic biological imaging. Many subcellular practices have been imaged with enhanced contrast. This instrument can achieve a high lateral resolution of 250 nm. Figure 2c illustrates the sampled images of lipids, proteins, and nucleic acids in fibroblast cells by using an ultraviolet-localized mid-infrared PAM [88]. However, it lacks an optical sectioning capability. Theoretically, the axial resolution of PAM is controlled with the bandwidth of a photoacoustic signal and the central frequency of an ultrasonic transducer [89]. Studies have been performed to improve the axial resolution of PAM without losing its lateral accuracy. For instance, a Grueneisen relaxation photoacoustic microscope (GR-PAM) delivering two identical laser pulses has been proposed [90], where the axial resolution can be improved to 2.3 \(\upmu\)m, and the lateral resolution was 410 nm. Figure 2d illustrates the optically sampled images of red blood cells at three different depths and their 3D reconstruction model. Another study has developed an isometric PAM based on a transparent micro-ring resonator ultrasonic detector, whose axial resolution was 2.12 \(\upmu\)m [91]. In addition, an axial resolution improvement method has been established on the basis of configuration modification [92]. Figure 2e shows the dual-view PAM and sampled images of a mouse brain slice. Two split lasers are focused onto the specimens, and photoacoustic waves are collected with the ultrasonic transducer. This design can improve the axial resolution to 1.8 \(\upmu\)m. Generally, due to its relative low axial resolution, PAM is more frequently to measure specimen profiles in 2D sampled planes instead of 3D space. Besides, PAM has several other limitations that hinder its applications in biological imaging. First, the sampling speed of PAM is low. There is a trade-off between resolution and sampling speed, that is, it takes several minutes to measure an area of 1 m \(\times\) 1 m\({}^{2}\) with an acceptable optical resolution using using OR-PAM [93]. Second, PAM requires complex light configurations, and there are almost no commercial tools available, which limits the accessibility of researchers who do not have expertise in this field. Finally, the potential damage from deposited laser energy is another issue that should be avoided in high-resolution imaging [94; 95]. Figure 2: Photoacoustic imaging. (**a**) Two operation modes of PAM. Adapted with permission from Reference [86]; (**b**) Time-lapse PAM measurement of microrobot movement in the intestine.