# Advanced Biological Imaging for Intracellular Micromanipulation: Methods and Applications
## 2 Image Sampling Methods
### State-of-the-Art Microscopes
#### 2.1.3 Light Sheet Fluorescence Microscope (LSFM)  
LSFM is another instrument with a long-term measurement capability. A light sheet generated from a thin laser sampling beam is focused only on a portion of specimens, and fluorescence is then collected perpendicularly from the illumination axis [70], as shown in Figure 1g. The highly efficient excitation and collection scheme ensure that the phototoxic and photobleaching effects of LSFM are minimized compared with those of CFM and TPFM [71]. Specifically, exposure light energy was found to be 5600 and 10\({}^{6}\) times less than that of CFM and TPFM, respectively [72]. Therefore, LSFM is suitable for such applications requiring long-term measurement and careful exposure. Morphology changes of Drosophila embryos at different biological stages, such as germ band retraction, segment formation, and dorsal closure, were imaged with a LSFM [73]. Cardiomyocytes were imaged based on a customized LSFM for up to 1.5 days to investigate the holistic cell behaviors sculpting the four-chambered mammalian heart [74]. It should be noted that sampled images of LSFM are invulnerable to out-of-focus blurs, and they can exhibit more detailed features than normal wide-field microscopes [75]. Figure 1h shows the mitochondria visualization of HeLa cells labeled with the mitochondrial import receptor subunit TOM-20 by using a LSFM [76]. The image demonstrates a better contrast for LSFM than for wide-field illumination. The lateral resolution of LSFM is regulated primarily by the numerical aperture of the detection objective, while the axial resolution is additionally determined by the thickness of the light sheet [77]. Specifically, LSFM has a comparable lateral resolution as CFM. A single-objective LSFM with a resolution about 300 nm was proposed, which was based on the concept that combining objective with a coverslip-walled immersion chamber. Parallel recording of 16 cell lines and their cellular responses to drug treatment was conducted with this instrument [78]. Utilizing a LSFM with a scheme of two orthogonal detection and illumination objectives, the neural wiring during _Caenorhabditis elegans_ brain development was imaged with a lateral resolution of 330 nm [79]. However, the axial resolution of LSFM is not as high as CFM, which is typically not better than 1 \(\upmu\)m [72; 80; 81; 82]. It is worthwhile to note that the complicated sample preparation is a problem for LSFM, because its orthogonal configuration hinders standard Petri dishes and multi-well plates for sample preservation [83]. In such a narrow space, the integration of LSFM with other manipulation tools is a huge challenge. Therefore, its applicability in intracellular micromanipulation is greatly limited.  
Figure 1: State-of-the-art microscopes and their applications in biological imaging. (**a**) Schematic of CFM. Adapted with permission from Reference [43]; (**b**) CFM-sampled images of HeLa cells (green) and porous CaCO\({}_{3}\) particles (yellow). Adapted by with permission from Reference [44]; (**c**) Three-dimensional distribution of polystyrene beads in human hepatocellular carcinoma (HCC) cells.  
Adapted with permission from Reference [46]; **(d)** CFM 3D reconstruction image of an injected cell. Adapted with permission from Reference [47]; **(e)** Schematic of a typical TPFM. Adapted with permission from Reference [63]; **(f)** Long-term motility measurement of transferred T cells with TPFM. Adapted with permission from Reference [58]; **(g)** Basic schematic of LSFM. Adapted with permission from Reference [70]; **(h)** Comparision of mitochondria visualization between LSFM and WFFM. Adapted with permission from Reference [76].