Table 1.

Characteristics and application of advanced imaging techniques.

	Characteristics	Advantages	Limitations	Application
Intravital imaging with multi-photon microscopy	Observe dynamic organisms in live animals in a spatiotemporal manner; Simultaneous absorption of multiple photons in multiphoton microscopy guarantees the excitation at the focal plane	Live animal imaging; No out-of-focus light; Less damaging to the tissue; Extended observing time and deep penetration within tissues	Multiphoton microscopy provides a lower resolution than confocal microscopy	Many research areas, such as immunology, tumor biology and neurobiology.
Atomic force microscopy (AFM)	Surface analysis tool for obtaining high- resolution nanoscale images. Offer information on physical properties (size, morphology, surface texture and roughness); Measure forces (adhesion strength, magnetic forces and mechanical properties)	Compared with scanning electron microscopy, AFM does not require special treatments (metal/carbon coatings); Work in both air and vacuum; Provide a true 3-D surface profile	Lateral resolution is not sufficient for detailed structural studies.	Biochemistry and biophysics applications (structure of biological molecules, cellular components), materials science and nanotechnology applications
Spinning disk confocal microscopy (SDCM)	Use a rotating disk with thousands of pinhole apertures, thousands of emission light scan the specimen simultaneously. Images are taken at a focal plane and out- of-focus lights are discarded	Compared with laser scanning confocal microscopy, SDCM has lower light levels; More efficient fluorescent detection; More accurate cell physiology	Inability to adjust the pinhole size to alter the optical sectioning strength; Pinhole cross-talk effect creates background signals.	Imaging fast dynamic processes and live specimens
Super resolution microscopy (SRM)	STORM reconstructs super-resolution image by combining the high-accuracy localization information of individual photo-switchable fluorophores.	Standard organic fluorescent dyes; Simple instrument and highest resolution on an optical imaging system in biological application.	Extensive post- acquisition image processing for image reconstruction	Detect molecular interaction and dynamics, visualize nanoscale structures with optical techniques, study
	STED switches off the fluorophores out of the diffraction limited excitation focus. Fluorescence from the excited dye molecules in the center of the focus is detected and form the high-resolution images.	Simple and fast acquisition process; Deep tissue imaging; Fast acquisition without the need for additional data processing	Increased photobleaching	fundamentals of biology through single molecule fluorescence.
Light sheet microscopy (LSM)	Thin sheet of light orthogonal to the detection plane scans plane by plane, collects the fluorescence signal of the observed region to reconstruct 3D images.	Compared with confocal microscopy, LSM reduces photobleaching effects; High signal-to-noise ratio and fast scanning rate, useful to image large scale specimens.	Transparent sample required.	Embryonic development, whole brain neural activity, immune cell interaction and motility.
Total internal refection fluorescence microscopy (TIRFM)	Use laser light at an angle to only excite fluorophores that are located near the cell membrane interface, and restrict the zone of observation to the plasma membrane or just beneath it.	Offers a much reduced background fluorescence	Total internal refection occurs only at the interface, more suitable for generating optical sections of 2D but not 3D images	Intracellular cargo transport, actin dynamics near the plasma membrane, and focal adhesions in living cells.