Table 1.

A summary of the benefits and limitations of imaging methods used to characterize 3D cellular structure. There are many exemplary case-uses for each method that have contributed significantly to our basic understanding of cell structure and function. For brevity, only a few examples are provided in the above table. Indications for potential variations in experimental setup are indicated in parenthesis in the Approach column.

Approach	Resolution Range	Benefit	Limitation	Reference Examples
Electron Microscopy (scanning, block-face, fluorescence correlative)	4–8 nm in approximately 1,000 μm3 tissue volume [55]	Can image in the context of tissue, keeping cell-to-cell contacts and neighboring vasculature intact for improved interpretations	Requires laborious serial sectioning which limits the number of cells and conditions that can be investigated. May also require plastic embedding or chemical fixation.	[28,29,56,57]
Cryo-electron tomography (thin periphery of cell, thin lamella of interior of cell, single-cell multiple distinct tomogram acquisitions)	2–5 nm in 180–250 nm thick samples	Provides high resolution details on 3D cellular organization within windows of the cell. Allows for in-situ structural biology.	Require use of thin lamella sections of cells that may not accurately reflect cellular organization due to polarity and uneven distributions of cellular contents	[[58], [59], [60]]
Fluorescence Microscopy (confocal, super-resolution)	70–250 nm in a single cell	Provides live cell dynamics and cellular rearrangements	Requires specific labeling strategy limiting unbiased discovery	[[61], [62], [63], [64]]
Soft X-Ray Tomography (cells cryo-fixed on a grid or in solution within capillary. Fluorescence correlative)	Up to 35 nm in 10 μm diameter cell	Produces unbiased and high-throughput 3D reconstructions of intact cells	Requires dissociated tissue for single cell analysis, limited to static timepoints, and limited access to facilities with ideal capabilities	[16,19,27,65]