Table 1.
Imaging techniques and applications for platelet research in vitro.
| Imaging method | Key points | Resolution | Platelet/cell imaging applications | Limitations | References |
|---|---|---|---|---|---|
| Conventional/ Bright-field/Widefield |
Uses visible light or high intensity light sources to illuminate a sample |
L = 200–300 nm Ax = 500–800 nm |
Thrombus formation >Microfluidics Large platelet aggregates |
Low resolution Not suitable for single cell evaluation Limited by wavelength of light and NA of objective lens |
(50) |
| Confocal/CLSM | Uses light to illuminate a sample through a pinhole to improve optical resolution Uses spatial filtering to block out-of-focus light |
L = >200 nm (reflection) >250 nm (fluorescence) |
Thrombus formation Platelet spreading >Surface receptor information Healthy controls vs. patients differences > wild type mice vs. knock out mice differences |
Fluorescence label Surface area and receptors data Minimal information on cytoskeleton |
(50–52) |
| QPM/DHM | Generates quantitative measurements from shifts in phase | L = >270 nm | Volumetric measurements of thrombus formation | No receptor profile details Requires complex post-image analysis |
(53–56) |
| CLEM/3D cryoEM | Approaching atomic level analysis of ultrastructural changes, adhesion, and granule secretion | L = < 1 nm | Platelet secretion; Megakaryocyte positioning in sinusoids and platelet production (applied in intravital setting) | Samples need to be mounted on a grid; precise solvent requirements | (57–60) |
| STED | Confocal excitation beam overlaid by a depletion beam to inhibit fluorescence emission at target area of interest | L = 50–60 nm | Platelet protein distribution when co-incubated with cancer cells Platelet protein storage |
Deconvolution required Need specific STED dyes Decreased scan step size + increased acquisition time |
(61–64) |
| SMLM SIM PALM (d) STORM PAINT |
Illumination that relies on single molecule switching by stochastic excitation Switching on/off of a fluorescent molecule or through excitation |
L = >20 nm Ax = >50 nm |
Platelet cytoskeleton proteins Actin nodules/tubulin Megakaryocyte structure and function Synapses Platelet receptor co-localization and receptor clustering |
Computer power/software and storage Vast number of data points Post-data analysis and complex image reconstruction Specific photoswitchable and activatable fluorescence labels |
(31, 65–67) |
A non-exhaustive list of imaging techniques used to study platelet spreading, function, receptor profiles, and platelet protein/cytoskeletal protein organization in vitro. Rows highlighted in blue are examples of microscopy approaches that operate at nanoscopic/super resolution limits of diffraction. L, Laterally; Ax, Axially; NA, numerical aperture; CLSM, Confocal Laser Scanning Microscopy; QPM, Quantitative Phase Microscopy; DHM, Digital Holographic Microscopy; CLEM, Correlative light-electron microscopy; cryoEM, Cryogenic Electron Microscopy; SPIM, Selective Plane Illumination Microscopy; STED, Stimulated Emission Depletion; SMLM, Single-Molecule Localization Microscopy; SIM, Structured Illumination Microscopy; PALM, Photo-Activated Localization Microscopy; STORM, Stochastic Optical Reconstruction Microscopy; PAINT, Point Accumulation for Imaging Nanoscale Topography.