Table 2 Advantages and disadvantages of techniques listed in the manuscript
Technique | Advantages | Disadvantages |
|---|---|---|
In vivo fluorescence imaging | • Simple, cheap, user friendly techniques • High spatial resolution (~200 nm in x,y,) with high sensitivity cameras • Development of FarRed and NIR probes allow greater tissue visualization with much less damage whilst imaging • High sensitivity (10–12 to 10–15μm/L) | • Use of a probe generally required which may have repercussions on stem cell physiology • Photo-toxicity to tissue and depth resolved imaging still an issue • Vectors employed to introduce reporter genes are still under scrutiny for safety and efficacy of use in clinical trials |
QPM | • Accurate quantitative visualisation of phase changes within cells | • No depth-resolving capabilities |
ODT | • Depth-resolving capabilities, resolution of up to 1 μm | • Low penetration depth (a few hundred microns), not suitable for real-time imaging (slow techniques) |
DHM | • Imaging of a 3D volume with a single exposure, structural and phase imaging, and also flexibility for image processing. Resolution almost as in conventional microscopy | • Relative complexity (more complicated optical set up), limitation on coherent properties of the light source, on environmental conditions (vibrations, etc.) |
SESF and srSESF | • High (nano-scale, ~10 nm demonstrated) sensitivity to structural alterations within object and super- resolution imaging | • More complicated optical set up, for example for detailed quantitative analysis of the structure an imaging spectrometer or swept light source is needed |
OCT | • Improved image resolution (morphological and functional information) of depth-resolved images • Can be combined with other imaging techniques for multimodal imaging • Suitable for clinical translation | • Penetration depth is limited ~2 mm into tissue • Spatial resolution is typically limited to ~10 μm, making this technique unsuitable for cell imaging • Limited molecular sensitivity of tissue |
OCM | • Enhanced penetration depth compared to standard confocal microscopy; dramatically improved resolution over OCT imaging (up to 1 micron) | • Small penetration depth (compared with OCT) |
nsOCT | • Depth-resolved images with high sensitivity (~30 nm demonstrated experimentally) | • Resolution and penetration depth are approximately the same as conventional OCT |
OCPM | • Quantitative phase information with high sensitivity, useful for 3D intracellular imaging | • Small depth of field |
PAI | • Capable of collecting molecular and spatial information from the tissue using endogenous contrast alone • Greater sensitivity than OCT and confocal imaging • Suitable for clinical translation • The ratio of the imaging depth to the best spatial resolution is roughly a constant of 200 | • Sometimes requires the use of biocompatible labelling materials such as gold or silver nanoparticles |
Confocal reflectance microscopy | • High spatial resolution images achievable (diffraction limited ~200 nm) • Can work in combination with other modes of microscopy including fluorescence and OCT | • Lack of specific light reflecting probes for confocal microscopy when used in reflectance mode |
Super-resolution microscopy (nanoscopy) | • Images created have a higher spatial resolution that normal diffraction limited techniques. (STED x.y resolution ~20–100 nm, PALM and STORM x.y ~20–50 nm) • Increased localization and clarity of intracellular structures due to increased resolution | • Fluorophores or fluorescent markers must be used. Potential for photo bleaching of the sample under study • Expensive equipment • Currently most super resolution techniques are not suitable for live cell imaging • Refractive index variations in the substrate can cause distortions which when translated to the nanoscale can be significant |
Microcomputed tomography | • Can generate defined structural images with increased all round resolution (100 μm in x,y and z dimensions) • Suitable for clinical translation | • Exposure to ionizing radiation which can cause DNA damage • Not suitable for soft tissues |
Radionuclide imaging | • Only low doses of labels need to be employed due to the high sensitivity of the probes • Good tissue penetration of the probe • Suitable for clinical translation • Fair sensitivity (10–8 to 10–9μm/L) | • Exposure to ionizing radiation which can cause DNA damage • Half-life of the probe must be considered |