**Acknowledgements**

single cell motility [261, 263]; cell proliferation [264]; cell cycle and cell lineage analysis [107]; changes in mitotic and interphase duration [141]; cell-cell contacts [52]; studying specific cells and tissues [265] and specific intracellular processes such as transcription [99] or morphogenesis [266]; colocalization of cells and intracellular markers [184]; tracking cellular organelles [258]; highlighting the certain cell type within tissues or mixed cell cultures [267]; clustered, overlapping or dying cells [268]; *in toto* imaging of developing organisms, tissues and organs [241] and assessing development and selection of embryos for *in vitro* fertilization [269, 270].

**2.3. TLM for assisted reproductive technology and its promise for clinical medicine**

motility analysis [283].

50 Cell Culture

**3. Conclusion**

TLM is emerging as a promising clinical technique for selecting embryos for transplantation, although the discussion is still under way whether TLM may become an alternative to preimplantation genetic screening [271, 272]. The so-called morphokinetic analysis [273] by TLM is aimed to assess the number, development and quality (viability) of embryos by monitoring cleavage anomalies, multinucleation [274] or specific cell cycle kinetics [274, 275] and cleavage divisions [276], aneuploidy [277, 278], which is considered as a key causal factor of delays in embryonic development toward a blastocyte [278], and even chromosomal abnormalities [279]. Although more clinical research is required to finally prove that TLM can identify the best embryo for transfer and has an advantage over the conventional incubation of embryos [280], TLM is under consideration for patenting as a method for selecting embryos for implantation [281, 282]. TLM can also be used for sperm

One of the potential medical applications of TLM is the assessment of *ex vivo* engineered cells for cell therapy of degenerative and inherited disorders and other human pathologies like cancer [284–288]. TLM can also be used for diagnostics, for example, for detecting abnormalities in cell behavior in human dystrophic muscle cultures [289] or estimating tumor malignancy [290] in drug discovery [291], for testing gene therapeutic agents [292] and for evaluating side effects of antibiotics [293] and efficacy of chemotherapeutics [294, 295]. TLM is a valuable tool for understanding the pathogenesis of certain disorders, such as dysplastic erythroblast formation of erythroblasts from the patient with congenital dyserythropoietic anemia [296], thrombus formation [224], IgE-mediated mast cell degranulation and recovery [297], imaging of disease progression in deep brain areas using fluorescence microendoscopy

TLM is a powerful and versatile tool in modern biological research, with the immense potential for future clinical applications. One of the probably underexplored features of TLM is its promise to further characterize heterogeneity of cells within tissues [144], in particular, stem/progenitor cells and differentiating cells [299] as well as cancer cells [300]. Some of the above-mentioned methods are associated with unavoidable costs (expensive equipment, such as lenses, filters and sensors, and their damage due to high humidity

[298], reprogramming in induced pluripotent cells [110] and other applications.

We thank Mr. Joffry Maltha (CytoSMART Technologies) for assistance with preparation of this manuscript. The work was partially supported by the National Cancer Institute, USA, Award Number R37CA229417.
