**2. Time-lapse microscopy: from making movies to bedside**

#### **2.1. Versatility of TLM**

In this part, we will briefly review some selected publications, which highlight the rapid development of TLM as a versatile discovery tool within the broad scope of modern biology and medicine. Importance of TLM as a new method in biological research was highlighted by Burton [5]. The progress of tissue culture methods, phase-contrast microscopy (see below) and real-time imaging by TLM enabled scientists to overcome the major limitation of traditional microscopy; preparation of very thin transparent samples, which required tissue fixation and did not make it possible to investigate living cells, let alone, and biological processes over time in the same sample. Early reports demonstrated the feasibility of TLM for comparative studies of cultured cells [6–8] and for monitoring living blood and lymph cells [1], cell division [9, 10] and reaction of cells to varying contents of electrolytes in perfusion chambers [11]. TLM was helpful to decode the process of multinucleation in the developing skeletal muscles [12] and to describe the variable cytotoxic response toward allografts [13, 14].

TLM is a suitable tool to monitor *cell motility and migration*, including quantitative assessment of migration, such as the number of migrating cells and the distance [15–20]. In multicellular organisms, the directed and coordinated cell migration (chemotaxis) occurs during embryonic development, tissue regeneration and inflammatory response [21], while cancer cells migrate into surrounding tissues and the vasculature. To monitor chemotaxis, TLM can be used together with the Dunn chamber [21–23]; Boyden chamber [24, 25]; Bridge chamber [26]; LOCOMOTIS, the motility tracking system [27] and other types of chambers for cell visualization and TLM applications [28–30]. TLM was employed to study embryonic stem cells [31]; hematopoietic progenitor cells [20, 32–34]; mesenchymal stem cells [35]; activated lymphocytes forming lymphocytes colonies [36]; primordial germ cells, a migratory cell population that will eventually give rise to the gametes [37–39]; the migration route of progenitor cells in cell cultures obtained from live chicken embryos [40, 41]; microglial cells [42–44]; olfactory cells from schizophrenia patients [45]; neurons [46]; chemokines that drive migration of megakaryocytes from the proliferative osteoblastic niche within the bone marrow to the capillary-rich vascular niche, which is an essential step for platelet production [47]; migration of osteoclasts toward bone surfaces [48]; motility of cultured endothelial cells to study remodeling of their intercellular junctions [49]; generation of a complete polarized epithelial monolayer by the epithelial cells of mammary gland [17]; movement of cancer cells that were cultured under hypoxic conditions [50] and treated with salinomycin [24]; individual cell motility in fibroblastoid L929 cells [51]; human osteosarcoma MG-63 cells [52]; B35 neuroblastoma cells transiently expressing GFP and C6 glioma cells after staining with Hoechst 33258 [16] and motility of L5222 leukemia cells within the mesentery and migration of induced pluripotent cells during their early reprograming [53]. Of note, most studies are devoted to neural stem cells [18, 19, 54–63] due to growing clinical importance.

science and medicine. For this review, we focused on mammalian cell cultures, although TLM can also be efficiently employed to study prokaryotic cells and unicellular microorganisms. In the absence of up-to-date comprehensive review on TLM advances, our aim was to familiarize the readers with the current advances of TLM methodology and provide for the reference guide to the most interesting reports where TLM has been utilized both for biological research

In this part, we will briefly review some selected publications, which highlight the rapid development of TLM as a versatile discovery tool within the broad scope of modern biology and medicine. Importance of TLM as a new method in biological research was highlighted by Burton [5]. The progress of tissue culture methods, phase-contrast microscopy (see below) and real-time imaging by TLM enabled scientists to overcome the major limitation of traditional microscopy; preparation of very thin transparent samples, which required tissue fixation and did not make it possible to investigate living cells, let alone, and biological processes over time in the same sample. Early reports demonstrated the feasibility of TLM for comparative studies of cultured cells [6–8] and for monitoring living blood and lymph cells [1], cell division [9, 10] and reaction of cells to varying contents of electrolytes in perfusion chambers [11]. TLM was helpful to decode the process of multinucleation in the developing skeletal muscles

TLM is a suitable tool to monitor *cell motility and migration*, including quantitative assessment of migration, such as the number of migrating cells and the distance [15–20]. In multicellular organisms, the directed and coordinated cell migration (chemotaxis) occurs during embryonic development, tissue regeneration and inflammatory response [21], while cancer cells migrate into surrounding tissues and the vasculature. To monitor chemotaxis, TLM can be used together with the Dunn chamber [21–23]; Boyden chamber [24, 25]; Bridge chamber [26]; LOCOMOTIS, the motility tracking system [27] and other types of chambers for cell visualization and TLM applications [28–30]. TLM was employed to study embryonic stem cells [31]; hematopoietic progenitor cells [20, 32–34]; mesenchymal stem cells [35]; activated lymphocytes forming lymphocytes colonies [36]; primordial germ cells, a migratory cell population that will eventually give rise to the gametes [37–39]; the migration route of progenitor cells in cell cultures obtained from live chicken embryos [40, 41]; microglial cells [42–44]; olfactory cells from schizophrenia patients [45]; neurons [46]; chemokines that drive migration of megakaryocytes from the proliferative osteoblastic niche within the bone marrow to the capillary-rich vascular niche, which is an essential step for platelet production [47]; migration of osteoclasts toward bone surfaces [48]; motility of cultured endothelial cells to study remodeling of their intercellular junctions [49]; generation of a complete polarized epithelial monolayer by the epithelial cells of mammary gland [17]; movement of cancer cells that were cultured under hypoxic conditions [50] and treated with salinomycin [24]; individual cell

**2. Time-lapse microscopy: from making movies to bedside**

[12] and to describe the variable cytotoxic response toward allografts [13, 14].

and clinical purposes.

46 Cell Culture

**2.1. Versatility of TLM**

TLM allows investigators to visualize and characterize *cell-cell contacts* [46, 52, 64–71]. The most interesting reports are concerned with the contacts between the various types of stem/ progenitor cells as well as the tumor-environment cell interactions: the importance of proper cell-cell contacts level for their correct positioning and cell polarity during organogenesis [39], glial-neuronal interactions [72–74], interactions between microglia and brain tumors [75], between astrocytes and neural progenitor cells [42], between mesenchymal stem cells and human myoblasts [76], dendritic cells [77], endothelial cells [78], cancer cells [79], extracellular matrix molecules [80], between erythroblastic islands in bone marrow [81], between neural progenitor cells [62, 82–84], between neural cells and hematopoietic stem cells that migrate to the central nervous system [85], hematopoietic stem cells and stromal cells [20], endothelial progenitor cells and cardiac myocytes [86], between induced pluripotent cells during the early reprogramming phase [53], vesicle traffic through intercellular bridges between prostate cancer cells [87] and synaptic contacts [88–94].

*Cell division* and *cell death* can be well investigated with TLM [50, 52, 95–97]. Division and growth of both labeled [96, 98–100] and non-labeled [101, 102] cells in culture [52, 95, 98, 103–105] and tissue slices [106], including monitoring of a single cell [95, 99, 107–112], can be observed and assessed with TLM. The fluorescent ubiquitination-based cell cycle indicator (FUCCI) system can effectively label individual G1, S/G2/M and G1/S-transition phase nuclei as red, green and yellow, respectively, to visualize the real-time cell cycle transitions in living mammalian cells [113–116]. Microinjection of complementary RNA to cyclin B1 was reported as a tool for TLM studying meiosis [117]. Real-time imaging was employed to monitor nuclear envelope breakdown, which is one of the major morphological changes during mitosis [118] and apoptosis [119]; nucleolar assembly after mitosis [120]; tracking of template DNA strands during mitosis [121, 122]; preferred mitotic orientation of daughter cells, which is important for their following self-organization and tissue formation [123, 124]; interkinetic nuclear migration toward the apical surface in epithelial cells [125, 126]; multinucleation of skeletal muscle cells [12]; asymmetric division of stem cells [127–129]; identification and characterization of cell division genes by combining RNA interference, time-lapse microscopy and computational image processing [130]; cytokinesis [131, 132]; cleavage furrow [133]; abscission by using TLM in combination with electron microscopy [134, 135] and mitotic synchronization in the cell population [136]. The observations related to *cellular senescence* and various forms of *cell death* include re-entry into the cell cycle [10, 124, 137–139] and variable frequency of divisions [140]; changes in mitotic and interphase duration [141–147]; short G1 phase, which is a distinctive feature of mouse embryonic stem cells [148]; delayed G2 phase [149]; *neosis*, the term used for karyokinesis via nuclear budding followed by asymmetric, intracellular cytokinesis [150]; secretion of exosomes with anti-apoptotic microRNAs [151]; apoptosis [119, 152–156]; phagocytosis of apoptotic cells [157]; necrosis [158]; autophagy [159]; mitotic catastrophe [52, 143] and phototoxicity [160].

TLM can also be used to study *intracellular dynamics* of subcellular organelles [161, 162], natural cellular proteins and reporters, introduced nanoparticles and even physiological effects of small inorganic molecules and gases. Time-lapse imaging was used to monitor and quantify movements and changes in mitochondria [163–165]; Golgi apparatus [166]; centrosomes and microtubules [167–170]; centromeres [171]; cellular membrane [172]; dendritic spines [173]; dynamics of interkinetic nuclear migration [174, 175]; intercellular uptake and distribution of nano-sized (less than 100 mkm) ceramic particles [176]; intracellular translocation of p65 and IkappaB-alpha proteins [177]; intracellular distribution of integrin beta1 and F-actin [178]; fluctuations in Notch signaling to maintain neural progenitors [179]; re-localization of PP1gamma, which is implicated in multiple cell cycle-related processes including regulation of chromosome segregation and cytokinesis [180]; movement of the replication origin region of the chromosome during the cell cycle in *Bacillus subtilis* [181]; dynamics of 53BP1 protein in DNA-damage response [182]; measuring gene dynamics with luciferase as a reporter [183]; colocalization of MAP kinases in mitochondria [184]; clustering of acetylcholine receptor on myotubes [185]; multiple chromosomal populations of topoisomerase II [186]; focal points for chromosome condensation and decondensation [187]; intracellular calcium dynamics [188, 189] and single-cell time-lapse imaging of intracellular O2 [190].

they appear darker or brighter. This is the most common TLM technique that is used since 1950s [1, 6, 7, 11] for studying different types of cells and microorganisms both alone and in combination with electron microscopy [220–222]. The so-called differential interference contrast (DIC) microscopy (Nomarski microscopy) also produces high-contrast images of transparent nonstained biological objects, and it has been broadly used for TLM [223–226]. Fluorescent TLM dating back in 1950s TLM [9, 227] can be used nowadays with fluorescent proteins-reporters [207, 228–231], fluorescent nanoparticles [232, 233] and membrane dyes [160, 234, 235]. As the further proof of TLM flexibility, we present some reports where TLM is combined with other advanced microscopy techniques: multiplexed or multifield (recording of many fields simultaneously) TLM [236, 237], confocal TLM [156, 171, 207, 238–242], multi-photon TLM [58, 243–245], the so-called four-dimensional imaging (three-dimensional images over time) [242, 246], time-lapse bioluminescence analysis [247], Forster resonance energy transfer (FRET) microscopy [248], time-lapse optical coherence tomography [249–251], *in toto* imaging to image and track every single cell movement and division during the development of organs and tissues [241] and other innovative approaches [50, 252]. TLM can be used to monitor not only cultured cells (cell population and single cell [109] but also living cells in tissue slices up to a depth of 60 micrometers in brain slices, in regions where cell bodies remain largely uninjured by the tissue preparation and are visible in their natural environment [229, 253]. For real-time observation of corneal cells in a living mouse, a novel microscope system was designed, which consists of an upright fluorescence microscope for visualization of corneal cells, a mouseholding unit for immobilization of the animal and the eye and a set of gimbals which permit

Time-Lapse Microscopy

49

http://dx.doi.org/10.5772/intechopen.81199

observation of a wide area of corneal surface without refocusing [254].

TLM would not be possible without an *automated image analysis*, which is used to extract meaningful data from the bulk of images. Automated cell tracking faces problems associated with high cell density; cell mobility; cell division; multiple cell parameters such as object size, position or texture; cell lysis or overlap of cells [255]. A variety of algorithms, including *segmentation* (the process of partitioning a digital image into multiple sets of pixels or segments) algorithms, have been developed, and they are constantly improving. For most datasets, a *preprocessing* step is needed before information can be extracted. Irregular illumination and shading effects can be removed by using a *background subtraction method*. Other commonly used techniques include *contrast enhancement* and *noise filtering* [256]. In some cases, *registration* is needed to align subsequent image frames and compensate for unwanted movements. Global movements can be caused by movement of the specimen or imaging equipment, but local deformations in the specimen might also have to be corrected for. This is especially the case when considering TLM of living animals, which is heavily affected by breathing and heartbeat [257]. At higher magnifications, when studying intracellular dynamics, cell migration itself might also be considered an unwanted movement that has to be corrected [258]. *Object detection* is a set of techniques to separate objects of interest from the background. The objects of interest can be cells or intracellular particles [130, 259]. Basic segmentation techniques can be sufficient to detect individual cells, although more advanced techniques are still being developed to cope with increasingly complex data [260, 261]. Finally, several *analysis* techniques are available to quantify the different types of cell behavior over time, for example, *trajectory analysis* for assessing trajectory length and directional persistence [262]. By now, various algorithms are designed for quantifying and tracking cell migration [3] and

Although TLM is mostly used with cultured mammalian cells and live cells in tissues, the significant number of reports indicates that TLM could be employed to observe and study prokaryotic cells and other unicellular and multicellular organisms as well as viruses. Here, we mention only few examples, such as time-lapse imaging of growth, cell-cell contacts and formation of spherical granules in *E. coli* [191–194]; time-lapse visualization of bacterial colony morphologies in the special bacterial chamber MOCHA [195]; screening and assessing effects of antibiotics, such as antibiotics-bacteria interactions [196–199] and studying yeasts [200–202] and viruses [203–207]. The smaller microorganisms, analogously to intracellular structures, usually require higher magnification and more sophisticated microscopic equipment.
