**2.2. TLM technical approaches**

TLM monitoring of mammalian cells usually requires the inverted microscope, which is fully or partially enclosed by a cell incubator (environmental chamber), a partly sealed transparent box that maintains the temperature, humidity and even partial gas (carbon dioxide) pressure, protects cultured cells from the light and allows the investigator to manipulate with the microscope in order to choose the field of view and adjust other imaging parameters [208–210]. The TLM chambers and devices underwent significant improvements over the time, from the simple glass tissue chambers and manual capturing sequences of images to the automated high-resolution microscopes and sophisticated computerized equipment for long-term TLM observations [154, 162, 211–219]. The up-to-date portable live cell culture monitor (CytoSMART Technologies, Eindhoven, The Netherlands) works within the regular CO2 incubator. The culture flask (T-flask, Petri dish, wells or any other transparent vessel) is positioned onto the lens of the device; the field of view is chosen by the investigator, and the cell growth and migration can be monitored and analyzed in the real-time mode by accessing the cloud [52].

The *phase-contrast* method of imaging is based on the ability of materials with a different refractive index to delay the passage of the light through the sample by different amounts, so that 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 observation of a wide area of corneal surface without refocusing [254].

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

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.

Technologies, Eindhoven, The Netherlands) works within the regular CO2

can be monitored and analyzed in the real-time mode by accessing the cloud [52].

TLM monitoring of mammalian cells usually requires the inverted microscope, which is fully or partially enclosed by a cell incubator (environmental chamber), a partly sealed transparent box that maintains the temperature, humidity and even partial gas (carbon dioxide) pressure, protects cultured cells from the light and allows the investigator to manipulate with the microscope in order to choose the field of view and adjust other imaging parameters [208–210]. The TLM chambers and devices underwent significant improvements over the time, from the simple glass tissue chambers and manual capturing sequences of images to the automated high-resolution microscopes and sophisticated computerized equipment for long-term TLM observations [154, 162, 211–219]. The up-to-date portable live cell culture monitor (CytoSMART

ture flask (T-flask, Petri dish, wells or any other transparent vessel) is positioned onto the lens of the device; the field of view is chosen by the investigator, and the cell growth and migration

The *phase-contrast* method of imaging is based on the ability of materials with a different refractive index to delay the passage of the light through the sample by different amounts, so that

[190].

incubator. The cul-

[188, 189] and single-cell time-lapse imaging of intracellular O2

**2.2. TLM technical approaches**

48 Cell Culture

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 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].

within the incubator), non-natural impacts on living cells by the high excitation energy of lasers and bleaching/degradation of the fluorochromes over time, which influences quantification of long-running processes. However, the growing number of reports about new improvements and advances in TLM techniques and TLM-related applications that provide valuable information, which is not imageable by other techniques, makes it possible to conclude that the era of microcinematography in biomedical research has just begun.

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,

, Kai Ding3

4 Department of Biological & Environmental Sciences, Troy University, Troy, AL, USA

[1] Klausewitz W. Cytodiagnostic studies on living blood and lymph cells of some Amphibia by means of micro-time lapse film and phase contrast microscopy. Zeitschrift

[2] Baker M. Cellular imaging: Taking a long, hard look. Nature. 2010;**466**(7310):1137-1140 [3] Svensson CM, Medyukhina A, Belyaev I, Al-Zaben N, Figge MT. Untangling cell tracks: Quantifying cell migration by time lapse image data analysis. Cytometry Part A: The

Journal of the International Society for Analytical Cytology. 2018;**93**(3):357-370

and Alexander V. Kofman<sup>4</sup>

\*

Time-Lapse Microscopy

51

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

**Acknowledgements**

**Conflict of interest**

None declared.

**Author details**

John L. Collins1

**References**

Award Number R37CA229417.

, Bart van Knippenberg2

\*Address all correspondence to: akofman@troy.edu

1 University of Tennessee at Martin, Martin, TN, USA

2 Cytosmart Technologies BV, Eindhoven, The Netherlands

3 Johns Hopkins University School of Medicine, Baltimore, MD, USA

für Zellforschung und Mikroskopische Anatomie. 1953;**39**(1):1-35
