Time-Lapse Microscopy

Time-lapse microscopy is a powerful, versatile and constantly developing tool for realtime imaging of living cells. This review outlines the advances of time-lapse microscopy and refers to the most interesting reports, thus pointing at the fact that the modern biology and medicine are entering the thrilling and promising age of molecular cinematography.


Introduction
Originally described as time-lapse cinemicrography (microphotography) [1], the modern time-lapse microscopy (TLM) emerged as a powerful and continuously improving tool for studying the cellular processes and cell-cell interactions with the applications ranging from fundamental aspects of molecular and cell biology to medical practice. The related time-lapse photography is more relevant to observing non-microscopic objects, such as plants and landscapes. TLM is the technique of capturing the sequence of microscopic images at regular intervals. TLM allows scientists to observe cellular dynamics and behavior of the population of living cells as well as of the single living cell within the population [2,3]. Live cell imaging and the first nonsophisticated TLM techniques were pioneered at the very beginning of the twentieth century [4]. However, to be visible in the light microscope, the cells are to be subjected to fixation and staining, the processes that kill the cells. Introduction of phase-contrast microscopy in 1940s, development of fluorescent and multidimensional microscopy, flow cytometry and computational tools made live cell imaging a widespread approach and prompted scientists to consider TLM as an essential technique that carries an enormous promise for basic biological 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 and clinical purposes.

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].
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][192][193][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][197][198][199] and studying yeasts [200][201][202] and viruses [203][204][205][206][207]. The smaller microorganisms, analogously to intracellular structures, usually require higher magnification and more sophisticated microscopic equipment.

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][209][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][212][213][214][215][216][217][218][219]. The up-to-date portable live cell culture monitor (CytoSMART Technologies, Eindhoven, The Netherlands) works within the regular CO 2 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][221][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][224][225][226]. Fluorescent TLM dating back in 1950s TLM [9,227] can be used nowadays with fluorescent proteins-reporters [207,[228][229][230][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][239][240][241][242], multi-photon TLM [58, [243][244][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][250][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 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].

TLM for assisted reproductive technology and its promise for clinical medicine
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 motility analysis [283].
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][285][286][287][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 [298], reprogramming in induced pluripotent cells [110] and other applications.

Conclusion
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 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.