\r\n\tThis book aims to cover following topics: i) the effects of long-term environmental change past events on turtle diversification and their evolutionary responses to climate change, ii) responses (developmental, physiological and behavioural) of extant species to environmental stressors, iii) impacts of changing environmental conditions on life history traits (growth patterns, sexual maturation, reproduction, longevity), iv) thermal environment change, biogeographic distribution and ecological niche modeling, v) environmental variation, population and community dynamics, and population level-response modeling, and vi) impacts of future global environmental change (climate change, alongside habitat destruction and fragmentation and overexploitation,) on population future trend and viability and their implications for informing adaptive conservation management strategies.
",isbn:null,printIsbn:"979-953-307-X-X",pdfIsbn:null,doi:null,price:0,priceEur:0,priceUsd:0,slug:null,numberOfPages:0,isOpenForSubmission:!1,hash:"ec7c5f39f89066d7d788873d669bf740",bookSignature:"Prof. Mohammed Znari and Dr. Mohamed Naimi",publishedDate:null,coverURL:"https://cdn.intechopen.com/books/images_new/8155.jpg",keywords:"Climate-mediated species diversification, turtle fossil record, evolutionary adaptation, Environmental stressors, growth, sexual maturation and reproduction, fluctuating asymmetry, homeostatic adaptations, ecological niche modelling , population and community dynamic, conservation strategies",numberOfDownloads:null,numberOfWosCitations:0,numberOfCrossrefCitations:0,numberOfDimensionsCitations:null,numberOfTotalCitations:null,isAvailableForWebshopOrdering:!0,dateEndFirstStepPublish:"August 29th 2019",dateEndSecondStepPublish:"September 19th 2019",dateEndThirdStepPublish:"November 18th 2019",dateEndFourthStepPublish:"February 6th 2020",dateEndFifthStepPublish:"April 6th 2020",remainingDaysToSecondStep:"2 years",secondStepPassed:!0,currentStepOfPublishingProcess:5,editedByType:null,kuFlag:!1,biosketch:null,coeditorOneBiosketch:null,coeditorTwoBiosketch:null,coeditorThreeBiosketch:null,coeditorFourBiosketch:null,coeditorFiveBiosketch:null,editors:[{id:"223073",title:"Prof.",name:"Mohammed",middleName:null,surname:"Znari",slug:"mohammed-znari",fullName:"Mohammed Znari",profilePictureURL:"https://mts.intechopen.com/storage/users/223073/images/system/223073.jpg",biography:"Mohammed Znari, has PhDs in Ecology (from Pierre & Marie Curie University, Paris, France, 1988 and Cadi Ayyad University, Marrakech, Morocco, 1999). He is a full Professor of ecology and conservation biology at the Faculty of Science – Semlalia, Marrakech. His research interests are morphometrics, systematics and molecular phylogeography, physiological ecology, ecology and life history along with conservation ecology in various taxa including turtles, steppe-land birds and mammals. He has been involved in several international projects and obtained several international fellowships and research-conservation grants. He is also the curator of vertebrate zoology at the Natural History Museum of Marrakech. He was a member of the International Committee of the World Congress of Herpetology and he is currently a vice-President of the Moroccan Herpetological Society. He has produced dozens of publications in peer-reviewed journals and contributions in international scientific meetings.",institutionString:"Cadi Ayyad University",position:null,outsideEditionCount:0,totalCites:0,totalAuthoredChapters:"1",totalChapterViews:"0",totalEditedBooks:"0",institution:null}],coeditorOne:{id:"292272",title:"Dr.",name:"Mohamed",middleName:null,surname:"Naimi",slug:"mohamed-naimi",fullName:"Mohamed Naimi",profilePictureURL:"https://mts.intechopen.com/storage/users/292272/images/system/292272.jpg",biography:"Mohamed NAIMI has a PhD in Animal Biology, Ecology, Conservation Biology and Environment (Cadi Ayyad University –UCA- Marrakech, Morocco). He has been an Assistant professor at Sultan Moulay Slimane University - SMSU - Beni-Mellal, Morocco since 2016. He is a member of the Polyvalent Laboratory for Research and Development (LPVRD) at SMSU and associate member of the Biodiversity and Ecosystems Dynamics Laboratory and the Natural History Museum of Marrakech –UCA-. His research interests are trophic ecology, developmental and reproductive biology, morphometry and physiological ecology (on various taxa, including aquatic animals, herpetofauna and terrestrial mammals). He has participated in several international projects and received different scholarships. He has produced several papers in peer-reviewed journals and communications in international scientific meetings. Before joining the SMSU, he held several positions in the external services of the Marine Fisheries Department in Morocco.",institutionString:"University of Sultan Moulay",position:null,outsideEditionCount:0,totalCites:0,totalAuthoredChapters:"0",totalChapterViews:"0",totalEditedBooks:"0",institution:null},coeditorTwo:null,coeditorThree:null,coeditorFour:null,coeditorFive:null,topics:[{id:"5",title:"Agricultural and Biological Sciences",slug:"agricultural-and-biological-sciences"}],chapters:null,productType:{id:"1",title:"Edited Volume",chapterContentType:"chapter",authoredCaption:"Edited by"},personalPublishingAssistant:{id:"250240",firstName:"Nino",lastName:"Popovic",middleName:null,title:"Mr.",imageUrl:"https://mts.intechopen.com/storage/users/250240/images/7088_n.png",email:"nino@intechopen.com",biography:"As an Author Service Manager my responsibilities include monitoring and facilitating all publishing activities for authors and editors. From chapter submission and review, to approval and revision, copyediting and design, until final publication, I work closely with authors and editors to ensure a simple and easy publishing process. I maintain constant and effective communication with authors, editors and reviewers, which allows for a level of personal support that enables contributors to fully commit and concentrate on the chapters they are writing, editing, or reviewing. I assist authors in the preparation of their full chapter submissions and track important deadlines and ensure they are met. I help to coordinate internal processes such as linguistic review, and monitor the technical aspects of the process. As an ASM I am also involved in the acquisition of editors. Whether that be identifying an exceptional author and proposing an editorship collaboration, or contacting researchers who would like the opportunity to work with IntechOpen, I establish and help manage author and editor acquisition and contact."}},relatedBooks:[{type:"book",id:"6418",title:"Hyperspectral Imaging in Agriculture, Food and Environment",subtitle:null,isOpenForSubmission:!1,hash:"9005c36534a5dc065577a011aea13d4d",slug:"hyperspectral-imaging-in-agriculture-food-and-environment",bookSignature:"Alejandro Isabel Luna Maldonado, Humberto Rodríguez Fuentes and Juan Antonio Vidales Contreras",coverURL:"https://cdn.intechopen.com/books/images_new/6418.jpg",editedByType:"Edited by",editors:[{id:"105774",title:"Prof.",name:"Alejandro Isabel",surname:"Luna Maldonado",slug:"alejandro-isabel-luna-maldonado",fullName:"Alejandro Isabel Luna Maldonado"}],productType:{id:"1",chapterContentType:"chapter",authoredCaption:"Edited by"}},{type:"book",id:"1591",title:"Infrared Spectroscopy",subtitle:"Materials Science, Engineering and Technology",isOpenForSubmission:!1,hash:"99b4b7b71a8caeb693ed762b40b017f4",slug:"infrared-spectroscopy-materials-science-engineering-and-technology",bookSignature:"Theophile Theophanides",coverURL:"https://cdn.intechopen.com/books/images_new/1591.jpg",editedByType:"Edited by",editors:[{id:"37194",title:"Dr.",name:"Theophanides",surname:"Theophile",slug:"theophanides-theophile",fullName:"Theophanides Theophile"}],productType:{id:"1",chapterContentType:"chapter",authoredCaption:"Edited by"}},{type:"book",id:"3161",title:"Frontiers in Guided Wave Optics and Optoelectronics",subtitle:null,isOpenForSubmission:!1,hash:"deb44e9c99f82bbce1083abea743146c",slug:"frontiers-in-guided-wave-optics-and-optoelectronics",bookSignature:"Bishnu Pal",coverURL:"https://cdn.intechopen.com/books/images_new/3161.jpg",editedByType:"Edited by",editors:[{id:"4782",title:"Prof.",name:"Bishnu",surname:"Pal",slug:"bishnu-pal",fullName:"Bishnu Pal"}],productType:{id:"1",chapterContentType:"chapter",authoredCaption:"Edited by"}},{type:"book",id:"3092",title:"Anopheles mosquitoes",subtitle:"New insights into malaria vectors",isOpenForSubmission:!1,hash:"c9e622485316d5e296288bf24d2b0d64",slug:"anopheles-mosquitoes-new-insights-into-malaria-vectors",bookSignature:"Sylvie Manguin",coverURL:"https://cdn.intechopen.com/books/images_new/3092.jpg",editedByType:"Edited by",editors:[{id:"50017",title:"Prof.",name:"Sylvie",surname:"Manguin",slug:"sylvie-manguin",fullName:"Sylvie Manguin"}],productType:{id:"1",chapterContentType:"chapter",authoredCaption:"Edited by"}},{type:"book",id:"371",title:"Abiotic Stress in Plants",subtitle:"Mechanisms and Adaptations",isOpenForSubmission:!1,hash:"588466f487e307619849d72389178a74",slug:"abiotic-stress-in-plants-mechanisms-and-adaptations",bookSignature:"Arun Shanker and B. 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Physical and chemical agents can affect cell health and metabolism. These agents may cause toxicity on cells via different mechanisms such as destruction of cell membranes, prevention of protein synthesis, irreversible binding to receptors, inhibition of polydeoxynucleotide elongation, and enzymatic reactions [1]. In order to determine the cell death caused by these mechanisms, there is a need for cheap, reliable and reproducible short-term cytotoxicity and cell viability assays.
In vitro cell viability and cytotoxicity assays with cultured cells are widely used for cytotoxicity tests of chemicals and for drug screening. Application of these assays has been of increasing interest over recent years. Currently, these assays are also used in oncological researches to evaluate both compound toxicity and tumor cell growth inhibition during drug development. Because, they are rapid, inexpensive and do not require the use of animals. Furthermore, they are useful for testing large number of samples. Cell viability and cytotoxicity assays are based on various cell functions such as cell membrane permeability, enzyme activity, cell adherence, ATP production, co-enzyme production, and nucleotide uptake activity [1].
It is important to know how many viable cells are remaining and/or how many cells are dead at the end of the experiment. A broad spectrum of cytotoxicity and cell viability assays is currently used in the fields of toxicology and pharmacology. The choice of assay method is crucial in the assessment of the interaction type [3].
Although there are different classifications for cytotoxicity and cell viability assays, in this chapter, these assays are classified according to measurement types of end points (color changes, fluorescence, luminescent etc.).
Dye exclusion: Trypan blue, eosin, Congo red, erythrosine B assays.
Colorimetric assays: MTT assay, MTS assay, XTT assay, WST-1 assay, WST-8 assay, LDH assay, SRB assay, NRU assay and crystal violet assay.
Fluorometric assays: alamarBlue assay and CFDA-AM assay.
Luminometric assays: ATP assay and real-time viability assay.
The proportion of viable cells in a cell population can be estimated in various methods. The simplest and widely used one of the methods is dye exclusion method. In dye exclusion method, viable cells exclude dyes, but dead cells not exclude them. Although the staining procedure is quite simple, experimental procedure of large number of samples is difficult and time consuming [4]. Determination of membrane integrity is possible via dye exclusion method. A variety of such dyes have been employed, including eosin, Congo red, erythrosine B, and trypan blue [5, 6]. Of the dyes listed, trypan blue has been used the most extensively [7, 8, 9, 10].
If dye exclusion assays are used, following factors must be considered (i) lethally damaged cells by cytotoxic agents may require several days to lose their membrane integrity, (ii) the surviving cells may continue to proliferate during this time, and (iii) some lethally damaged cells are not appear to be stained with dye at the end of the culture period, because they may undergo an early disintegration. Factors (ii) and (iii) may cause an underestimate of cell death when the results of the assay are based on percent viability expression [11, 12, 13].
Dye exclusion assays have unique advantages for chemosensitivity testing. They are comparatively simple, require small numbers of cells, are rapid, and are capable of detecting cell kill in nondividing cell populations. Further investigations into the possible role of these assays in chemosensitivity testing are warranted [11]. However, none of these dyes is recommended for use on monolayer cell cultures but rather they are intended for cells in suspension; thus monolayer cells must first trypsinized [6].
This dye exclusion assay is used to determine the number of viable and/or dead cells in a cell suspension. Trypan blue is a large negatively charged molecule. Trypan blue dye exclusion assay is based on the principle that live cells possess intact cell membranes that exclude this dye, whereas dead cells do not. In this assay, adherent or nonadherent cells are incubated with serial dilutions of test compounds for various times. After the compound treatment, cells are washed and suspended. Cell suspension is mixed with dye and then visually examined to determine whether cells take up or exclude dye. Viable cells will have a clear cytoplasm, whereas dead cells will have a blue cytoplasm [14, 15]. Number of viable and/or dead cells per unit volume is determined by light microscopy as a percentage of untreated control cells [15, 16].
While the staining procedure is quite simple, it is difficult to process large number of samples concurrently, particularly where the exact timing of progressive cytotoxic effects is required [4]. Furthermore, trypan blue staining cannot be used to distinguish between the healthy cells and the cells that are alive but losing cell functions. Therefore, it is not sufficiently sensitive to use for in vitro cytotoxicity testing. Another disadvantage of trypan blue is toxic side effect of this dye on mammalian cells [20].
Erythrosine B, also known as erythrosine or Red No. 3, is primarily used as food coloring agent [20, 21]. Erythrosine B has already been introduced as a vital dye for counting viable cells. Principle of this dye exclusion assay is similar to trypan blue dye exclusion assay principle. Although erythrosine B is an alternative bio-safe vital dye for cell counting; it is not widely used to count viable or dead cells.
Principle of colorimetric assays is the measurement of a biochemical marker to evaluate metabolic activity of the cells. Reagents used in colorimetric assays develop a color in response to the viability of cells, allowing the colorimetric measurement of cell viability via spectrophotometer. Colorimetric assays are applicable for adherent or suspended cell lines, easy to perform, and comparably economical [22, 23]. Commercial kits of colorimetric assays are available from several companies and generally experimental procedures of these assays are available in kit packages.
MTT (3-(4,5-dimethylthiazol-2-yl)-2–5-diphenyltetrazolium bromide) assay is one of the most commonly used colorimeteric assay to assess cytotoxicity or cell viability [24]. This assay determines principally cell viability through determination of mitochondrial function of cells by measuring activity of mitochondrial enzymes such as succinate dehydrogenase [18]. In this assay, MTT is reduced to a purple formazan by NADH. This product can be quantified by light absorbance at a specific wavelength.
Additional control experiments should be conducted to reduce false-positive or false-negative results that caused by background interference due to inclusion of particles. This interference could lead to an overestimation of the cell viability. This can often be controlled by subtraction of the background absorbance of the cells in the presence of the particles, but without the assay reagents [18, 26].
The MTS assay (5-(3-carboxymethoxyphenyl)-2-(4,5-dimethyl-thiazoly)-3-(4-sulfophenyl) tetrazolium, inner salt assay) is a colorimetric assay. This assay is based on the conversion of a tetrazolium salt into a colored formazan by mitochondrial activity of living cells. The amount of produced formazan is depend on the viable cell number in culture and can be measured with spectrophotometer at 492 nm.
A colorimetric method based on the tetrazolium salt XTT (2,3-bis(2-methoxy-4-nitro-5-sulphophenyl)-5-carboxanilide-2H-tetrazolium, monosodium salt) was first described by Scudiero et al. [34]. While MTT produced a water-insoluble formazan compound which required dissolving the dye in order to measure its absorbance, the XTT produces a water-soluble dye. The procedure of XTT is simply for measuring proliferation and is therefore an excellent solution for quantitating cells and determining their viability. XTT is used to assay cell proliferation as response to different growth factors. It is also used for assaying cytotoxicity.
This assay is based on the ability reduction of the tetrazolium salt XTT to orange-colored formazan compounds by metabolic active cells. Orange-colored formazan is water soluble and its intensity can be measured with a spectrophotometer. There is a linear relationship between the intensity of the formazan and the number of viable cells. The use of multiwell plates and a spectrophotometer (or ELISA reader) allows for study with a large number of samples and obtaining results easily and rapidly. The procedure of this assay includes cell cultivation in a 96-well plate, adding the XTT reagent and incubation for 2–24 hours. During the incubation time, an orange color is formed and the intensity of color can be measured with a spectrophotometer [34, 35].
WST-1 (2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2
To perform the assay, the WST-1 reagent that is ready-to-use is added directly into the media of cells cultured in multiwell plates. The cultures are then given 30 minutes–4 hours to reduce the reagent into the dye form. The plate is then immediately read at 450 nm with a reference reading at 630 nm [37].
WST-8 assay is a colorimetric assay for the determination of viable cell numbers and can be used for cell proliferation assays as well as cytotoxicity assays. WST-8 (2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2
LDH (lactate dehydrogenase) cytotoxicity assay is a colorimetric method of assaying cellular cytotoxicity. LDH Cytotoxicity Assay Kit can be used with different cell types not only for assaying cell-mediated cytotoxicity but also for assessment of cytotoxicity mediated by toxic chemicals and other test compounds. The assay measures the stable, cytosolic, lactate dehydrogenase (LDH) enzyme quantitatively. This enzyme releases from damaged cells. LDH is an enzyme that is normally found within the cell cytoplasm. When cell viability reduced leakiness of the plasma membrane increase and therefore LDH enzyme is released into the cell culture medium. The released LDH is measured with a coupled enzymatic reaction that results in the conversion of a tetrazolium salt (iodonitrotetrazolium (INT)) into a red color formazan by diaphorase. In the first step, LDH catalyze conversion of lactate to pyruvate and thus NAD is reduced to NADH/H+. In a second step, catalyst (diaphorase) transfers H/H+ from NADH/H+ to the tetrazolium salt 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyltetrazolium chloride (INT), which is reduced to red formazan [38, 39].
The LDH activity is determined as NADH oxidation or INT reduction over a defined time period. The resulting red formazan absorbs maximally at 492 nm and can be measured quantitatively at 490 nm.
The detergent Triton X-100 is commonly used as positive control in the LDH assay to determine the maximum LDH release from the cells. In addition, well-known membranolytic particles such as crystalline silica can be used as a positive control in LDH assay [40].
SRB (Sulforhodamine B) assay is a rapid and sensitive colorimetric method for measuring the drug-induced cytotoxicity in both attached and suspension cell cultures. This assay as first described by Skehan and colleagues was developed for use in the disease-orientated, large-scale anticancer drug discovery program of the National Cancer Institute (NCI) that was launched in 1985. SRB is a bright pink aminoxanthene dye with two sulfonic groups. Under mildly acidic conditions, SRB binds to protein basic amino acid residues in TCA-fixed (trichloroacetic acid) cells to provide a sensitive index of cellular protein. SRB assay is also used to evaluate colony formation and colony extinction [43].
The neutral red uptake (NRU) assay is also one of the most used colorimetric cytotoxicity/cell viability assay. This assay was developed by Borenfreund and Puerner [44]. This assay was based on the ability of viable cells to take up the supravital dye neutral red. This weakly cationic dye penetrates cell membranes by nonionic passive diffusion and concentrates in the lysosomes. The dye is then extracted from the viable cells using an acidified ethanol solution and the absorbance of the dye is measured using spectrophotometer.
Neutral red uptake depends on the capacity of cells to maintain pH gradients through the ATP production. At physiological pH, net charge of the dye is zero. This charge enables the dye to penetrate the cell membranes. Inside the lysosomes, there is a proton gradient to maintain a pH lower than that of the cytoplasm. Thus, the dye becomes charged and is retained inside the lysosomes. When the cell dies or pH gradient is reduced, the dye cannot be retained. In addition, the uptake of neutral red by viable cells can be modified by alterations in cell surface or lysosomal membranes. Thus, it is possible to distinguish between viable, damaged, or dead cells [44]. Lysosomal uptake of neutral red dye is a highly sensitive indicator of cell viability. The assay can quantitate cell viability and measure cell replication, cytostatic effects or cytotoxic effects depending on the seeding density [45]. Absorbance is measured at 540 nm in multiwell plate reader spectrophotometer.
Adherent cells detach from cell culture plates during cell death. This feature can be used for the indirect assessment of cell death and to determine differences in proliferation rate upon stimulation with cytotoxic agents. One simple method to detect maintained adherence of cells is crystal violet assay. In this assay, crystal violet dye binds to proteins and DNA of viable cells, and thus, attached cells are stained with this dye. Cells lose their adherence during cell death and are subsequently lost from the population of cells, reducing the amount of crystal violet staining in a culture. Crystal violet assay is a quick and reliable screening method that is suitable for the examination of the impact of chemotherapeutics or other compounds on cell survival and growth inhibition [47].
Fluorometric assays of cell viability and cytotoxicity are easy to perform with the use of a fluorescence microscope, fluorometer, fluorescence microplate reader or flow cytometer, and they offer many advantages over traditional dye exclusion and colorimetric assays. Fluorometric assays are also applicable for adherent or suspended cell lines and easy to use. These assays are more sensitive than colorimetric assays [52, 53, 54]. Commercial kits of fluorometric assays are available from several companies and generally experimental procedures of these assays are available in kit packages.
alamarBlue assay is also known as resazurin reduction assay. The alamarBlue assay is based on the conversion of the blue nonfluorescent dye resazurin, which is converted to the pink fluorescent resorufin by mitochondrial and other enzymes such as diaphorases [53].
Resazurin is a phenoxazin-3-one dye and cell permeable redox indicator that can be used to monitor viable cell number with protocols similar to those utilizing the tetrazolium compounds [55]. It is known to act as an intermediate electron acceptor in the electron transport chain between the final reduction of oxygen and cytochrome oxidase by substituting for molecular oxygen as an electron acceptor [52]. It is a nontoxic and cell permeable compound. Color of this compound is blue and it is nonfluorescent. After entering cells, resazurin is reduced to resorufin. Resorufin is red in color and highly fluorescent compound. Viable cells convert continuously resazurin to resofurin, increasing overall fluorescence and color of the cell culture medium. The quantity of produced resofurin is related to the number of viable cells. Ratio of viable cells can be quantified using a microplate reader fluorometer equipped with a 560 nm excitation/590 nm emission filter set. Resofurin can also be measured by absorbance changes, but absorbance detection is not often used because absorbance detection is less sensitive than fluorescence measurement.
The incubation period required to generate a sufficient fluorescent signal above background is usually about 1–4 hours, depending on metabolic activity of the cells, the cell density per well and other conditions such as the culture medium type [54].
CFDA-AM (5-carboxyfluorescein diacetate, acetoxymethyl ester) is another fluorogenic dye that is used for cytotoxicity determination. It is indicator for plasma membrane integrity. The dye CFDA-AM is nontoxic esterase substrate that can be converted by nonspecific esterases of viable cells from a membrane permeable, nonpolar, nonfluorescent substance to polar, fluorescent dye, carboxyfluorescein (CF). The conversion of CFDA-AM to CF by the cells indicates the integrity of plasma membrane, since only an intact membrane can maintain the cytoplasmic milieu which is needed to support esterase activity [56].
Measurement of a conserved and constitutive protease enzyme activity of viable cells is used as a good indicator of cell viability. A cell permeable fluorogenic protease substrate (glycylphenylalanyl-aminofluorocoumarin; GF-AFC) has been recently developed to selectively detect protease activity that is restricted to viable cells [59]. The GF-AFC substrate can penetrate viable cells. In these cells, cytoplasmic aminopeptidase activity removes the gly and phe amino acids to release aminofluorocoumarin (AFC) and produce a fluorescent signal proportional to the number of viable cells [54].
When cells die, this protease activity is rapidly loss. Therefore, this protease activity is a selective marker of the viable cell population. The signal generated from this assay approach has been shown to correlate well with other established methods of determining cell viability such as an ATP assay [54].
Luminometric assays provide fast and simple determination of cell proliferation and cytotoxicity in mammalian cells. These assays can be performed in a convenient 96-well and 384-well microtiter plate format and detection by luminometric microplate reader [54, 60, 61]. A remarkable feature of the luminometric assays is the persistent and stable glow-type signal produced after reagent addition. This attribute can be harnessed to produce both viability and cytotoxicity values from the same well [59]. Commercial kits of luminometric assays are available from several companies and generally experimental procedures of these assays are available in kit packages.
ATP (adenosine tri-phosphate) represents the most important chemical energy reservoir in cells and is used for biological synthesis, signaling, transport, and movement processes. Therefore, cellular ATP is one of the most sensitive end points in measuring cell viability [62]. When cells damaged lethally and lose membrane integrity, they lose the ability to synthetize ATP and the ATP level of cells decreases dramatically [54, 63]. The ATP assay is based on the reaction of luciferin to oxyluciferin. Enzyme luciferase catalyzes this reaction in the presence of Mg2+ ions and ATP yielding a luminescent signal. There is a linear relationship between the intensity of luminescent signal and ATP concentration [61] or cell number [64].
The ATP assay chemistry can typically detect fewer than 10 cells per well, and therefore, it has been widely used 1536-well plate format.
Recently, a new approach is developed to measure viable cell number in real time [60]. In this assay, an engineered luciferase derived from a marine shrimp and a small molecule prosubstrate is used. The pro-substrate and luciferase are added directly to the cell culture medium as a reagent. The pro-substrate is not a substrate of luciferase. Viable cells with an active metabolism reduce the pro-substrate into a substrate, which used by luciferase, to generate a luminescent signal. The assay can be performed in two formats: continuous read and endpoint measurement. In the continuous read format, the luminescent signal can be repeatedly recorded from the sample wells over an extended period to measure the number of cells in “real time” [54, 60].
A broad spectrum of cytotoxicity and cell viability assays is currently used in the fields of toxicology and pharmacology. An ideal assay for
The author would like to thank Asst. Prof. Tülay AŞKIN ÇELİK for her helpful advice in this chapter.
This chapter includes the details of solution based routes to deposit thin films which includes spray coating, dip coating, spin coating and inkjet printing processing. The contribution of different experimental parameters such as solution viscosity, surface tension, droplet size, substrate material & temperature, nature of solution are discussed briefly.
\nSpin coating is a quick and common route to deposit thin films on substrates with primary advantage of ease to produce very uniform films. The solution of a specific material is spun at high speeds, the centripetal force and the surface tension of the liquid together create an even covering on the substrate. The excessive solvent is evaporated, and spin coating results in a thin film ranging from a few nanometers to a few microns in thickness. Spin coating technique is used to coat small substrates from a few mm square to a metre or more in diameter. The key advantage of spin coating technique is the simplicity and relative ease to set up the process, coupled with the thinness and uniformity.
\nSpin coating consists of three major stages, solution dispensing, rotation dominated thinning and solvent evaporation as shown in \nFigure 1\n. The rotation pulls the liquid (solution) coating into an even covering and then evaporates to leave the desired material on the substrate in an even distribution. The high spin speeds and the high airflow leads to fast drying, which in turn results in high consistency at both macroscopic and nano length scales. However, the fast drying times lead to lower performance for certain processes, which requires time to self-assemble and/or crystallize.
\nSchematic of spin coating.
Spin coating also relatively low throughput process due to an inherently batch (single substrate) process compared to roll-to-roll processes. Despite these drawbacks, spin coating is usually the starting point and benchmark for most academic and industrial processes that require a thin and uniform coating. Spin coating process can be broadly divided into 4 main stages.
\n\n
\n
\n
\n
All above processes are repeated several times to research desired film thickness. The solution casting and drying stages of are an integral and crucial part of the spin coating process, which contributes to the key process such as stacking/crystallization, phase separation and aggregation. Precise control of these processes is critical as the characteristics of deposited thin films not only depend upon morphology (thickness, uniformity) but also on deposition processing. In general, the spin speed speeds of >1000 rpm is recommended for industrial processing to ensure the high uniformity. However, spin speeds down to 200 rpm can be employed for laboratory scale deposition which might slow down the drying process but allow additional time for self-assembly.
\nIn case of slow spinning, the solvent begins to evaporate as solvent is dispensed across the substrate and produces internal currents resulted in formation of highly ordered well assembled thin films by slowing down the evaporation rate. But, the coating across the surface is usually highly uneven with a typical “coffee staining” effect. However, it is possible to obtain a high level of nanoscale order by spinning at very lower speeds. Slow drop casting without rotation is a good way to deposit highly ordered thin films at the nanoscale but at the expense of uniformity.
\nIn general, the thickness of a spin coated thin film is proportional to the inverse of the spin speed squared. However, the relation does not always apply and use to predict the film thickness without experimental data. Usually a test film is grown and measured. By using the data point(s) obtained from test film, spin thickness curve can be plotted with a reasonable accuracy. The spin speed can then be adjusted to obtain the right film thickness. The exact thickness of a deposited thin film is subjected to many factors as the material concentration and solvent evaporation rate which in turn depends upon the solvent viscosity, vapor pressure, temperature and local humidity etc. Therefore, the spin thickness curves for specific solution is commonly determined empirically [1].
\nThe following parameters play very crucial role during spin coating process of thin film deposition.
\n\n
\n
\n
In a dynamic dispense, the substrate is first set on spinning and allowed to acquire desired speed and then the solution is dispensed at the center of the substrate. The centripetal force pulls the liquid away from the middle of substrate across the entire area before dries up. The dynamic dispense is more suitable due to precise controlling and better substrate-to-substrate variation. In this process, the solvent has less time to evaporate before the spinning start. Therefore, the ramp speed and dispense time is less critical as the substrate has been reached to the desired rpm. The dynamic dispense requires less solution in general although depends upon the wetting properties of the surface.
\nThe major setback of the dynamic dispense is the incomplete substrate coverage in case of low spin speeds below 1000 rpm and viscous solutions due to insufficient centripetal force to pull the liquid across the surface. The lower rotation speed enhances the probability that the solution may be dispensed before the substrate has completed a full rotation. For the majority of spin coating above 1000 rpm, a dynamic dispense is recommended as standard process. A dynamic dispense process can be performed at speeds all the way down to around 500 rpm. However it becomes more difficult to get complete surface coverage.
\n\n
\n
The dip coating is a facile, simple, the low cost and the high quality coating processing used for industrial as well as laboratory applications. The dip coating is commonly used for optical coatings such as in the production of automotive rear mirrors and large area antireflective coating for solar control glasses [4]. [The dip coating process involves immersing of a substrate into the solution of coating materials and then withdraw the solution. The process can be defined as deposition of aqueous-based liquid phase onto the surface of substrate using a solution. Generally, the required material is dissolved in solutions and directly coated on the substrate surface, then the sedimentary (solvent) wet coating is evaporated to get dry film. The dip-coating process involves complex chemical and physical multi-variable parameters. The film thickness and morphology depends on immersion time, withdrawal speed, dip-coating cycles, density and viscosity, surface tension, substrate surface and evaporation conditions of coating solutions. Photo-assisted dip-coating is used to control the evaporation process of coated solution and the irradiation effect facilitate the film disposition. To increase the uniformity and thickness of films, the multi-layered dip-coating is applied [5].
\nDip coating consists of four basic steps as immersion, dwelling, withdrawal, and drying as shown in \nFigure 2\n. In first stage, the substrate is immersed into a solution to be deposited until it is completely covered with liquid. The substrate is then withdrawn after a short interval. During the withdrawal process, a thin layer of the solution residues on the substrate surface. Once the substrate is fully withdrawn, the solution from the deposited film starts to evaporate and leaves behind a dry film. The deposited material undergoes a chemical or physical change.
\nSchematic of dip coating in lab.
The withdrawal and drying stages are critical stages to determine the properties of the deposited film. The withdrawal stage involves the interaction of different sets of forces. These forces are categorized into two categories, entraining forces and draining forces. Draining forces draw the liquid (solution) back to the bath from substrate surface. On the other hand, the entraining forces keeps the solution onto the substrate surface. The balance between these two sets of forces determines wet film thickness coated onto the substrate. During the withdrawal stage, the formation of the wet film can be broken into four regions as illustrated in \nFigure 3\n.
\nDifferent regions during dip coating film formation.
In the static meniscus region, the shape of the meniscus is determined by the balance of the hydrostatic and capillary pressures. Whereas the dynamic meniscus region occurs around the stagnation point where the entraining forces and draining forces are in equilibrium. In constant thickness zone, the wet film achieves a given thickness. The dynamic meniscus and the flow of solution in this region determine the wet film thickness. The transition between the dynamic and static meniscus happens within the boundary layer. Beyond the boundary layer, the draining forces are significantly higher as compared to the viscous forces and the balance between the capillary and hydrostatic pressure governs the meniscus shape.
\nDip coating typically has three different stages for drying, drying front during coating, the falling rate period and the constant rate period. The simplest drying stages are the constant and the falling rate periods. The constant rate period occurs within the constant thickness zone which involves the evaporation of solvent at the surfaces of wet film across whole area. The only exception is the edges of the substrate, where the drying front occurs. Over the time after deposition, most of the solvents is evaporated from the wet film and left a gel-like film. This is when the falling rate period starts. In falling rate period, small amount of solvent left trapped within the gel material and the evaporation is determined by the diffusion of solvent towards the surface. The drying front appears at the interface between the wet film and substrate. Due to the large surface to volume ration, the evaporation occurs much faster at the surface which leads to the formation of a wet film with higher concentration. The more complex drying stage occurs at the drying front.
\nSeveral theoretical formulas were established to predict the thickness of deposited films, such as the Landau-Levich theory, via the following equation:
\nwhere \n
In recent years, spray coating has emerged as a viable approach for low-cost deposition of solution-processed thin films. Spray coating is a large-area, high-throughput, inexpensive, and industrially scalable process that can be used to create thin films of material which conform to the shape of the substrate. Spray coating involves ejecting fine liquid particles of smart materials by a jet stream of carrier gas onto the substrate as illustrated in \nFigure 4\n. Spray coating is a contact-free approach suitable for any substrate material and is particularly appropriate for low temperature processing [6]. The dynamics of spray droplet impingement on a substrate surface is a complex fluid mechanics problem subjected to different details, such as spreading, splashing, rebounding, coalescence and interaction with other droplets, drying phenomena, wetting/dewetting, and etc. Substrate properties such as roughness, permeability and surface energy also contribute significantly towards the droplet spreading and surface wetting. Spray coating on a permeable and rough surface hinders droplet spreading and increases the chance of splashing. Solution absorption by the substrate may also slow down droplet spreading. Therefore, droplet impact dynamics, such as droplet size and velocity, requires adjustment to enhance spreading and surface coverage on rough and permeable surface. The functionality of deposited thin film has a direct dependence on structure, morphology, roughness, and integrity of the stacked thin solid films [7].
\nSchematic of spray coating.
The atomization process involves liquid breakup by the application of mechanical energy, which results in the production of a spray consist of micron-size drops. The solution characteristics such as liquid–vapor surface tension, viscosity, and density has direct influence on the atomization process. Atomization takes place when the dynamic pressure of an external force normally applied by a gas exceeds the internal pressure of the liquid droplet. These properties also affect morphological uniformity of deposited thin films. The increase in density hinder the movement of particulates and improve the morphological uniformity of a sprayed film [6].
\nThin film formation by spray coating can be achieved by two different routes: one is by a drop-by-drop film formation approach, where the thin solid film is grown by impingement of a large number of individual droplets on the substrate surface that dry upon impact to cover the entire area. In second approach, the film is formed by immediate merging of impacted droplets and converted to a liquid film and to a solid film upon drying. The desired good quality solid film can be grown through drying of a liquid film if the spray process conditions such as the spray flow rate, and substrate temperature are optimized [7]. The influence of different experimental parameters on the thin film growth via spray coating is discussed as follow.
\n\n
\n
\n
\n
\n
\n
Inkjet printing is a relatively novel process compared to industrial printing and other thin film coating technologies. Inkjet printing involves delivering of a small volume of a fluid material, typically in the picoliter to nanoliter range, onto the substrate surface [9]. Inkjet printers comprises of three basic parts, the motion stage, control systems attached with the print heads, and vision system. The printer heads are connected directly to the cartridge filled with solutions. Inkjet printing systems typically require three mechanical degrees of freedom, one rotational (θ stage) and two translational (X and Y stages) to generate 2D patterns align with previously printed patterns and realize facilely stacked structures. The vision system is employed for substrate alignment and to observe ejected droplets in flight to monitor the droplets. Whereas, the control systems is used to optimize the stage and the printer head temperature, which can directly influence the substrate temperature and the dropping velocity, respectively [10].
\nTwo different approaches are used to translate ink on the substrate surface: thermal and piezoelectric approach as depicted in \nFigure 5\n. The print cartridges consist of a series of tiny chambers and each chamber is attached with a heater over the nozzle. In case of thermal printing approach, a pulse of current is applied to the heater which leads to a rapid vaporization of the ink and build up pressure which push the droplet of ink out of nozzle [11]. The internal temperature creates a bubble in the cartridge and facilitates the single droplet formation of ink out of the nozzle via volume expansion. The negative pressure inside the cartridge after the droplet is ejected, draws new ink inside the reservoir [10]. In piezoelectric approach, the ink is ejected out from a nozzle through a sudden quasi-adiabatic reduction of the chamber volume via piezoelectric action. In the initial state, the ink in the printer head is in equilibrium state. Upon applying a voltage pulse signal to the piezoelectric element, the ink is ejected out due to the volume expansion inside the printer head. As the kinetic energy overcome the threshold, an in-flight droplet is generated and flights towards the target surface. In the sequentially applied opposite voltage, the ink in the printer head refills, and the whole process is repeated.
\nSchematic of inkjet printing (a) thermal printing. (b) Piezoelectric printing.
Several thousand droplets are delivered to substrate in every second and pinter head also moves at the same time. Thermal printing is simple in design and low cost approach, however, it is confined to vaporizable inks to form bubble. The elevated operating temperature makes it inappropriate for polymer-based printing. Therefore, piezoelectric printing is widely used than thermal printing [12].
\nInk viscosity and surface tension are crucial parameters during inkjet printing process. The low value of viscosity is required to allow the ink to fill the chamber as well as nozzle and the high surface tension enough to hold the ink in the nozzle without dripping [13]. To create droplets via thermal nozzles, the inks must be heat-compatible and sensitive to the volume contraction/expansion depending on the temperature. The piezoelectric nozzles contain a piezoelectric film along the wall of a reservoir. The deformation of the film generates the mechanical volume expansion in response to applied voltage pulses and as result the ink is ejected in response to the pressure generated by the piezoelectric element [10]. Typical inks used with a piezoelectric printing require a viscosity of 0.5–40 cp and a surface tension of 20–70 dyne/cm as the piezoelectric transducer only generates limited power, and printing high viscosity ink is difficult [14]. The piezoelectric nozzles have relatively better resolution and require lower temperature, which enables more precise operation to deposit thin film and do not suffer from ink degradation concerns and temperature-sensitive solvent choice. However thermal nozzles are typically less expensive and widely used in commercial printers [10].
\nStable drop formation without satellite droplets after ejection from the nozzles is also important to obtain well-defined printed patterns on a substrate. The droplet velocity and volume are strongly depend on the pulse width and amplitude. The size of the droplets increases linearly with the size of the nozzle. However, the fine droplets generates high resolution and high surface morphology of final printed film. Usually nozzle size, droplets shape and fly direction are determined by the manufacture company and these parameters depend on the composition of the ink, especially on the solvent. Surface tension and viscosity are the primary physical properties that determine the shape and droplet-tail of in-flight droplets, and satellite droplet formation [10].
\nSpin coating, dip coating and spray coating are very common techniques and widely used to deposit thin films in research laboratories as well as industries. The spin coating is one of the important route for lab scale due high reproducibility and its suitability over a wide viscosity range. It is a quick and easy approach to grow uniform film at small scale from a few nanometers to a few micrometers in thickness. However, it is not suitable for scale up in industry due to its high material consumption and the restriction to large area. Dip coating is a commonly used method for mirror coating and dye processing as it can provide easy and fast deposition thin films over a large area. The key advantages is the large area processing and the thickness of film can be controlled by withdraw speed and viscosity of solution. Dip coating requires a high volume of coating solution and large tanks. Despite of long deposition life (several months) only about 20% of the solution can be used. The spray coating technique is able to access a broad spectrum of fluids, and offers the opportunity to tune the system to deposit any kinds of solution and obtain the desired film thickness. It is reproducible, and have great potential for large scale production. Inkjet printing is a powerful and cost-effective technique for deposition of liquid inks with high accuracy. The special characteristics offered by inkjet printing includes additive patterning, reducing materials consumption, non-contact deposition, low cost and capability of large area. Moreover, inkjet printing is capable of deposition a given material on a substrate that has pre-existing patterns, where contamination or damage of patterns would be induced with other deposition processes. However, to deposit materials on substrates, the solution or ink must be compatible with the print head and the viscosity should within a specific range.
\nThe author would like to thank Ibnu Sina Institute for Scientific and Industrial Research (ISI-SIR), Universiti Teknologi Malaysia (UTM) for providing facilities. This research work has supported by Tier 1 Grant.
\nIn our mission to support the dissemination of knowledge, we travel throughout the world to present our publications, support our Authors and Academic Editors at international symposia, conferences, and workshops, as well as to attend business meetings with science, academic and publishing professionals. We are always happy to meet our contributors in our offices to discuss further collaborations. Take a look at where we’ve been, who we’ve met and where we’re going.
",metaTitle:"IntechOpen events",metaDescription:"In our mission to support the dissemination of knowledge, we travel worldwide to present our publications, authors and editors at international symposia, conferences, and workshops, as well as attend business meetings with science, academia and publishing professionals. We are always happy to host our scientists in our office to discuss further collaborations. Take a look at where we’ve been, who we’ve met and where we’re going.",metaKeywords:null,canonicalURL:"/page/events",contentRaw:'[{"type":"htmlEditorComponent","content":"16-18 June 2020 Smart University Forum #SUF20, Ankara, Turkey
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