Primary DTV standards.
\r\n\tFurther development of geophysical methods in the direction of constructing more and more adequate models of media and phenomena necessarily leads to more and more complex problems of mathematical geophysics, for which not only inverse, but also direct problems become significantly incorrect. In this regard, it is necessary to develop a new concept of regularization for simultaneously solving a system of heterogeneous operator equations.
\r\n\r\n\tCurrently, the study of processes associated not only with geophysics and astrophysics but also with biology and medicine requires even more complication of interpretation models from non-linear and heterogeneous to hierarchical. This book will be devoted to the creation of new mathematical theories for solving ill-posed problems for complicated models.
",isbn:"978-1-83962-592-3",printIsbn:"978-1-83962-591-6",pdfIsbn:"978-1-83962-593-0",doi:null,price:0,priceEur:0,priceUsd:0,slug:null,numberOfPages:0,isOpenForSubmission:!0,hash:"d93195bb64405dd9e917801649f991b3",bookSignature:"Prof. Olga Alexandrovna Hachay",publishedDate:null,coverURL:"https://cdn.intechopen.com/books/images_new/8253.jpg",keywords:"Ill-Posed, Inverse Problems, Geophysics, Seismic, Electromagnetic, Thermal, Magnetic, Medicine, \r\nMathematical, Algorithms, Hierarchical, Nonlinear, Historical Description, Regularization",numberOfDownloads:null,numberOfWosCitations:0,numberOfCrossrefCitations:0,numberOfDimensionsCitations:null,numberOfTotalCitations:null,isAvailableForWebshopOrdering:!0,dateEndFirstStepPublish:"October 7th 2019",dateEndSecondStepPublish:"October 28th 2019",dateEndThirdStepPublish:"December 27th 2019",dateEndFourthStepPublish:"March 16th 2020",dateEndFifthStepPublish:"May 15th 2020",remainingDaysToSecondStep:"2 months",secondStepPassed:!0,currentStepOfPublishingProcess:3,editedByType:null,kuFlag:!1,editors:[{id:"150801",title:"Prof.",name:"Olga",middleName:"Alexandrovna",surname:"Hachay",slug:"olga-hachay",fullName:"Olga Hachay",profilePictureURL:"https://mts.intechopen.com/storage/users/150801/images/system/150801.jpg",biography:"EDUCATION BACKGROUND:\r\nAstrophysics (1969) Ural State University \r\nPh. D. «The inverse problem for electromagnetic research of one-dimensional medium » (1979) IZMIRAN, Russian Academy of Sciences, Moscow\r\nProf: «Mathematical modeling and interpretation alternating electromagnetic fields in the heterogeneous crust and earth’s Mantle » (1994) Moscow State University Geological Faculty.\r\nLanguage: Fluent in Russian, English, German. \r\nEMPLOYMENT: HISTORY\r\nFrom 1969 scientific member of the Institute of Geophysics UB RAS\r\n1995-2004 Chief of the group of seismic and electromagnetic research. Elaboration of new common methods for searching the structure and the state of the upper crust Elaboration new theory of interpretation of electromagnetic and seismic fields and realizing it in new programs. From 2002-main scientific researcher of the Institute of geophysics UB RAS From 2008 –leader scientific researcher of the Institute in the frame of the laboratory of borehole geophysics.",institutionString:"Ural Branch of the Russian Academy of Sciences",position:null,outsideEditionCount:0,totalCites:0,totalAuthoredChapters:"4",totalChapterViews:"0",totalEditedBooks:"0",institution:{name:"Ural Branch of the Russian Academy of Sciences",institutionURL:null,country:{name:"Russia"}}}],coeditorOne:null,coeditorTwo:null,coeditorThree:null,coeditorFour:null,coeditorFive:null,topics:[{id:"15",title:"Mathematics",slug:"mathematics"}],chapters:null,productType:{id:"1",title:"Edited Volume",chapterContentType:"chapter",authoredCaption:"Edited by"},personalPublishingAssistant:{id:"305835",firstName:"Ketrin",lastName:"Polesak",middleName:null,title:"Mrs.",imageUrl:"https://mts.intechopen.com/storage/users/305835/images/9351_n.png",email:"ketrin@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. 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Venkateswarlu",coverURL:"https://cdn.intechopen.com/books/images_new/371.jpg",editedByType:"Edited by",editors:[{id:"58592",title:"Dr.",name:"Arun",surname:"Shanker",slug:"arun-shanker",fullName:"Arun Shanker"}],productType:{id:"1",chapterContentType:"chapter",authoredCaption:"Edited by"}}]},chapter:{item:{type:"chapter",id:"52035",title:"Implementation of Video Compression Standards in Digital Television",doi:"10.5772/64833",slug:"implementation-of-video-compression-standards-in-digital-television",body:'\nVideo compression technology is technology which allows you to record video in such a way that they take up less memory space and allows for the video to be a little different from the original, when playing. Reducing data (data compression) is possible because the image contains redundant (same) information [1]. Compression is the process of reducing the number of bits that are used to encode individual picture elements.
\nIn digital television, parameters for digital video signal with compression and without compression are given by recommendation ITU-R BT.601 [2]. In broadcasting, transmission with lower speed requires less bandwidth and transmitter with lower power. Recording signals, using compression, reduces the required capacity of storage media, and it is directly proportional to the size of compression. For archival purposes that significantly reduces the required space and cost of the archive.
\nTechniques for accomplishing a reduction in the video size are mostly confined to compress individual frames of content and techniques of writing changes and differences between frames. Videos are usually composed of three types of frames: I-frames (intra-frames), P-frames (predicted-frames) and B-frames (bidirectional-frames). The difference between different types of frames is only in the write mode and read mode (the interpretation). During the playing (displaying), each frame is shown as a normal image regardless of recording technique of the video format. Intraframe or spatial compression is technique in the video compression for reducing the size of individual frames. Interframe or temporal compression is a video compression technique that achieves a reduction in size of similar series of frames [3].
\nThe development of digital telecommunications allows the use of high-definition television (HDTV) besides standard-definition television (SDTV). HDTV is a technology that offers significantly higher quality of picture and sound than the traditional display technology did (analog PAL, NTSC, SECAM, SDTV and digital). Since the resolution is higher, the image is sharper, less blurry and the content is closer to reality. HDTV offers smoother movement, detailed and more vibrant colors, and there is a very high-quality multichannel sound that makes viewing experience even better. Table 1 shows the basic characteristics of the primary digital TV standards.
\nDTV | \nResolution | \nAspect ratio | \nNumber of frames per second | \n
---|---|---|---|
HDTV | \n1920 × 1080 | \n16:9 | \n25p, 30p, 50i | \n
1280 × 720 | \n16:9 | \n25p, 30p, 50i | \n|
SDTV | \n720 × 576 | \n16:9 | \n25p, 30p, 50i | \n
720 × 576 | \n4:3 | \n25p, 30p, 50i | \n
Primary DTV standards.
HDTV offers two quality signals: 720 and 1080 are the basic tags, which can be added to either the letter “i” or the letter “p”, which means the ways of drawing the image (i = interlaced—draws every other line; p = progressive—draws the line-by-line). The “heights” of image are 720 and 1080, and the width is 1280 or 1920 pixels. The number of images per second is specified next to the tag, for example. 720p50 indicates a resolution of 1280 × 720, progressively rendering images and 50 frames per second [4].
\nWithout compression, digital video signal would contain an enormous amount of data. For example, the standard digital video signal according to CCIR standards has 25 frames per second, resolution of 720 × 576 pixels, and each pixel is represented by 24 bits (3 bits for each color component). Transmission of uncompressed video signals requires channel capacity of 216 Mb/s. Video-definition HDTV signal requires six times bigger channel capacity of about 1.5 Gb/s. In multimedia systems, problems occur during the storage of digital video signal. That is why different algorithms are used to compress video signals. Compression ratio depending on the algorithm used for compression (MPEG-2, MPEG-4, etc.) can be different. The required bit rate for MPEG-2 standard used to transfer HD signal is about 20 Mb/s, and for SDTV, resolution 720 × 576, line is about 4 Mb/s. If we are using the MPEG-4 standard then for the same quality, twice lower is required signal strength. European broadcasters mainly use MPEG-2 standard, although lately MPEG-4 standard is increasingly used [5, 6].
\nTable 2 shows the flows of compressed television signals that are used in practice in a broadcast, obtained from the MPEG-2 and MPEG-4 standards.
\nStandards for video compression | \nTV video resolution | \nBit rates compressed video signals (Mb/s) | \n
---|---|---|
MPEG-2 | \nSDTV | \n2–4 | \n
HDTV | \n15–20 | \n|
MPEG-4 | \nSDTV | \n1.5–2 | \n
HDTV | \n6–8 | \n
The flows of compressed video/audio signals for certain standards.
Ultra-high-definition television (UHDTV) includes 4K UHDTV (2160p) and 8K UHDTV (4320p), which represents the two digital video formats proposed by NHK Science & Technology Research Laboratories and approved by the International Telecommunications Union (ITU). Minimum resolution of this format is 3840 × 2160 pixels [7]. Digital TV program consists of three components: video, audio and service data as shown in Figure 1.
\nComponents of digital television.
Service information, which contains additional information such as teletext and specific information of network, including an electronic program guide (EPG), is generated in digital form and does not require coding. Encoders compress the data by removing irrelevant or redundant parts of the image and sound signals and perform a reduction operation to produce separate video and audio packets of elementary streams.
\nIn 1990, due to the need for storage and playback of moving images and sound in digital format for multimedia applications on various platforms, ISO has formed an expert group called Motion Picture Experts Group (MPEG).
\nIn order to enable the interconnection of equipment from different manufacturers, standards for compression and transmission of video signals are defined. Among them, the best known are H.261, H.263 and H.264 for videoconferencing transmission, videophone and distribution of digital material via the web, as well as the MPEG standards (MPEG-1, MPEG-2, MPEG-4, MPEG-7, MPEG-21), which are intended for standardization of multimedia systems and digital television.
\nOther standard developed by the MPEG group is ISO/IEC IS 13818: Generic Coding of Moving Pictures and Associated Audio, so-called MPEG-2 standard. It is aimed to professional digital television [8, 9], adopted in 1999, produced on the disadvantages of the standard MPEG-1. It is compatible with MPEG-1 standard, using the same tools and adding some new.
\nBasic innovations with the MPEG-2 standard are as follows: an increased bit rate, picture formats with and without thinning, scalability of quality and time, improved methods of quantization and coding, etc. Since it is primarily designed for TV signal compression, MPEG-2 standard allows the use of both types of image scanning: progressive scanning and scan by line spacing. In the compression process, all three types of pictures can be coded as I, P and B pictures. Standard encoder structure comprises a mixture of I, P and B frames in a way that I frame appears after every 10–15 frames, and two B frames between two adjacent I frame. Usually, a group of pictures (GOP) has one I frame or more P and B frames.
\nSince the complete syntax of the MPEG-2 standard is complex and difficult for practical implementation on a single silicon chip, the MPEG-2 standard defines five subsets of the full syntax, called profiles, which are designed for a variety of applications. These are simple (simple) profile, main (main) profile, signal-to-noise ratio (SNR) scalable profile, spatial scalability (spatial scalable) and high profile (high) profile. Later, another is created, 4:2:2 profile, and definition of another (multiview) profile is in progress.
\nThe profile is defined by four levels, which regulate the choice of available parameters during the hardware implementation. The levels determine the maximum bit rate, and according to the bit rate the speed of transmission of TV programs and resolutions of the system are chosen, and they are, on the other hand, determined by the number of samples per line, number of lines per image and the number of frames per second. There are four levels: high level (HL) H14L (H 1440 level), main level (ML) and low level (LL) [2]. Parameter limitations by levels are shown in Table 3 [3].
\nLevel | \nMaximum pixel number | \nMaximum line number | \nMaximum frames/s | \n
---|---|---|---|
Low level | \n352 | \n288 | \n30 | \n
Main level | \n720 | \n576 | \n30 | \n
H 1440 level | \n1440 | \n1152 | \n60 | \n
H 1920 level | \n1920 | \n1080 | \n60 | \n
Limits of parameters in the levels of MPEG-2 standard.
Simple profile is designed to simplify the transmitter encoder and receiver decoder, with reductions in binary rate (transfer speed), and the inability bidirectional prediction (B pictures do not exist) supports only I and P prediction. As such it is suitable only for low-resolution terrestrial television. The maximum bit rate is 15 Mb/s.
\nThe main profile is the optimal compromise between compression ratio and price. It supports all three types of prediction I, P, B, which automatically leads to the complexity of the encoder and decoder. Main profile supports all four levels, with a maximum bit rates of 4, 15, 60 and 80 Mb/s, for low, main, high-1440 and 1920 high level, respectively. The majority of broadcast applications are scheduled for operation in the main profile. Terrestrial digital TV uses the main profile and main level (MP and ML). SNR scalable supports profile only for low and main levels with a maximum bit rate of 4 and 15 Mb/s, respectively.
\nSpatially scalable profile supports only high-1440 level with a maximum flow rate of 60 Mb/s, of which 15 Mb/s is part of the base layer. It allows the transfer of basic image quality depending on the spatial resolution (spatial) or quantization accuracy, with addition of supporting information (enhanced layer). This allows simultaneous broadcasting of a program in elementary and higher resolution, so that in case of difficult reception conditions the signal of lower quality can be received (lower resolution) instead of higher. They are intended for extended-definition TV (EDTV).
\nHigh profile (also known as professional) is designed for later use with hierarchical coding for applications with extremely high definition (HDTV—high-definition TV) in format sampling 4:2:2 or 4:2:0. High profile supports the main, high-1440 and 1920 high level, with a maximum flow rates of 20, 80 and 100 Mb/s, respectively. The flow of the base layer is 4, 20 and 25 Mb/s, respectively.
\n4:2:2 profile has been introduced to allow working with color images in 4:2:2 format, which is necessary for studio equipment. Although, during the development of MPEG-2 standard, studio uses have not been taken into account, it showed that the MPEG-2 standard is suitable for this purpose. 4:2:2 profile has allowed the use of existing tools for coding and in studio applications, which requires a higher bit rate.
\nMultiview profile (MVP) is introduced in order to enable efficient coding of two video sequences derived from two cameras which are recording the same scene, and which are set at a slight angle (stereovision). This profile also uses existing tools for encoding, but with a new purpose. There is also reverse compatible decoder which means a higher level still can play lower level profile, while compatibility in the opposite direction is not possible. Present stage of development uses a combination profile and level of main profile at main level. Maximum number of pixels that can theoretically be transmitted by MPEG-2 encoder is 16,383 × 16,383 = 22,657,689.
\nVideo and audio encoders transmit signal in the main stream. Raw uncompressed audio and video parts of the signal, known as presentation units, are located in the encoder for receiving video and audio access units. Video access unit can be I, P and B coded picture. Audio access units are containing encoded information for a few milliseconds of sound window: 24 ms (layer II), and 24 or 8 ms in the case of the layer III. The video and audio access units form the elementary streams in a respective manner. Each elementary stream (ES) is then divided into packets to form a video or audio packetized elementary stream (PES). Service and other data are similarly grouped into their PES. PES packets are then divided into smaller 188-bit transport packages [2, 10].
\nTo gain access to the transfer of MPEG-2 signal, data streams must be multiplexed. With multiplexing, the following is obtained:\n
portable data stream (TS = transport stream)—designed to transmit signals to terrestrial, cable and satellite connections,
programming data stream (PS = program stream)—designed for storing digital data on DVD or other storage space.
Multiplexing of audio and video signals is necessary in order to enable their joint transmission, and properly decode and display. The multiplexing hierarchy determined by MPEG-2 standard can be divided into:\n
basic data stream (ES = elementary stream),
packetized basic data stream (PES = packetized elementary stream),
portable (TS) or program data stream—PS (Figure 2).
Programming flow obtained by multiplexing includes packages resulting from one or more elementary streams belonging to one program. It can contain one stream of the video signal, and more data streams of an audio signal.
\nAll packages have certain common components that are grouped into three parts: header, data and control data [10, 11].
\nPackets of the program stream have a variable length, which causes difficulties when the decoder needs to recognize the exact beginning and the end of the package. To make this possible, the packet header contains information of the length of the package. PES packet can vary in length up to a maximum of 64 KB, while the typical length is about 2 KB. The part that follows the header contains the access unit as parts of the original elementary stream. At the same time, there is no obligation to equalize the start of access units with a start of information part (payload). According to that a new access unit can start at any point in the information part of PES packets, there is also the possibility that a few small access units can be contained in one PES packet.
\nObtaining transmission and programming data flow.
The most important components of the header are as follows:\n
starting prefix code (3 bits),
starting code of a flow (1 bit),
start time stamp,
PTS (33 bits),
decoding time stamp (DTS; 33 bit).
PTS and DTS cannot be included in each PES packet, as long as they are being involved in at least 100 ms in the transport data stream (DTV), or every 700 ms in the programming data stream (DVD). DTS indicates the time required for deleting or decoding access unit. Within the headers, some other fields that contain important parameters are included, such as the length of the PES packet, the length of the header and whether the PTS and DTS fields are present in the package. Among this, there are several other optional fields, a total of 25, which can be used to transfer additional information about packetized elementary stream, such as the relative priority and copyright information.
\nMPEG-4 is a generic standard for coding audiovisual information, and it was presented in 1998 under the label ISO/IEC 14496 [12]. In this standard, video and audio signals are characterized by interactivity, high degree of compression, and universal access, and this standard has a high level of flexibility and expandability.
\nThe algorithms that are implemented in MPEG-4 standard represent scene as a set of audiovisual objects, among which there are some hierarchical relations in space and time. In all previous standards for compression of video, image has been seen as a unified whole. In this standard, we are meeting with the concept of video object, thereby to distinguish two types of visual objects—natural and synthetic visual objects.
\nAt the lowest hierarchical level are primitive media objects, such as, for example, static images (fixed background scenes), visual objects (a person who speaks no background), and audio facilities (voice of the speaker). This approach brings an increase in compression ratio, increased interactivity and enables the integration of objects’ different nature such as natural image or video, graphics, text and sound.
\nMPEG-4 standard has inherited the MPEG-2 standard. Each MPEG standard consists of several parts (Parts). Each part covers a certain aspect and area of use. Thus, for example, MPEG-4 Part 2 is used for video coding (such as DivX and Quicktime 6), MPEG-4 Part 10/H.264 represents an Advanced Video Coding (AVC), and it is used in areas with high-definition content such as HD broadcasting and storage, HD formats such as HD-DVD and Blue-ray discs [13]. MPEG-4 Part 3 Advanced Audio Coding (AAC) is a part for high-quality audio coding.
\nThe first inheritor to MPEG-2 format was MPEG-4 Part 2, which is published by ISO in 1999. As in the case of the MPEG-2, coding efficiency is strictly related to the complexity of the source material and the encoder implementation. MPEG-4 Part 2 is defined for applications in the field of multimedia in small bit rates, but it is in further expanded for applications in the field of broadcasting. Formal subjective evaluation has shown that the gain of the efficient coding with MPEG-4 Part 2, compared to the MPEG-2, is between 15 and 20%. For Digital Video Broadcasting (DVB) applications, this gain is not enough to justify the destabilization and destruction of MPEG-2 codec (which are used by DVB systems)—considering that the MPEG-4 Part 2 is not compatible with MPEG-2.
\nFollowing the example of MPEG-2 standard, MPEG-4 standard supports both ways scanning images, progressive and interlaced scanning. Spatial resolution of luminance component can be expressed in blocks ranging from 8 × 8 pixels, up to 2048 × 2048 pixels. For presentation of video signal in color, this standard is using a conventional Y Cb Cr color coordinate system with weighing 4:4:4, 4:2:2, 4:1:1 and 4:2:0. Each component is represented with 4–12 bits per image pixel. Different temporal resolution is supported, as well as an infinitely variable number of frames per second [2].
\nAs it was the case in the previous MPEG standards, the macroblock presents basic unit in which data of video signal are transmitted. Macroblock contains coded information about the shape, motion and texture (color) of the pixels. There is a wide range of bit rate from 5 Kb/s to 38.4 Mb/s, but it is optimized for use in three ranges of bit rate: <64 Kb/s, 64 Kb/s to 384 Mb/s and 384 Kb/s to 4 Mb/s. Also are supported constant bit rate and variable bit rate.
\nEach video object can be coded in one or more layers, which allows it variable resolution (scalability) encoding. Also, each video object is discretized in time so that each time samples representing a video object plane (VOP) [2, 13, 14]. Time samples of video object are grouped into group of video object planes (GOV).
\nPrevious video coding standards such as MPEG-2 and MPEG-4 Part 2 have been established and are used in the areas of videoconferencing over mobile TV and broadcasting TV content in standard/high definition, up to the application of very high quality, such as applications for professional digital video recorders and digital cinema—digital images on the big screen. But with the spread of digital video applications and its use in new applications such as advanced mobile TV or broadcast HDTV signal, requirements for effective representation of the video image are increased to the point where the previous standards for video coding cannot keep pace.
\nThe new MPEG-4 Part 10 (MPEG-4.10) standard of video compression is the result of efforts of the Joint Video Team (JVT), which includes members of the Video Coding Expert Group (VCEG) and the Motion Picture Experts Group (MPEG), which is the reason for naming it twice (H.264 and MPEG-4.10). Standard is also commonly referred to as H.264/Advanced Video Coding (AVC).
\nThis standard, registered under the number ISO-IEC14496-10, provides a significant increase in compression efficiency in regard to MPEG-2 (gain of at least 50%) [12]. This efficiency is of particular importance for high-definition television (HDTV), which in the MPEG-2 requires a bit rate of at least 15–18 Mb/s.
\nH.264 showed significant improvement in coding efficiency, a significant improvement when it comes to resistance to errors, as well as increased flexibility and area of use compared to their predecessors. A change was added in the MPEG-4.10 (H.264/AVC), the so-called FRExt (FREkt) amendment, which further extended the area of use to areas such as mobile TV, internet broadcasting, distribution and professional studio and postproduction [15]. Table 4 [16] shows the usage scenarios and compression in bits supplied with the H.264 codec, and Table 5 [16] shows the characteristics of the H.264 standard level.
\nUsing | \nResolution and frame rates | \nBit rate | \n
---|---|---|
Mobile content (3G) | \n176 × 144, 10–24 fps | \n70–180 Kb/s | \n
Internet/standard definition | \n640 × 480, 24 fps | \n2–3 Mb/s | \n
High definition (HDTV) | \n1280 × 720, 25p, 30p | \n7–8 Mb/s | \n
Full high definition (full HDTV) | \n1920 × 1080, 25p, 30p | \n10–12 Mb/s | \n
Different scenarios of use H.264 standard.
H.264 consists of two layers: layer for video encoding, designed for effective representation of video coding layer (VCL) and network-flexible layer network abstraction layer (NAL), which converts VCL video content in formats suitable for transmission over a variety of transport layers or storage media.
\nH.264 level | \nResolution | \nFrame rate | \nMax. compressed bit rate (non-FRExt profile) maximum | \nMaximum number of reference frames | \n
---|---|---|---|---|
1 | \nQCIF | \n15 | \n64 Kb/s | \n4 | \n
1b | \nQCIF | \n15 | \n128 Kb/s | \n4 | \n
1.1 | \nCIF or QCIF | \n7.5 (CIF)/30 (QCIF) | \n192 Kb/s | \n2 (CIF)/9 (QCIF) | \n
1.2 | \nCIF | \n15 | \n384 Kb/s | \n6 | \n
1.3 | \nCIF | \n30 | \n768 Kb/s | \n6 | \n
2 | \nCIF | \n30 | \n2 Mb/s | \n6 | \n
2.1 | \nHHR (480i or 576i) | \n30/25 | \n4 Mb/s | \n6 | \n
2.2 | \nSD | \n15 | \n4 Mb/s | \n5 | \n
3 | \nSD | \n30/25 | \n10 Mb/s | \n5 | \n
3.1 | \n1280 × 720p | \n30 | \n14 Mb/s | \n5 | \n
3.2 | \n1280 × 720p | \n60 | \n20 Mb/s | \n4 | \n
4 | \nHD formats (720p or 1080i) | \n60p/30i | \n20 Mb/s | \n4 | \n
4.1 | \nHD formats (720p or 1080i) | \n60p/30i | \n50 Mb/s | \n4 | \n
4.2 | \n1920 × 1080p | \n60p | \n50 Mb/s | \n4 | \n
5 | \n2k × 1k | \n72 | \n135 Mb/s | \n5 | \n
5.1 | \n2k × 1k or 4k × 2k | \n120/30 | \n240 Mb/s | \n5 | \n
Levels of H.264 standard.
Video coding layer (VCL) for MPEG-4.10 (H.264/AVC) codec is in a some way similar to the previous video codecs such as MPEG-2 [15]. Figure 3 shows a block diagram of coder.
\nStructure of H.264 video coder.
The coded video sequence in the H.264 consists of a series of encoded pictures. The coded image may represent either the entire frame or one field, as was the case with the MPEG-2 codec. Overall, it can be considered that the video frame comprises two fields: the field at the top and the field at the bottom. If the both fields of a given frame were taken at various time points, the frame is called interlaced scan frame; otherwise, it is called a progressive scan frame.
\nThanks to the evolution of technology, which has enabled us to have a resolution of video material from 4K and higher reality, the evolution of video coding is inevitable, so it can keep up the step. HEVC/H.265 video coding (High Efficiency Video Coding) [17], is the fruit of cooperation between ISO/IEC Moving Picture Experts Group (MPEG) and ITU-T Video Coding Experts Group (VCEG) standardization organization, which brings better performance than the previous coding standards, as well as H.264/AVC, and the biggest advantage of the new standard is up to 50% more efficient compression compared to H.264 and support for 8K UHD resolution. This means that the video material of the same quality will occupy at half encoding less space with HEVC than the H.264/AVC coding, thanks to better algorithms and analysis of the video material which eventually brings better coding efficiency.
\nDirect predecessor of this standard is H.264/MPEG-4 Advanced Video Coding (AVC). HEVC seeks to replace its predecessor by using a generic syntax that could be customized to newer emerging applications. He wants to achieve several goals, such as code efficiency, adaptability to different systems of transport, resilience on errors and implementation with parallel processing in a multiprocessor’s architecture.
\nH.265/HEVC is a hybrid video coding algorithm based on blocks. The basic coding algorithm is a hybrid of intraprediction, interprediction and transformational coding. For representation of a color video signal, H.265/HEVC standard uses YCbCr color space in format 4:2:0. Each sample of the individual components of the color space is represented with a resolution of 8–10 bits per sample, in coding and decoding. Video image is progressively scanned in a rectangular format dimensions W × H, where W represents a width, H height of the image for the luma component. Chrominance components for color format 4:2:0 are scanned in a rectangular format dimensions W/2 × H/2 [17, 18].
\nH.265/HEVC standard has kept hybrid architecture of previous coding MPEG standard for video encoding. A significant difference in approach lies in the fact that the previous H standards—video coding are based on macroblocks, H.265/HEVC standard for encoding uses the adaptive quadtree structure based on Coding Tree Unit (CTU). Basically, the quadtree structure is composed of various blocks and units.
\nA block is defined as a matrix of samples of different sizes, while the unit includes a luma block and corresponding chrominance blocks together with the syntax necessary for their coding. With the further division of structure coding units are obtained and also the coding blocks.
\nDecoding of quadtree structure does not represent a significant additional burden because it can easily be switch into a hierarchical structure by using z-scan. Predictable modes for interframe encoded CU are using non-square PU, which requires the necessary support for decoding in the form of additional logic in the decoder which performs multiple conversions between the raster scan, and the scan-scan. In terms of preserving the speed of bit rates, with the encoder side, there is a simple algorithm to analyze the structure of the tree to determine the optimal share of the blocks [7]. CTU sizes are 16 × 16, 32 × 32 and 64 × 64 pixels.
\nProfile is defined by a set of coding tools or algorithms which, if used, ensure compatibility of the output coded bit stream with standard applications that belong to this profile, or have similar functional requirements. Level refers to the limitations of the current stream bits that define memory and resource requirements of the decoder. These restrictions are maximum number of samples and the maximum number of samples per second that can be decoded (sample rate), the maximum image size, maximum bit rate (how many bits can decoder spend per second of video record), minimum compression ratio, size of the buffer memory and so on. In HEVC standard, only for the purposes of diversity from some applications, in bit rate and buffer memory, which are used to store the encoded image (control flow information), were defined two layers: a basic “Main” and demanding “High”. Currently, the draft of HEVC has defined a single profile “Main” and expectation is more defined profiles. Goal is to reduce the number of profiles, so that there will be maximum compatibility between devices, and also, due to the fact that sometimes services are separated, for example, for broadcasting TV signals, mobile services and video on demand, the goal is convergence to devices that will support all of them together.
\nUltra-high-definition television (UHDTV or UHD) includes 4K UHDTV (2160p) and 8K UHDTV or Super Hi-Vision (4320p), which are two digital video formats proposed by NHK Science & Technology Research Laboratories and defined and approved by the International Telecommunication Union (ITU) [17].
\nFull high definition (FHD) indicates that the image with 1920 pixels set in the 1080 lines. UHD includes twice the number of pixels and lines in its basic version, which can be called a Quad Full HD because it has four times more pixels than Full HD. Basically, there are two UHD resolution, 3840 × 2160 and 7680 × 4320 for easier identification is often called the first UHD-1 (4K) and the other UHD-2 (8K).
\nNumber of 3840 pixels in one row consist UHD 4K, while Full HD consist from 1920 lines. The point is that the nomenclature “No. K” it was taken from formats works with theatrical distribution; on the other side, UHD starts to use as commercial term. When you see a movie that has a 2K resolution film, it will be 2048 pixels’ resolution in 1080 lines, and in the case of 4K projection, it will be a resolution of 4096 × 2160 pixels [19].
\nUHD brings many benefits, but also the kind of disadvantages like any new technology, especially in its beginning. The benefits of higher resolution logically have a greater amount of information on the screen and therefore a more realistic view, especially on the diagonals that are larger than 140 cm (55″) and where full HD resolution loses the impression of high sharpness. This is why manufacturers have presented the first UHDTVs in diagonals of 84″ (213 cm) that they would now be available in smaller dimensions—140 cm (55″) and 165 cm (65″). UHD on smaller diagonals does not have much sense because the density of information is too large and the average viewing distances further details cannot be seen in relation to the full HD content.
\nMany parameters have effect on the realism of images, and among them resolution is not most important element. Number of pixels has a smaller impact on how we experience the image of other parameters, such as increased dynamic range, the range and depth of color, as well as the number of frames per second. UHD used Rec. 2020 standard color range in contrast to the definition used by Rec. 709 standard. Rec. 2020 defines a bit depth of either 10 bits per sample or 12 bits per sample. Rec. 2020 specifies the following frame rates: 120p, 119.88p, 100p, 60p, 59.94p, 50p, 30p, 29.97p, 25p, 24p, 23.976p [18]. Table 6 [18] provides an overview of the main characteristics of images in HDTV, 4K and 8K UHDTV.
\n\n | HDTV | \n4K UHDTV | \n8K UHDTV | \n
---|---|---|---|
Pixels × number of lines | \n1280 × 720 p 1440 × 1080 i 1920 × 1080 p(i) | \n3840 × 2160 | \n7680 × 4320 | \n
Mpixels/frame | \n0.922 1.6 2.1 | \n8.3 Progressive | \n33.2 Progressive | \n
Aspect ratio | \n16:9 | \n16:9 | \n16:9 | \n
Frame rate | \n25, 50, … fps 30 fps +24 fps | \n25, 50, … fps 30, 60, 120 fps +24 fps | \n25, 50, … fps 30, 60, 120 fps +24 fps | \n
Bit depth | \n8 or 10 bits | \n10 or 12 bits | \n10 or 12 bits | \n
Viewing distance | \n3 × H (30°) | \n1.5 × H (60°) | \n0.75 × H (100°) | \n
The characteristics of different digital TV formats.
In the period from May 15 to June 16, 2006, in Geneva, held a Regional Conference on Radio Communications (RRC-06), organized by the International Telecommunication Union (ITU), with the aim of establishing a new international agreement and the associated frequency plan for the digital broadcasting of radio and television programs. The conference RRC-06 Final Act were adopted (Final Acts) which contain a new agreement Geneva 2006 (GE06), which enables the introduction of complete digital terrestrial television broadcasting in the planning zone. All European countries have pledged that no later than June 17, 2015, the switch to digital broadcasting of radio and television signals, and perform analog switch off (ASO). In many countries, it is already implemented as ASO [20].
\nEuropean countries have adopted the standard Digital Video Broadcasting-Terrestrial (DVB-T) and DVB-T2. The first concepts DVB-T were adopted in 1993, and the first final version in 1997. It involves the transmission of digitized audio and video content via terrestrial broadcasting technology in the VHF and UHF band using conventional system transmitter and corresponding receiver [21].
\nDVB-T2 is an enhanced version of the DVB standard for terrestrial broadcasting. Compared with DVB-T, DVB-T2 offers a significantly lower sensitivity to noise and interference and provides 30–50% greater flow of data which is particularly suitable for HDTV [22].
\nVideo compression standards of DVB-T standards used in different countries are shown in Table 7 [23]. The number of national multiplex (MUX) is given, local and regional non-represented. When digital terrestrial TV transmission started and year ASO was executed are presented. Data were collected from the official websites of national regulatory agencies and providers of digital terrestrial transmission.
\nCountry | \nMUX | \nDVB standard | \nStart | \nASO | \n|
---|---|---|---|---|---|
\n | \n | DVB-T | \nDVB-T2 | \n\n | \n |
Andora | \n6 | \nMPEG-2 | \n– | \n2005 | \n2007 | \n
Austria | \n6 | \nMPEG-2 | \nMPEG4 for pay TV and HD | \n2004 | \n2010 | \n
Belgium | \n2 | \nMPEG-2 | \n– | \n2002 | \n2011 | \n
Bulgaria | \n3 | \nMPEG-4 | \n– | \n2004 | \n2013 | \n
Croatia | \n5 | \nMPEG-2 | \nMPEG-4 for pay TV | \n2002 | \n2010 | \n
Cyprus | \n4 | \nMPEG-4 | \n– | \n2010 | \n2011 | \n
Czech Republic | \n3 | \nMPEG-2 | \nMPEG-4 for experimental HD | \n2000 | \n2012 | \n
Denmark | \n6 | \nMPEG-4 | \nMPEG-4 for pay TV | \n2003 | \n2009 | \n
Estonia | \n4 | \nMPEG-4 | \nMPEG-4 for HD | \n2004 | \n2010 | \n
Finland | \n9 | \nMPEG-2 | \nMPEG-4 | \n1999 | \n2007 | \n
France | \n8 | \nMPEG-2 | \nMPEG-4 for pay TV and HD | \n2005 | \n2011 | \n
Germany | \n5 | \nMPEG-2 | \n– | \n2002 | \n2008 | \n
Hungary | \n3 | \nMPEG-4 | \n– | \n2004 | \n2013 | \n
Ireland | \n2 | \nMPEG-4 | \n– | \n2006 | \n2012 | \n
Italy | \n22 | \nMPEG-2, MPEG-4 | \nMPEG-4 tests | \n1998 | \n2012 | \n
Latvia | \n7 | \nMPEG-4 | \n– | \n2002 | \n2010 | \n
Lithuania | \n5 | \nMPEG-4 | \n– | \n2003 | \n2012 | \n
Luxemburg | \n4 | \nMPEG-2 | \n– | \n2002 | \n2010 | \n
Macedonia | \n7 | \nMPEG-4 | \n– | \n2004 | \n2013 | \n
Netherlands | \n5 | \nMPEG-2 | \n– | \n1998 | \n2006 | \n
Norway | \n5 | \nMPEG-4 | \n– | \n1999 | \n2009 | \n
Poland | \n3 | \nMPEG-4 | \n– | \n2001 | \n2013 | \n
Portugal | \n1 | \nMPEG-4 | \n– | \n2009 | \n2012 | \n
Slovakia | \n4 | \nMPEG-2, MPEG-4 | \nMPEG-4 tests | \n2009 | \n2012 | \n
Slovenia | \n2 | \nMPEG-4 | \n– | \n2001 | \n2010 | \n
Spain | \n8 | \nMPEG-2, MPEG-4 | \n– | \n1999 | \n2010 | \n
Sweden | \n7 | \nMPEG-2, MPEG-4 | \nMPEG-4 | \n1999 | \n2007 | \n
Switzerland | \n4 | \nMPEG-2 | \n– | \n2000. | \n2008 | \n
United Kingdom | \n6 | \nMPEG-2 | \nMPEG-4 for HD | \n1998. | \n2012 | \n
Albania | \n10 | \nMPEG-2, MPEG-4 | \nMPEG-4 for HD | \n2004 | \n– | \n
Belarus | \n3 | \nMPEG-4 | \nMPEG-4 for pay TV | \n2004 | \n– | \n
Greece | \n8 | \nMPEG-2, MPEG-4 | \n– | \n2006 | \n2015 | \n
Iceland | \n3 | \nMPEG-2 | \nMPEG-4 | \n2005 | \n2015 | \n
Moldova | \n2 | \nMPEG-4 | \nMPEG-4 | \n2003 | \n2015 | \n
Montenegro | \n1 | \nMPEG-4 | \nMPEG-4 | \n2014 | \n2015 | \n
Romania | \n3 | \nMPEG-4 | \nMPEG-4 adopted | \n2005 | \n2015 | \n
Russia | \n2 | \n– | \nMPEG-4 | \n2005 | \n– | \n
Serbia | \n1 | \n– | \nMPEG-4 | \n2005 | \n2015 | \n
Turkey | \n1 | \n– | \nMPEG-4 | \n2006 | \n– | \n
Ukraine | \n4 | \n– | \nMPEG-4 | \n2007 | \n– | \n
Video compression standards of digital terrestrial TV transmission in Europe.
From Table 2, it can be seen that countries that have moved completely to digital broadcasting mainly used DVB-T standard, or used in parallel and DVB-T2, while countries that are transitioning to digital transmission opted for the DVB-T2 standard. A small number of countries using DVB-T standard include MPEG-2 compression, mainly for free-to-air (FTA). Compression standard MPEG-4, due to savings in capacity, mainly used for encrypted channels, i.e., pay TV and HDTV. An increasing number of countries that use the DVB-T standard are planning to in the near future switch to an enhanced DVB-T2 standard.
\nNumber of UHD content is not large, but their number is growing rapidly. Many cameras are now able to record materials and above 4K resolution, such as RED Epic camera which can record approximately 5K resolution or 5120 × 2700 pixels, as well as the Sony F65 8K camera recording at a resolution of 8192 × 4320 pixels. The first 4K UHD facilities were available over broadband services (Netflix and YouTube) to 2013 and in 2014 started the first experimental TV channels that broadcast 4K UHD controversial content. Sporting events in 2012, 2013 and 2014 were the first UHD content broadcast via satellite. Pioneers in the distribution of 4K UHDTV are the Japanese public broadcaster NHK and KBS Korean TV [24]. The leading satellite companies took part in the distribution of UHD Eutelsat, SES Astra, Measat, Eutelsat, and Hispasat. Although in tests carried out with video H.264/AVC, HEVC is mainly used today. Table 8 [25] provides an overview of the number of SDTV, HDTV and 4K UHDTV that may be received from the satellite to the various transmission parameters.
\nSatellite transmission | \nCarrier data rate (Mbps) | \nNumber of channels | \n||
---|---|---|---|---|
SDTV (p25/p30) | \nHDTV (p25/p30) | \n4K UHDTV (p50/p60) | \n||
DVB-S, QPSK, FEC 3/4 | \n38 | \n4–5 in MPEG-2 | \n4–5 in MPEG-4 | \n– | \n
DVB-S2, 8PSK, FEC 5/6 | \n72 | \n24 + in MPEG-4 | \n7–9 in MPEG-4 14–18 in HEVC | \n2–5 in HEVC | \n
DVB-S2, 16APSK, FEC 2/3 | \n79 | \n– | \n7–9 in MPEG-4 15–19 in HEVC | \n1 in MPEG-4 3–5 in HEVC | \n
DVB-S2X, 16APSK, FEC 3/4 | \n83 | \n– | \n8–10 in MPEG-4 16–20 in HEVC | \n1 in MPEG-4 3–5 in HEVC | \n
DVB-S2X, 16APSK, FEC 135/180 | \n99 | \n– | \n9–12 in MPEG-4 19–24 in HEVC | \n1 in MPEG-4 3–6 in HEVC | \n
Number of satellite SSTV, HDTV and 4K UHDTV channels for the various transmission parameters.
Initial tests 4K UHDTV in digital terrestrial television systems were carried out in 2012 in Japan and South Korea by KBS and NHK still using unstandardized HEVC video compression. Later tests were done in other countries. Table 9 [26] gives the basic test characteristics of 4K UHDTV in digital terrestrial television (DTT) networks in the world.
\n\nTechnicolor has successfully conducted tests to broadcasting terrestrial 4K UHDTV content. Broadcasting used American ATSC 3.0 standard, trough Sinclair Broadcast transmitter [27]. Technically speaking, this is the world premiere of the use of scalable HEVC (SHVC) video coding, MPEG-H compression standards, as well as MMT MPEG A/V standards. The test was performed in the Sinclair Broadcast experimental facility. The new technology allows you to receive signals via conventional antenna, as well as through mobile and tablet devices.
\nBased on technological profiles and the typology of the various countries, the report predicts that the demand of end users and the transition to the new standards take between 3 and 12 years old. Markets that were among the first to adopt new technologies will likely take between three and six years to the current broadcasting possibilities of yarn on a combination of DVB-T2 MPEG4/HEVC, SDTV/HDTV/UHDTV (4K). The third profile that refers to the HDTV/UHDTV (4K/8K) is supposed to happen between 2023 and 2030. The report says that the DTT platform is currently threatened due to scarcity of radio spectrum, as well as plans for the redistribution of 700 MHz range, which will reduce the available capacity by an average of 30%.
\nCountry | \nMultiplex capacity (Mbit/s) | \nSignal bit rate (Mbit/s) | \nVideo encoding standard | \nPicture standard | \n
---|---|---|---|---|
Republic of Korea | \n<35.0 | \n25–34 | \nHEVC Main 10 | \n3840 × 2160p 60 frames/s 8 bits or 10 bits/pixel | \n
France | \n40.2 | \n22.5 17.5 | \nHEVC | \n3840 × 2160p 50 frames/s 8 bits/pixel | \n
Spain | \n36.72 | \n35 | \nHEVC | \n3840 × 2160p 50 frames/s 8 bits/pixel | \n
Sweden | \n31.7 | \n24 | \nHEVC | \n3840 × 2160p 29.97 frames/s 8 bits/pixel | \n
United Kingdom | \n40.2 | \nVariable (35) | \nHEVC | \nMixture of 3840 × 2160p 50 frames/s and 3840 × 2160p 59.94 frames/s 8 or 10 bits/pixel | \n
Czech republic | \n– | \n– | \nHEVC | \n3840 × 2160p | \n
Overview of the characteristics 4K UHDTV tested in the DTT.
In addition to broadcast 4K UHDTV channels in satellite and terrestrial digital networks during 2015 in the world has launched several UHDTV services in Internet Protocol Television (IPTV) and Over-The-Top (OTT) systems.
\nBesides the ultra HD format, there is also Super Hi-Vision 8K for whose development and promotion are in charge of the Japanese public broadcaster NHK. Super Hi-Vision format was able to show 120 frames per second and a resolution of 7680 × 4320 pixels which corresponds to the format of 32 megapixels. This format offers four times the resolution of 4K format and 16 times higher than HD. Table 10 [26] gives the basic test characteristics of 8K UHDTV in DTT networks in the world.
\nCountry | \nMultiplex capacity (Mbit/s) | \nSignal bit rate (Mbit/s) | \nVideo encoding standard | \nPicture standard | \n
---|---|---|---|---|
Japan | \n91.8 | \n91.0 | \nMPEG-4 AVC/H.264 | \n7680 × 4320p 59.94 frame/s 8 bits/pixel | \n
Republic of Korea | \n50.47 | \n50.0 | \nHEVC | \n– | \n
Overview of the characteristics 8K UHDTV tested in the DTT.
Digital Video Broadcasting Consortium in July 2014 adopted the basic parameters UHDTV transmission, and defined development plan of distribution UHDTV in stages, as shown in Table 11 [25].
\n\n | 4K UHDTV—Phase 1 | \n4K UHDTV—Phase 2 | \n8K UHDTV | \n
---|---|---|---|
Deployment | \n2015 | \n2018 | \n2020 | \n
Resolution | \n3840 × 2160 | \n3840 × 2160 | \n7680 × 4320 | \n
Frame rate | \np50/p60 | \np100/p120 | \np100/p120 | \n
Dynamic range | \nHDR preferred | \nHDR mandatory | \nHDR mandatory | \n
Color space | \nRec. 2020 | \nRec. 2020 | \nRec. 2020 | \n
Color sampling | \n4:2:0, 4:2:2 | \n4:2:0, 4:2:2 | \n4:2:0, 4:2:2, 4:4:4 | \n
Color bit depth | \n10 bits | \n10/12 bits | \n10/12 bits | \n
Video encoding | \nHEVC Main 10 | \nHEVC Main 10 | \nHEVC Main 10 | \n
Audio format | \n5.1 | \nBeyond 5.1 | \nObject based | \n
Audio codec | \nOpen | \nTBD | \nNext-generation audio codec | \n
Viewing angle | \n66° | \n66° | \n100° | \n
Viewing distance | \n1.5 picture height | \n1.5 picture height | \n0.75 picture height | \n
Development plan of distribution UHDTV.
The advantages brought by compression of the TV signal are as follows: reducing the frequency range of telecommunication channel which transmits TV signal, reducing the memory capacity required for recording images (storing images), access to data is reduced because the faster skips over the material, provided a data transfer in real time, it reduces the needed RAM memory and hardware becomes less expensive and leads to the miniaturization of hardware in the television. By reducing the number of bits, less power is required to broadcast; for example, if the transmitter of the same power is broadcasting analog and digital signal, for digital reception antenna of smaller diameter is required.
\nTo ensure reliable communication between users who use equipment and software from different manufacturers, standardization of methods of compression was carried out. So today, depending on the quality and use (television, multimedia services, videoconferencing, video telephony, etc.), there are several standards (JPEG, MPEG-1, MPEG-2, MPEG-4, H.261, H.263, H. 264, H.265, etc.).
\nSince it is a new technology that just catches the “momentum” toward global use, UHD is the future of television. Also, UHD offers the ultimate user experience and creates opportunities for the entire industry. 4K and 8K services will stimulate the growth of broadband, as well as the expansion of TV services in emerging markets. Consequently, the compression standard for TV in the near future will be HEVC/H.265.
\nThis work was done within the Erasmus Plus Capacity-Building projects in the field of Higher Education: “Implementation of the Study Program—Digital Broadcasting and Broadband Technologies (Master Studies)”, Project No. 561688-EPP-1-2015-1-XK-EPPKA2-CBHE-JP.
\nHistology is the branch of anatomy that focuses on the study of tissues of animals and plants. The term tissue refers typically to a collection of cells. In humans, organs comprise two or more tissue types, including epithelial, connective tissue, nervous, and muscular. The word “histology” stems from the Greek word “histos,” meaning web or tissue, and “logia,” meaning branch of learning. In brief, histological processing involves obtaining fresh tissue, preserving it (i.e., fixing it) in order to allow it to remain in as life-like a state as possible, cutting it into very thin sections (3–8 microns), mounting it on glass microscopic slides, and then staining the sections so that they can be observed under a microscope to identify different histological components within the tissue.
\nFor tissue removal, it is necessary to gather first the informed consent of the patient, as tissue taken from a live individual for diagnosis or treatment requires his/her consent. In other words, the patient must know at the time he/she consents, the purpose of tissue removal (e.g., diagnosis, research purposes, etc.) [1]. Similarly, harvesting tissue from an animal requires approval of the procedure by the institutional review board (Institutional Animal Care and Use Committee, IACUC) [2, 3]. An important first step in the histological process is tissue acquisition. This step can be achieved by means of traditional tissue dissection or endoscopic ultrasound (EUS)-guided fine needle aspiration [4]. If the former dissection method is chosen, it is important to ensure that sharp dissecting tools are used to minimize crushing the tissue while cutting for removal. The tissue should be kept moist (e.g., 0.85% saline, isotonic) while dissecting and trimming. The tissue should be trimmed 1–2 cm in width/length (but should not be more than 5 mm thick). There should be at least one to two cut sides for easy penetration of the fixative. It is important, at this stage, to determine the desired orientation of the tissue and that all tissue components are represented during this trimming stage, if possible [5].
\nIt is important to maintain cells in as life-like a state as possible and to prevent post-mortem changes as a result of putrefaction (destruction of tissue by bacteria or fungi) and autolysis (destruction of tissue by its own enzymes). In the latter case, as cells die, they release enzymes from their lysosomes and other intracellular organelles, which start to hydrolyze (i.e., break down or decompose by reacting with water) components of the tissue, such as proteins and nucleic acids with the help of proteases and nucleases, respectively. Cases of autolysis are most severe in tissues rich in enzymes (e.g., liver, brain, kidney, etc.) and are less rapid in tissues such as elastic fibers and collagen. Therefore, it is critical that fixation be carried out as soon as possible after removal of the tissues to prevent autolysis and putrefaction, as well as to prevent the tissue from undergoing osmotic shock, distortion, and shrinkage. Unfortunately, fixatives may, unintentionally, introduce artifacts which can interfere with interpretation of cellular ultrastructure [6–10].
\nAs fixation is typically the first step to prepare the tissue for microscopic, or other, analysis, the choice of fixative and fixation protocol is very important. The fixative acts to denature proteins by (i) coagulation (of secondary and tertiary protein structures to form insoluble gels), (ii) forming additive compounds (cross-linking end-groups of amino acids), or (iii) a combination of coagulative and additive processes. In addition, fixatives (iv) promote the attachment of dyes to particular cell components by opening up protein side groups to which dyes may attach, (v) remove bound water to increase tissue refractive index to improve optical differentiation, and (vi) alter the refractive index of tissues to improve contrast for viewing without staining. Prolonged fixation may result in the chemical masking of specific protein targets and prevention of antibody binding during immunohistochemistry protocols. In such cases, alternative fixation methods may be incorporated depending on the biological material. Therefore, there is no universal fixative which will serve all requirements. Each fixative has specific properties and disadvantages. There is no single fixative, or combination of fixatives, that has/have the ability to preserve and allow the demonstration of every tissue component. Some fixatives have only special and limited applications, while mixtures of two or more reagents may be necessary to employ the special properties of each. So, it is important to identify specifically which histological structures one is trying to demonstrate, as well as the effects of short-term and long-term storage of the tissues [6–10].
\nWhen tissue is fixed, it is important to keep the sample size small, if possible (i.e., 2–3 mm3), as increased thickness will retard fixative penetration. The volume of the fixative should be 20–25 times the volume of the tissue. The peritoneum or capsule around the tissue should be removed or pierced. The blood and mucus should be rinsed off with saline. The tissues should be cut with a new, sharp razor blade/scalpel, rather than scissors, as the latter could result in squeezing of the tissue, causing damage. Some tissues/organs (e.g., lung, eye, etc.) will require special handling to ensure that the fixative reaches all internal components. Care should be given to ensure that the specimen has one or more cut sides to guarantee good penetration of the fixative. Sometimes, an agitating instrument can be employed to ensure that the fixative reaches all surfaces. At no time should the tissue be allowed to dry out. Each fixative will have its own fixation time and post fixation treatment for best preservation of cellular detail. Typical fixatives, depending on the type of tissue and microscopy technique intended, may include, formalin, Zenker’s fixative, Bouin’s fixative, Helly’s fixative, Carnoy’s fixative, glutaraldehyde, osmium tetroxide, chromic acid, potassium dichromate, acetic acid, alcohols (ethanol, methanol), mercuric chloride, and acetone [5–10].
\nMicrowave fixation has been found to be useful in increasing the molecular kinetics giving rise to accelerated chemical reactions (i.e., faster fixation time, accelerated cross-linking of proteins). [11]. While conventional formalin-fixed, paraffin-embedded tissue offers superior cellular morphology and long-term storage, microwave-assisted tissue fixation with phosphate-buffered saline [12] of normal saline [13] offers the removal of the use of noxious and potentially toxic formalin fixation and a decrease in the turnaround time. In addition, staining of the microwave-fixed tissues was found to be sharper and brighter in most of the tissues than those obtained after conventional fixation [12, 14]. Interestingly, cold microwave irradiation procedures can offer rapid fixation and staining of tissues for electron microscopy and ultrastructural analysis [15].
\nFixatives can be classified on the basis of three main criteria: (i) action on proteins; (ii) types of fixative solution; and (iii) use [6–10, 16, 17].
Action on proteins
Fixatives can have two main actions on proteins. They can be coagulant or non-coagulant fixatives. Coagulant fixatives affect proteins in such a way that a coagulum (clot) forms (e.g., white of an egg when cooked). In contrast, non-coagulant fixatives result in a smoother “gel” formation. Cytoplasm is converted typically into an insoluble gel. In addition, while organelles are preserved, there is typically poor tissue penetration and artifacts are more likely to occur.
Types of fixative solution
There are two main types of fixatives: primary and compound. Primary fixatives consist of a single fixative in solution (e.g., may be in absolute form, such as absolute ethanol or 10% formalin). Compound fixatives consist of two or more fixatives in solution, such as Zenker’s, Helly’s, and Bouin’s fixatives.
Their use and mechanism of action
The intention of microanatomical fixatives is to preserve components of organs, tissues, or cells in spatial relation to each other. These fixatives are largely coagulant in nature (cell organelles are destroyed, typically), and used for light microscopy (e.g., neutral buffered formalin or NBF, Zenker’s, Bouin’s, and 10% formal saline). Cytological fixatives, on the other hand, preserve cellular structures or inclusions (e.g., mitochondria), often at the expense of even penetration and allow the tissues to be cut relatively easily. They are non-coagulant in nature and are used typically for electron microscopy. They can be further subdivided into nuclear (e.g., Carnoy’s) and cytoplasmic (e.g., Helly’s and 10% formal saline).
\n
Aldehydes include formaldehyde (formalin, when in its liquid form), paraformaldehyde, and glutaraldehyde. Tissues are fixed through cross-linking agents that react with proteins and nucleic acids in the cell (particularly lysine residues). Formaldehyde is a good choice for immunohistochemical studies, while formalin (10% neutral buffered formalin or NBF) is standard. The buffer prevents acidity in the tissues. Formaldehyde offers low levels of shrinkage and good preservation of cellular detail. This fixative is used routinely for surgical pathology and autopsy tissues requiring hematoxylin and eosin (H and E) staining [6–10, 16, 17]. Since formalin is toxic, carcinogenic, and a poor preserver of nucleic acids, there have been attempts to find a more suitable substitute; however, this has proved difficult [18].
Glutaraldehyde causes deformation of the alpha-helix structure in proteins, so it should not be used for immunohistochemistry staining. While it fixes very quickly, which makes it an excellent choice for electron microscopic studies, it provides poor penetration. It gives very good overall cytoplasmic and nuclear detail and is prepared as a buffered solution (e.g., 2% buffered glutaraldehyde). This fixative works best when it is cold and buffered and not more than 3 months old [6–10, 16, 17].
Oxidizing agents include permanganate fixatives, such as potassium permanganate, dichromate fixatives (potassium dichromate), osmium tetroxide, and chromic acid. While these fixatives cross-link proteins, they cause extensive denaturation [6–10, 16, 17].
Alcohols, including methanol and ethanol, and protein denaturants (acetic acid) are not used routinely as they cause brittleness and hardness to tissues. They are useful for cytologic smears, as they act quickly and provide good nuclear detail. Alcohols are used primarily for cytologic smears. They are fast acting, cheap, and preserve cells through a process of dehydration and precipitation of proteins. Methanol has been shown to be effective during immunostaining [6–10, 16, 17, 19].
Mercurials fix tissues by an unknown mechanism. They contain mercuric chloride which is a known component in fixatives such as B-5 and Zenker’s. These fixatives offer poor penetration and tissue hardness, but are fast and provide excellent nuclear detail, such as for visualization of hematopoietic and reticuloendothelial tissues (i.e., lymph nodes, spleen, thymus, and bone marrow). These fixatives must be disposed of carefully. Mercury deposits must be removed (dezenkerized) prior to staining, otherwise black deposits will occur in tissue sections [6–10, 16, 17].
Picrates include fixatives with picric acid, such as Bouin’s solution. These fixatives have unknown modes of action. The most common is Bouin’s alcoholic fixative. This fixative provides good nuclear detail and does not cause much hardness. It is recommended for fixation of testis, gastrointestinal tract, and endocrine tissues. This fixative has an explosion hazard in dry form, so it must be kept submerged in alcohol at all times [6–10, 16, 17].
Other factors affecting fixation
Buffering: Fixation is best performed at close to neutral pH (pH 6–8; formalin is buffered with phosphate at pH 7). Common buffers include: phosphate, bicarbonate, cacodylate, and veronal [6–10, 16, 17].
Penetration: Each fixative has its own penetration rate in tissues. While formalin and alcohol penetration are superior, glutaraldehyde is the worst. Mercurial fixatives are in between. The thinner the sections are cut, the better the penetration [6–10, 16, 17].
Volume: The volume of the fixative should be in at least a ratio of 10:1. Fixation can be enhanced if the fixative solution is changed at regular intervals and the specimen is agitated [6–10, 16, 17].
Temperature: If the temperature at which fixation is carried out is increased, it will yield an increased speed of fixation. Of course, too much heating of the fixative can result in cooking or creating tissue artifacts [6–10, 16, 17].
Concentration: The concentration of the fixative should be as low as possible, because too high a concentration may adversely affect the tissue and provide artifacts (formalin is best at 10%, while glutaraldehyde is best at 0.25–4%) [6–10, 16, 17].
Time interval: The faster the fresh tissue can be acquired and fixed, the better, as to minimize cellular organelle degradation and nuclear shrinkage, resulting in artifacts. The tissue should always be kept moist with saline [6–10, 16, 17].
Some animal tissues contain deposits of calcium salts which may interfere with sectioning, resulting in torn sections and damaged blades. Calcium compounds must be chemically removed (usually with an acid) before typical histological techniques can be used for the study of softer components. Tissues requiring decalcification include bone, teeth, and calcified cartilage [17, 20–22]. Pathological states include arteriosclerosis, tuberculosis, and several tumor types. Such tissues should be fixed prior to decalcification and washed for 12 hours in running water between fixation and decalcification. While decalcification agents remove typically calcium salts and do not interfere with staining reactions, they can cause minimal distortion to cells and connective tissue. The decalcifying agent should have a volume of 30–50 times that of the tissue and occasional agitation may be required to expedite this process. Heating should not be employed. The process is complete typically when bubbling has ceased. Over decalcification can cause a severe reduction (of what) in subsequent sectioning of the tissue. Some typical decalcifying agents include, nitric acid, Gooding and Stewart’s fluid, Rapid Bone Decalcifier (RDO), and chelating agents. More recently, new methods have been discovered to allow hard tissues to be decalcified faster [23].
\nAfter fixation, and to begin the dehydration step (i.e., removal of water), tissues are placed in progressively increasing concentrations of a dehydrating agent (e.g., 70, 85, 95, and 100%) which is typically ethanol. Methanol, isopropanol, and acetone are alternative options, depending on the tissue being processed. It is important to include two absolute alcohol (i.e., 100%) steps to ensure that all remaining water has been removed. The dehydration step is critical, as water is immiscible with most embedding media (i.e., paraffin wax). Therefore, the tissue must be exchanged between polar (e.g., water) and non-polar (e.g., organic reagents, such as xylene) agents. If the tissue is incompletely dehydrated, it is not possible to “clear” the tissue. When it is exposed to a subsequent clearing agent (e.g., xylene) the tissue remains opaque and appears milky. This will necessitate re-dehydration of the tissue. Dehydration will also remove some of the lipoidal material in the tissue. If the lipids are supposed to be visible, it will be necessary to use an appropriate fixative that will preserve the lipids prior to the dehydration step (e.g., osmium tetroxide) [7–10, 16, 17, 21, 22].
\nThe term “clearing” is related to the appearance of the tissue after it has been treated with a dehydrating agent. Many agents have a similar refractive index to that of the tissue, rendering the tissue “clear” or translucent. In this step, the dehydrating agent must be removed from the tissue and replaced with a solvent of wax. A clearing agent should be used when the dehydrating agent (e.g., ethanol) is not miscible with the impregnating medium/embedding agent (i.e., paraffin wax). It is a wax solvent and must be miscible with both the dehydrating and embedding agents. The selection of a suitable clearing agent should be based on the speed and ease of removal from the embedding media (i.e., the lower the boiling point the more rapid the removal), interaction with the tissue, flammability, toxicity, and cost. The clearing step can be more effective with the use of a vacuum system and should be carried out in a fume hood. Typical clearing agents include xylene, chloroform, toluene, benzene, dioxane, carbon tetrachloride, cedarwood oil, isoamyl acetate, methyl benzoate, methyl salicylate, and clove oil. Due to the potential hazards of some of these chemicals, others have been proposed, such as some vegetable oils, terpenes, and alkanes. Some histological protocols have the potential option of processing the tissue without the use of a clearing agent (e.g., xylene) as a safe alternative to exposure to the hazardous effects of these chemicals. One such protocol includes the use of isopropanol as a safer alternative [7–10, 16, 17, 21, 22, 24–30].
\nThe role of the infiltration agent is to remove the clearing agent from the tissue and to completely permeate the tissue with paraffin wax. This will allow the tissue to harden and produce a wax block from which thin histological sections can be cut. Ideally, the consistency of any solidified embedding medium should be the same as the specimen it encloses. Unfortunately, this rarely happens due to the wide variation in consistency of tissue and the large variety in embedding media. Paraffin wax is commonly used and heated to a temperature that is 2–3°C above its melting point. Any higher temperature will result in tissue hardening. The paraffin wax should be 20–25 times the volume of the tissue. Generally, the tissues are transferred directly from the clearing agent to pure paraffin, but sometimes with fragile specimens, it is necessary to use graded mixtures of clearing agent and paraffin. The duration and number of changes of paraffin necessary for impregnation vary with the size and consistency of the tissue. As exposure of the tissue to paraffin increases, it is more likely that shrinkage and hardening will occur. Complete infiltration is only possible after complete dehydration and complete clearing. The selection of paraffin depends on the nature of the tissue to be embedded and thickness of section required. A high melting point of the wax (e.g., 55–60°C) increases the hardness and decreases the thickness to which the tissue may be sectioned (e.g., 45–50°C is considered soft). Paraffin wax can be purchased in the form of tablets, pellets, or granules. Numerous substances can be added to the molten paraffin to modify its consistency and melting point. Typically, the process of infiltration occurs with the use of a tissue processing machine, although this can be carried out using a heated container maintained 2–3°C above the melting point of wax. If residual clearing agents remain in tissue or improper processing of the tissue has occurred, this will lead to difficulties with sectioning. Evaporation of the clearing agent, infiltration with paraffin wax, and removal of any air bubbles trapped in the specimen will be more completely alleviated if clearing and infiltration procedures are carried out at reduced pressure (under vacuum) [7–10, 16, 17, 21, 22, 24–28].
\nAfter the infiltration process has been completed, it is necessary to obtain a solid block containing the tissue. To accomplish this, it is necessary to first coat a stainless steel histological base mold of suitable size to fit the tissue with glycerol or “mold release” to prevent adherence of the wax block containing the tissue to the metal mold upon solidification. Pre-warming of the metal block is advised to prevent premature solidification of the wax block. In addition, using warmed forceps to help press the tissue against the base of the metal mold, in addition to reducing the chance of premature solidification, helps with this process. Prior to beginning the infiltration process, an embedding cassette should be placed on top of the mold and labeled with the name of the tissue, fixative, and date. If an embedding unit (machine) is being used, the combined unit should be dispensed two-thirds full with molten paraffin. The specimen should be oriented in the metal mold to ensure that the tissue will be cut in the correct plane of section. Alternatively, the mold can be filled slightly and the tissue can then be placed in the mold and positioned in the desired orientation at the base of the mold. The combined unit should then be set out on the cooling tray of the embedding unit (machine) and not disturbed until the wax has cooled and solidified completely. After sufficient time, the cassette and mold should be separated and the paraffin block should be placed in the microtome in preparation for sectioning. If the tissue has been thoroughly fixed, dehydrated, cleared, and infiltrated, tissues embedded in paraffin wax provide good cutting qualities. On average, paraffin blocks remain durable and retain their good cutting qualities and staining characteristics indefinitely [7–10, 16, 17, 21, 22, 24–28].
\nThe most common infiltrating agent and embedding medium is paraffin wax. Ester wax offers a lower melting point than paraffin wax and tends to be harder when solid, allowing this medium to be suitable for cutting thinner (i.e., 2–3 μm) sections with minimal tissue shrinkage.
\nWhen water-soluble waxes (i.e., polyethylene glycol waxes) are used, tissues are transferred directly from aqueous fixatives to wax for infiltration without dehydration or clearing. This results in less tissue shrinkage, but sectioning is more difficult than with paraffin wax. Tissue blocks must be kept in a dry atmosphere. If cellulose nitrate (i.e., celloidin/low-viscosity nitrocellulose) is chosen as an embedding medium, tissues must be dehydrated and embedded with solutions of cellulose nitrate dissolved in an alcohol/ether mixture. The solvent is allowed to evaporate to produce a tissue block of required consistency. No heat is applied using this method. This medium is used typically for large pieces of, for example, bone and brain tissues. Synthetic resins are used for preparing sections most typically for electron microscopy and light microscopy (0.5–2 μm sections), such as for undecalcified bone. Freeze-drying protocols can be applied when special staining techniques are used [7–10, 16, 17, 21, 22, 24–28].
\nMicrotomes are used to cut the tissue into thin sections for microscopic viewing. The type of specimen will determine the type of microtome to be used. Rotary microtomes are the most common microtomy instrument. The feed mechanism is achieved by turning a wheel at one side of the machine. While the knife is fixed and is secured in a knife holder, the object moves against the cutting surface of the knife, according to the thickness of section required. The knife holder allows the knife to be set at an oblique angle to the specimen. One complete rotation of the operating wheel is equivalent to one complete cycle. The downward motion of the knife reflects the cutting stroke, while the upward stroke reflects the return stroke and activation of the advance mechanism. The feed mechanism is activated by turning a wheel located on the side or top of the microtome. The tissue block is passed across the knife at every stroke to produce a section. Microtomes have a feed mechanism to advance the specimen (or knife) to a predetermined thickness for sectioning (i.e., typically 5–10 μm) and can produce serial sections [7–10, 16, 17, 21, 22, 24–28].
\nA cryostat or freezing microtome is used for obtaining thin sections of unfixed tissues. It can be used, additionally, for observing fatty tissues. The microtome is maintained at −15 to −20°C in a refrigerated chamber. The cabinet is designed to operate at −5 to −30°C. The tissue block can be mounted in a high-viscosity water-soluble gel, such as 1% glucose, gelatin, or cellulose on the platform and must be frozen immediately. An anti-roll plate is used to keep sections flat on the knife blade for direct mounting onto the slide. Sections are cut one at a time. When a section is cut, the anti-roll plate is lifted and a section is picked up from the surface of the knife and placed onto a slide using a camel hair brush. Sections are fixed in 5% acetic acid in absolute alcohol and then subsequently stained (e.g., with hematoxylin and eosin). Frozen sectioning is typically used for rapid preparation and diagnosis by a pathologist [7–10, 21, 22].
\nThere are many different types of microtome knives (e.g., stainless steel, carbide, diamond, glass, or disposable blades). Wedge-shaped stainless steel knives are used for most paraffin-embedded specimens. They must be kept clean and well-oiled or lubricated. The knife’s edge should be cleaned with a clearing agent with a soft, moistened cloth in a fume hood. As an alternative to wedge-shaped stainless steel knives, disposable blades provide an excellent cutting edge for paraffin sectioning and are available in different sizes and thicknesses. Glass, sapphire, and diamond knives are used for specimens embedded in hard resin plastic (e.g., epoxy, glycolmethacrylate). Diamond and sapphire knives tend to function better than glass knives, but are much more expensive. If a wedge-shaped stainless steel knife is used, it must be free of nicks and sharpened with a carborundum stone (manual sharpening) or by an automatic knife sharpener (with a glass wheel and with an abrasive). A process called stropping produces a finely polished, smooth, and even knife edge. The knife is secured at the desired angle place by adjusting holder screws [7–10, 16, 17, 21, 22, 24–28].
\nA thickness of 6 μm is standard for histological tissue sections. For highly cellular tissues (e.g., lymph nodes), 4 μm is used most often. For thicker sections, 10 μm is used. For neurological tissues and myelinated nerves, 6–20 and15–20 μm is used, respectively. The tissue block will be examined to establish how it needs to be oriented in the block holder. Excess paraffin should be trimmed away from each side of the tissue block to create a trapezoid shape. The longer edge should be parallel with the knife edge. The tissue block should be roughly cut by advancing the block manually and sectioning until the entire surface of the tissue is exposed [7–10, 16, 17, 21, 22, 24–28].
\nSection adhesives, such as gelatin, casein glue, starch, and albumin, can be used to aid in adhering sections to the slide prior to further processing, such as staining. Gelatin can be added to the water bath. The use of adhesives in the water bath promotes bacterial and fungal growth. Daily cleaning of the water bath with sodium hypochlorite solution (e.g., Clorox soap) is necessary to prevent contamination. Alternatively, a thin coat of albumin can be applied directly to the slide by dipping it into the solution or using your fifth finger (i.e., most ulnar and smallest finger). This latter process is referred to as “subbing.” A newer idea is to use “plus” (+) slides. Treatment of the slide with a reactive silicon or polylysine compound chemically changes the glass, such that it bears abundant amino groups, which ionize to provide a positively charged surface. Sections which contain a preponderance of anionic groups, such as carboxyls and sulfate-esters adhere strongly to this modified glass. When creating a ribbon (what is a ribbon), i.e., a series of adjacent tissue sections, the hand wheel should be turned at a slow and even speed. Rotating the wheel too rapidly will cause sections of unequal thickness. The floatation bath should be heated to a few degrees below the melting point of the paraffin wax. Tap, deionized, or distilled water can be used. The ribbon should be gradually lowered onto the flotation bath to eliminate wrinkles and entrapped air. Air bubbles may be removed with a camel’s hair brush or by submerging a slide under the ribbon. If the sections are wrinkling, a 70% alcohol solution can be added to the water bath prior to section collection. If necessary, sections may be separated, depending on their sizes, and each can be placed on a clean, pre-marked glass slide. Individual sections or tissue ribbons may be picked up by submerging a clean glass slide into the water bath at a ~45° angle, directly beneath the location of the section or ribbon. The slide should be lifted out of the water slowly to ensure that the sections lay on the slide. The slides should be drained vertically on a paper towel for several minutes before placing them onto a warming table (37–40°C). The slides should remain on the warming table, overnight, for 20–30 minutes at approximately 58°C or a few degrees below the melting point of the paraffin wax. Failure to drain the slides will create air bubbles under the tissue and decrease the section’s adhesion to the slide. Air bubbles produce section unevenness and staining artifacts, making the final preparation difficult to examine with the microscope. Once the desired sections have been cut, the block can be removed from the block holder and sealed with molten paraffin wax to ensure that the tissue will not dry out and become brittle (blocks can last for weeks, months, or years) [7–10, 16, 17, 21, 22, 24–28].
\nHistologists are confronted often with difficult tissue blocks that will not section easily. This may be the result of, for example, brittle or shrunken tissue, improperly infiltrated tissues, or sections with, for example, holes or scratches in them. If the tissue block appears to be brittle, a 10% diluted ammonium hydroxide solution may be applied (via soaking) to soften the tissue to prevent cracking and to more easily facilitate sectioning. If sections have holes in them, this can be indicative of incompletely infiltrated tissue. This may be alleviated by placing the tissue block back in the heated wax bath to melt it and then proceed to re-embed the block. If artifactual scratches or tears occur across the tissue sections, this may be indicative of flaws or dirt on the cutting edge of the knife and may be alleviated by repositioning or replacing the blade. Alternatively, other problems can occur if the tissue block appears to be too soft or too hard. If too soft, a remedy may be to place the block tissue side down on several sheets of Kimwipes or paper towel in the freezer (−15°C) or a refrigerator (0–4°C) (chilling times may vary), prior to sectioning. This technique will help to harden the wax so that it better matches the hardness of the infiltrated tissue and will result in more successful tissue sectioning. If too hard, a piece of wet cotton/Kimwipe may be placed in lukewarm water and then placed over the surface of the block (times may vary). This will allow the tissue to expand/swell and soften as it absorbs water. It should be noted, however, that with either too soft or too hard tissue blocks, these solutions are temporary and may allow only a few successful sections to be cut [31, 32].
\nStaining of tissue slides is carried out by reversing the embedding process in order to remove the paraffin wax from the tissue to allow water-soluble dyes to penetrate the sections. This process is referred to as “deparaffinization.” The tissue slides must be exposed to a clearing agent and subsequently taken through a descending alcohol series to water (also referred to as “bringing your slides to water”). Choosing the appropriate dye for a particular tissue slide is related to its ability to color otherwise transparent tissue sections and various cellular components of the tissue. The term “routine staining” includes the hematoxylin and eosin (i.e., H and E) stain. This stain is used routinely as it provides the pathologist or researcher with a detailed view of the tissue, clearly staining, for example, the cytoplasm, nucleus, and organelles. The term “special stains” refers to a large number of staining techniques, other than H and E, that allow the visualization of particular tissue structures, elements, or microorganisms that cannot be identified with H and E staining [6–10, 17, 21, 22, 24–28, 31–35]. Examples include, Masson’s trichrome (e.g., skin; identification of collagenous connective tissue), GMS silver stain (e.g., lung; identification of Pneumocystis or Aspergillus spp.), Periodic acid-Schiff (e.g., kidney; identification of high proportion of carbohydrates, such as glycogen, glycoproteins, and proteoglycans), Perl’s Prussian blue iron (e.g., liver; identification of ferric (Fe3+) iron in tissue preparations or blood and bone marrow smears), Ziehl-Neelsen (acid-fast bacillus) (e.g., lung; identification of acid fast bacilli), Alcian blue (e.g., intestine; identification of acid mucopolysaccharide and acidic mucins), Alcian blue and PAS (intestine; combination of staining properties of both Alcian blue and Periodic acid-Schiff for identification of similar tissue components), Gomori trichrome (blue or green) (e.g., submucosa, identification of muscle fibers, collagen, and nuclei) [36].
\nThis work was supported in part by grants from the National Institutes of Health (GM058264) and the National Science Foundation (1626326) to VDCS and from the National Science Foundation (1355034) and the Latham Trust Fund to TH.
\nThe authors declare that there are no conflict of interests regarding the publication of this chapter.
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