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Pixel art

Pixel Art Cat with Zoom-in Detail.svg made in a 64x64 Pixel art canvas. Includes a zoomed-in window in the bottom left corner to enhance the details of the cat's face.

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Pixel art is a form of digital art drawn with graphical software where images are built using pixels as the only building block. It is widely associated with the low-resolution graphics from 8-bit and 16-bit era computers, arcade machines and video game consoles, in addition to other limited systems such as LED displays and graphing calculators, which have a limited number of pixels and colors available. The art form is still employed to this day by pixel artists and game studios, even though the technological limitations have since been surpassed.

Most works of pixel art are also restrictive both in file size and the number of colors used in their color palette because of software limitations—to achieve a certain aesthetic or simply to reduce the perceived noise. Older forms of pixel art tend to employ smaller palettes, with some video games being made using just two colors (1-bit color depth). Because of these self-imposed limitations, pixel art presents strong similarities with many traditional restrictive art forms such as mosaics, cross-stitch and fuse beads.

The precise definition of pixel art is a subject of debate, but an artwork is usually considered as such if deliberate thought was put into each individual pixel of the image. Standard digital artworks or low-resolution photographs are also composed of pixels, but they would only be considered pixel art if the individual pixels were placed with artistic intent, even if the pixels are clearly visible or prominent (see Definition).

The phrases "dot art" and "pixel pushing" are sometimes used as synonyms for pixel art, particularly by Japanese artists. A much more popular variation is the term spriting, which sometimes refers to the activity of making pixel art elements for video games specifically. The concept most likely originated from the word sprite, which is used in computer graphics to describe a two-dimensional bitmap that can be used as a building block in the construction of larger scenes.

Definition
The majority of pixel artists agree that an image can only be categorized as pixel art when the pixels play an important individual role in the composition of the artwork, which usually requires deliberate control over the placement of each individual pixel. When purposefully editing in this way, changing the position of a few pixels can have a drastic effect on the image. Modern pixel art software incorporates tools that automatically place multiple pixels at once (such as fill tools, line tools, and brush tools), therefore defining pixel art as "art in which an artist has placed each individual pixel" is not accurate anymore. The following is a better way to interpret it: "The process that leads to the final artwork is less relevant than the final result. If the pixels play an important individual role in the final composition, it will be broadly regarded as a pixel art piece by most artists independently of the techniques that may have been implemented to achieve that result."

A common characteristic in pixel art is the low overall color count in the image. Pixel art as a medium mimics a lot of traits found in older video game graphics, rendered by machines capable of only outputting a limited number of colors at once. Additionally, many pixel artists are of the opinion that in most cases, using a large number of colors, especially when very similar to each other in value, is unnecessary, and detracts from the overall cleanliness of the image, making it look messier. Many experienced pixel artists recommend not using more colors than necessary.

Differences from digital art
As images get higher in resolution, pixels get harder to distinguish from each other, and the importance of their careful placement is diminished, to the point that the concept of pixel art falls apart. The exact point at which this occurs and the conditions for a piece to be reasonably called "pixel art" have been the source of great disagreement among professionals and enthusiasts.

In general, the construction of a pixel image can be very similar to that of a digital artwork, particularly during the early sketching phases. The part in which the biggest distinction is made is the final pixel-by-pixel polishing.

Differences from Oekaki
Oekaki is a form of digital art done at small resolutions that present many similarities with pixel art. However, in Oekaki, the placement of individual pixels is not considered as important compared to the general feel of the artwork, giving it a characteristic "messy" or jagged look.

Automation and algorithms
There are plenty of algorithms that have been used to facilitate the creation or editing of pixel art, such as using 3D shaders within software like Blender to generate complex effects or rotating objects, that are later imported into pixel art software.

There also exist many interpolation algorithms calibrated specifically for pixel art.

Origin

Some traditional art forms, like counted-thread embroidery (including cross-stitch) and some kinds of mosaic and beadwork, are very similar to pixel art and could be considered as non-digital counterparts or predecessors. These art forms construct pictures out of small colored units similar to the pixels of modern digital computing.

Some of the earliest examples of pixel art could be found in analog electronic advertising displays, such as the ones from New York City during the early 20th century, with simple monochromatic light bulb matrix displays extant circa 1937. Pixel art as it is known today largely originates from the golden age of arcade video games, with games such as Space Invaders (1978) and Pac-Man (1980), and 8-bit consoles such as the Nintendo Entertainment System (1983) and Master System (1985).

The term pixel art was first published in a journal letter by Adele Goldberg and Robert Flegal of Xerox Palo Alto Research Center in 1982. The practice, however, goes back at least 11 years before that, for example in Richard Shoup's SuperPaint system in 1972, also at Xerox PARC.

1970s
Because of the severe restrictions of early graphics, the first instances of pixel art in video games were relatively abstract. The low resolution of computers and game consoles forced game designers to carefully design game assets by deliberate placement of individual pixels, to form recognizable symbols, characters, or items. Simple function-based avatars (or player-surrogates) such as spaceships, cars, or tanks required a minimum of animation and computing power, while enemies, terrain, and power-ups were often represented by symbols or simple designs. Due to the limited hardware of the 1970s, abstraction, as in the case of Pong's relatively simple design, sometimes led to better game readability and commercial success than attempting more detailed representational art.

Although computers had been used to create art since the 1960s and microcomputers were used in the late 1970s and there are examples of digital art utilizing a more pixelated aesthetic, There is no well-known tradition of pixel art from the 1970s that differentiated between the deliberate placement of pixels or the aestheticization of individual pixels in contrast to other forms of digital painting or digital art. For this reason, one could argue that pixel art was not a recognized medium or artform in the 1970s.

1980s
In what is sometimes referred to as the golden age of video games or golden age of arcade video games, the 1980s saw a period of innovation in video games, both as a new artform and a form of entertainment. During the early 1980s, video game creators were mainly programmers and not graphic designers. Technological innovation led to market pressure for more representational and "realistic" graphics in games. As graphics improved, it became possible to replace hand-drawn game assets with imported pictures or 3D polygons, which contributed to pixel art developing as a separate art form.

Gradually, professional artists and graphic designers had a bigger impact in the video game industry. Sierra Entertainment released Mystery House, pixelled by Roberta Williams, and the King's Quest series; and Lucasfilm Games released games such as Maniac Mansion, Zak McKracken and the Alien Mindbenders, and Indiana Jones and the Last Crusade: The Graphic Adventure. Mark J. Ferrari, an artist at Lucasfilm Games, later said: "When I was first hired by Lucasfilm Games in 1987 to do artwork for their computer games, pixel graphics was not thought of by anyone as an 'art form'. The use of pixels was not an aesthetic choice – as it certainly is now. If anything, pixels were an unavoidable and very irksome obstacle to the creation of any 'real art' for use in the exciting but bewildering new realm of computer entertainment. There were no pixel artists then – at all! There were only traditional artists." Nevertheless, the aesthetic of 1980s video games had a major impact on contemporary and future pixel art, both in video games, the demoscene graphics and among independent artists. As computers became more affordable in the 1980s, software such as DEGAS Elite (1986) for the Atari ST, Deluxe Paint (1985) and Deluxe Paint 2 (1986) for the Commodore Amiga, and Paint Magic for the Commodore 64, inspired many later pixel artists to create digital art by careful placement of pixels. In the case of the Commodore 64 and the Amstrad CPC, some early pixel artists used joysticks and keyboards to pixel.

With the rise of the demoscene movement in Europe in the late 1980s, artists who were proficient with creating pixel art using 8-bit computers like the Commodore 64 and ZX Spectrum or 16-bit computers like the Atari ST began to publish their pixel art as demos. Demogroups would often include coders (programmers), musicians, and graphicians, where graphicians were a common name for graphic designers and/or pixel artists. Although some graphicians worked on adding art to cracked video games (cracktros), the demoscene contributed to artistic communities creating pixel art for its own sake as art. These were often shared via floppy disks that were handed from person to person or via the postal service. The golden age of the demoscene and its associated pixel art milieu, however, is often regarded as beginning in the early 1990s.

1990s
Before the 1990s, display systems were mostly based on a small 4-bit palette of imposed colors (16 fixed shades innate to each system, often incompatible with one another). The coming decade greatly improved the graphics standard with the appearance of increased color depth and indexed color palettes (For example, 512 colors for the Atari ST and the Mega Drive, 4,096 for the Amiga ECS, 32,768 for the Super Nintendo, and 16,777,216 for the Amiga AGA and the VGA mode of the PCs). During the 1990s, 2D games with manually painted graphics saw increasing competition from 3-dimensional games and games using pre-rendered 3D assets. Still, pixel art games like Flashback, The Secret of Monkey Island, The Chaos Engine, Street Fighter III: 3rd Strike, and The Legend of Zelda: A Link to the Past had a major influence on future artists in the game industry, the contemporary demoscene and the aesthetic of pixel art in later decades.

In addition to pixel art influenced by video games, pixel artists (or graphicians) in the demoscene continued to make pixel art for demos and cracktros. Some demoscene pixel artists active in the 1990s have cited movies and urban graffiti as important influences for their art, particularly in designing logos. In addition to copying sprites from existing video games, demoscene pixel artists also copied the work of popular artists and illustrators such as Ian Miller and Simon Bisley. Competition gradually became an increasingly important part of demoscene gatherings, including pixel art (graphics) competitions, and as teenagers and young adults were the major demographic in these gatherings, a lot of demoscene pixel art referenced familiar fantasy, science fiction and cyberpunk tropes. Demoscene competitions had a major effect in shaping the direction of pixel art. Prominent artists would look for ways to innovate, display superior technique, overcome technical restrictions, and in many cases aim for photorealism through anti-aliasing.

As the internet became more available in the 1990s, pixel artists and demosceners gradually began to spread their pixel art via websites, instant messaging, and online file sharing. While the demoscene was arguably most popular among young males, an online movement began in the late 1990s known as pixel dolls or 'dollz', which was popular among young females. The increased popularity of online chatting and personal webpages at a time before digital cameras were common led to an increased demand for personal representation through personalized avatars. These avatars often took the form of pixel dolls, being pixel art characters that could be outfitted with different clothing and accessories. As pixel dolls grew more and more common, many pixel artists gradually began to develop this as an independent artform.

2000s
The 2000s were a pivotal decade for pixel art establishing itself as an artform practiced around the world, separate from other forms of digital art. In particular, the Pixelation forum and Pixel Joint gallery are credited as the most influential English-speaking online communities, connecting pixel artists all over the world in a way that the mostly Western European demoscene movement had not. Pixelation was a web forum where artists could share pixel art to give and receive feedback. Its main focus was critiquing, developing skills and understanding of pixel art, more so than simply sharing art for the sake of admiration, entertainment, or competition. Pixel Joint is an online gallery where members can submit personal work and comment on other members' pixel art. It has features such as weekly challenges, a forum, a hall of fame, and a monthly top 10-pixel art competition based on member voting.

Over time, the overlapping but separate communities of Pixelation and Pixel Joint were engaged in online discourse about the nature of pixel art, inspiring widely shared tutorials and arguably contributing to a new paradigm that was different from the pixel art of the 1990s. A major concern was to establish pixel art as its own medium and/or art form, separate from other types of digital art, such as Oekaki. In particular, highly restricted palettes (e.g. a maximum of 8 or 16 colors) were argued by some to be a defining feature of pixel art. One example is the so-called "8 color gentlemen's club", consisting of pixel artists who celebrated pixel art with 8 colors. The use of transparent layers and smudging tools was also considered non-pixel art and unacceptable for the Pixel Joint gallery. Whereas the demoscene had, to a large degree, revolved around physical gatherings and groups of artists, musicians, and programmers collaborating, pixel art communities on Pixel Joint and Pixelation were based mostly on online interaction among individual artists. On Pixel Joint, pixel art made through collaboration between artists was not accepted in the gallery but considered a separate pursuit. The relatively strict ideas of what constituted acceptable pixel art, enforced by moderators on Pixel Joint, led to repeated conflict but also contributed to pixel art standing out as a separate medium at a time when many types of digital paintings were shared on the internet, on websites like DeviantArt. In particular, several prominent artists mention Cure's tutorials as significant for learning about pixel art as a separate art medium or art form.

After the golden age of demoscene pixel art in the 1990s, many notable pixel artists of that milieu took a break from pixel art in the 2000s - although some made an early transition to join Pixelation and Pixel Joint, such as Gas 13 and Tomic. While activity did decline, the demoscene continued to explore pixel art on platforms from the late 1980s and early 1990s, particularly using the Commodore 64 and Atari computers.

Pixel doll communities grew rapidly during the 2000s, in large part due to the continued growth of forums, message boards, and chat services. With websites like the Doll Palace, Eden Enchanted, DeviantArt, and many personal websites, pixel dolls were increasingly recognized as a new art form, with public feedback, competition, tutorials, best practices, and rules of conduct. The 2000s were arguably the height of pixel doll popularity, in part because of various online communities that made use of pixel dolls for avatars and the popularity of internet forums in an age before social media. Despite this, there was relatively little interaction between the pixel doll communities and other pixel art communities such as Pixel Joint, Pixelation, or the demoscene.

In video games for computers and consoles during the 2000s, pixel art was largely abandoned in favor of more modern graphics, particularly based on 3D. Many professional game artists who had been working with pixel art either left the industry or switched to other forms of digital art. This arguably contributed to pixel art establishing itself as an independent art form, practiced mostly by independent artists rather than game industry employees. There are a few notable exceptions, such as Habbo Hotel, which inspired a great amount of isometric pixel art, and Advance Wars, for Game Boy Advance.

2010s
The popularity of pixel art accelerated during the 2010s. One major contribution to this trend was the success of several 'retro' pixel art games such as Superbrothers: Sword & Sworcery EP (2011), Fez (2012), Papers, Please (2013), Shovel Knight (2014), Undertale (2015), Owlboy (2016), Stardew Valley (2016), Deltarune (2018), Celeste (2018), and Octopath Traveler (2018). Many of these games combined low-resolution game assets with non-pixel art (NPA) elements such as filters, high definition UI, or special effects. The increased attention on pixel art in media and the game industry coincided with the rapid growth of pixel art communities. Owlboy's art director, Simon Andersen, explained that he wanted to show the advantages of the pixel as an art medium, pushing the potential of the artistic technique and showcase pixel art done properly, without millions of colors or 3D captures. In contrast, the heavily stylized and abstract Superbrothers: Sword & Sworcery EP made extensive use of filters and non-pixel art graphics. Superbrothers: Sword and Sworcery also allowed players to share various in-game achievements on Twitter, the #sworcery hashtag went viral, contributing to the renewed interest in pixel art in video games.

Pixel Joint and Pixelation remained important communities with a growing number of contributors, but the 2010s saw the rise of new communities on Reddit, DeviantArt, Instagram, and Twitter. While Pixelation, in particular, had a core of very dedicated, often professional, pixel artists and Pixel Joint had moderators who accepted or rejected submissions to its online gallery, new pixel art communities in social media were naturally less cohesive, yet more open. Not only did social media contribute significantly to the continued growth of pixel art, and more traffic to older pixel art websites, but websites like Tumblr, Twitter, and Instagram also gave significant global attention to new pixel artists outside of the traditional pixel art communities, such as Waneella and Pixel Jeff.

During the 2010s, the demoscene arguably entered a silver age, as several older artists returned to their hobbies after a hiatus. In addition, the increasing popularity of pixel art in this decade also attracted new artists to the demoscene, who had not been active during the 1990s and 2000s. It is important to note that the demoscene and its graphics competitions never stopped, but the 2010s could nevertheless be considered a period of rejuvenation.

As advanced digital painting software like Photoshop became more widely available and easier to use, the pixel doll community split into a group of artists using traditional pixel art, known as pixel-shaders, and a group of artists using more advanced tools, known as tool-shaders. As internet forums lost popularity and drawn avatars were increasingly replaced by photos in internet communities, the pixel dolling community gradually became less active in this decade.

Perhaps because of the rise of pixel art in video games and social media, pixel art was also seen in other areas of popular culture and even made its way to public museums. Ivan Dixon and Paul Robertson received international attention for making a pixel art version of The Simpsons' introduction sequence. Eboy became well known for their isometric pixel art displays, often used for advertisement or sold as independent art. The work of pixel artists such as Octavi Navarro and Gustavo Viselner was featured in major newspapers and magazines. French urban artist Invader received international attention for his urban pixel art mosaics, seen around the world. Among 1980s-inspired synthwave artists and groups, pixel art was used to make music videos, such as Valenberg's work for Perturbator and a Gunship music video by pixel artists Jason Tammemagi, Gyhyom, Mary Safro, and Waneella. In 2012, the Smithsonian Institution museum of Washington created an exhibition called The Art of Video Games, attended by almost 700,000 people.

2020s
Although some commenters had predicted the decline of "retro" pixel art after a wave of pixel art games in the 2010s, pixel art has continued to remain popular in the current decade. One major contributing factor seems to have been the COVID-19 pandemic, as people around the world spent more time online, playing games, using social media and developing new hobbies. A second contributing factor seems to have been the rise of NFTs, as pixel art became an inexpensive way to produce large numbers of artworks. As the market for NFTs grew rapidly, many pixel artists embraced the new technology as a new source of income. Many notable professional artists also expressed skepticism and disapproval of NFTs, citing environmental factors, pyramid scheme claims, and money laundering as reasons for rejecting offers. One of the most profiled examples of pixel artists expressing opposition to NFTs is Castpixel, citing environmental effects and artificial scarcity. Furthermore, as is the case with other forms of digital art, pixel artists have also discovered many cases of plagiarism and fraud by actors who download pixel art without permission to sell as NFTs. Although the discussion around pixel art theft is not new, the issue has certainly become more controversial with the rise of NFTs.

Perhaps linked to the increased influence and attraction of social media platforms like Twitter, Mastodon, Reddit, and Instagram, older pixel art communities on Pixelation and DeviantArt declined in the 2020s. Discord became an important platform for pixel art communities like Pixel Joint, PAD (Pixel Art Discord), Lospec, and Trigonomicon. In the early fall of 2022, Pixelation announced the closing of its webforum and a transition to Twitter.

Pixel art continued to be a popular style in games across platforms, with releases such as Eastward (2021), Teenage Mutant Ninja Turtles: Shredder's Revenge (2022), Songs of Conquest (2022), Vikings on Trampolines (2022), and Loop Hero (2021), Pizza Tower (2023) Some argued that the recent wave of pixel art games, released thirty years after the release of the SNES, was largely influenced by nostalgia and freedom from publishers, responding to unoriginal stories in modern video game blockbusters and wanting to integrate contemporary themes, such as ecological concerns and representation of queer characters.

Techniques
Pixel art typically involves more careful and deliberate placement of pixels compared to other forms of digital art. Artists use different brush sizes (e.g. 1x1 or 2x2 pixels) and a variety of tools, such as free-hand/pencil, lines, and rectangles. One key difference between pixel art and other digital art is that pixel artists tend to apply a single color at a time, avoiding tools such as soft edges, smudging, and blurring. Agreeing on which tools and techniques are considered non-pixel art (NPA) is something that has caused considerable disagreement in the past. For example, applying layers with different filters that adjust hue or light values is considered a non-pixel art technique, but the resulting art may still be considered pixel art if there's a clear foundation of deliberate placement of pixels. To preserve the careful pixel placement, pixel art is preferably stored in a file format utilizing lossless data compression. The JPEG format, for example, is often avoided because of its compression, as it introduces visible artifacts in the image.

Different restrictions are central to pixel art, and these are often traced back to technical limitations of hardware such as Amiga, Commodore 64, NES, and early computers. The two most common restrictions are resolution and palette, and pixel artists often work with a significantly reduced canvas size and number of colors compared to other digital artists. (Lee, p. 276) Examples include Commodore 64 restrictions (320x200 resolution, 16 colors), original ZX Spectrum restrictions (256x192 pixels, 15 colors), and GameBoy restrictions (160x144 pixels, 4 colors).

Some artists start the process of pixelling by drawing line art, while others begin by blocking in simple shapes and big clusters. Traditionally, pixel art was done on a single layer, similar to painting on a canvas, but modern software permits work with multiple layers, potentially adding animation or transparency to the foreground while keeping a static background. To deal with the various restrictions of pixel art, such as reduced resolution or colors, pixel artists have traditionally employed several techniques specific to the art form. One widely used technique is dithering, commonly using noise or a repeating pattern such as a checkerboard or lines to create a third color when seen from a distance. Other common techniques include anti-aliasing (AA) where strong edges are softened by manually placing pixels, and sub-pixelling, changing the colors of pixels to create an illusion of movement.

Styles
Because pixel art is a flexible medium, pixel artists can often imitate styles from traditional media, such as impressionism or pointillism in oil painting or cross-hatching in embroidery. Pixel art is not a style itself, but a broad artistic medium. However, there are different styles within pixel art that revolve around pixel placement. One common distinction in style is the use of clusters. A cluster is a field of connected pixels with the same color. The opposite of a cluster is an isolated pixel (1x1) surrounded by pixels of other colors. Cluster size has become an important marker of pixel art styles, where some pixel art uses large clusters and avoids isolated (orphan) pixels, whereas other works use clusters of all sizes in combination with isolated pixels, or even rely primarily on isolated pixels and small clusters for a more grainy, noisy texture. Styles that rely more on small clusters and isolated pixels often have similarities with pointillism and impressionism, whereas pixel art with larger clusters and geometric shapes has similarities with cubism and suprematism. Although some artists have been known for championing a particular style, many pixel artists alternate between different styles in their works.

Genres and subjects
Pixel art is a flexible medium and shares traditions of figure drawing, landscapes, and abstract with traditional media. However, certain genres have emerged as particularly popular. These genres often overlap, but include:
 * Isometric: Pixel art drawn in a near-isometric dimetric projection. Originally used in games to provide a three-dimensional view without using any real three-dimensional processing. Normally drawn with a 1:2 pixel ratio along the X and Y-axis.
 * Sprites: Characters and game assets typically drawn in the fashion of platformer games and fighting games such as Streets of Rage or Street Fighter II or JRPGs (Japanese Roleplaying Game) such as Final Fantasy.
 * Pixel dolls: Also known as dollz. Sets of pixel art characters, usually with customizable appearance and sets of clothing. Often used as avatars for chatting and forums, or in dress-up games.
 * Mock-ups: Concepts for non-existing pixel art games, including a user interface and other game elements. Retro-style mock-ups based on modern games are called 'demakes'.

Software
Essentially all raster graphics editors can be used in some way for pixel art, some of which include features designed to make the process easier. See Comparison of raster graphics editors for a list of notable raster graphics editors. The following notable programs were designed specifically to facilitate drawing pixel art.

title: Pixel art


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Flow cytometry

Flow cytometry (FC) is a technique used to detect and measure the physical and chemical characteristics of a population of cells or particles.

In this process, a sample containing cells or particles is suspended in a fluid and injected into the flow cytometer instrument. The sample is focused to ideally flow one cell at a time through a laser beam, where the light scattered is characteristic to the cells and their components. Cells are often labeled with fluorescent markers so light is absorbed and then emitted in a band of wavelengths. Tens of thousands of cells can be quickly examined and the data gathered are processed by a computer.

Flow cytometry is routinely used in basic research, clinical practice, and clinical trials. Uses for flow cytometry include:
 * Cell counting
 * Cell sorting
 * Determining cell characteristics and function
 * Detecting microorganisms
 * Biomarker detection
 * Protein engineering detection
 * Diagnosis of health disorders such as blood cancers
 * Measuring genome size

A flow cytometry analyzer is an instrument that provides quantifiable data from a sample. Other instruments using flow cytometry include cell sorters which physically separate and thereby purify cells of interest based on their optical properties.

History
The first impedance-based flow cytometry device, using the Coulter principle, was disclosed in U.S. Patent 2,656,508, issued in 1953, to Wallace H. Coulter. Mack Fulwyler was the inventor of the forerunner to today's flow cytometers – particularly the cell sorter. Fulwyler developed this in 1965 with his publication in Science. The first fluorescence-based flow cytometry device (ICP 11) was developed in 1968 by Wolfgang Göhde from the University of Münster, filed for patent on 18 December 1968 and first commercialized in 1968/69 by German developer and manufacturer Partec through Phywe AG in Göttingen. At that time, absorption methods were still widely favored by other scientists over fluorescence methods. Soon after, flow cytometry instruments were developed, including the Cytofluorograph (1971) from Bio/Physics Systems Inc. (later: Ortho Diagnostics), the PAS 8000 (1973) from Partec, the first FACS (fluorescence-activated cell sorting) instrument from Becton Dickinson (1974), the ICP 22 (1975) from Partec/Phywe and the Epics from Coulter (1977/78). The first label-free high-frequency impedance flow cytometer based on a patented microfluidic "lab-on-chip", Ampha Z30, was introduced by Amphasys (2012).

Name of the technology
The original name of the fluorescence-based flow cytometry technology was "pulse cytophotometry" (German: Impulszytophotometrie), based on the first patent application on fluorescence-based flow cytometry. At the 5th American Engineering Foundation Conference on Automated Cytology in Pensacola (Florida) in 1976 – eight years after the introduction of the first fluorescence-based flow cytometer (1968) – it was agreed to commonly use the name "flow cytometry", a term that quickly became popular.

Flow cytometers

Modern flow cytometers are able to analyze many thousands of particles per second, in "real time" and, if configured as cell sorters, can actively separate and isolate particles with specified optical properties at similar rates. A flow cytometer is similar to a microscope, except that, instead of producing an image of the cell, flow cytometry offers high-throughput, automated quantification of specified optical parameters on a cell-by-cell basis. To analyze solid tissues, a single-cell suspension must first be prepared.

A flow cytometer has five main components: a flow cell, a measuring system, a detector, an amplification system, and a computer for analysis of the signals. The flow cell has a liquid stream (sheath fluid), which carries and aligns the cells so that they pass single file through the light beam for sensing. The measuring system commonly uses measurement of impedance (or conductivity) and optical systems – lamps (mercury, xenon); high-power water-cooled lasers (argon, krypton, dye laser); low-power air-cooled lasers (argon (488 nm), red-HeNe (633 nm), green-HeNe, HeCd (UV)); diode lasers (blue, green, red, violet) resulting in light signals. The detector and analog-to-digital conversion (ADC) system converts analog measurements of forward-scattered light (FSC) and side-scattered light (SSC) as well as dye-specific fluorescence signals into digital signals that can be processed by a computer. The amplification system can be linear or logarithmic.

The process of collecting data from samples using the flow cytometer is termed "acquisition". Acquisition is mediated by a computer physically connected to the flow cytometer, and the software which handles the digital interface with the cytometer. The software is capable of adjusting parameters (e.g., voltage, compensation) for the sample being tested, and also assists in displaying initial sample information while acquiring sample data to ensure that parameters are set correctly. Early flow cytometers were, in general, experimental devices, but technological advances have enabled widespread applications for use in a variety of both clinical and research purposes. Due to these developments, a considerable market for instrumentation, analysis software, as well as the reagents used in acquisition such as fluorescently labeled antibodies have been developed.

Modern instruments usually have multiple lasers and fluorescence detectors. The current record for a commercial instrument is ten lasers and 30 fluorescence detectors. Increasing the number of lasers and detectors allows for multiple antibody labeling, and can more precisely identify a target population by their phenotypic markers. Certain instruments can even take digital images of individual cells, allowing for the analysis of fluorescent signal location within or on the surface of cells.

Fluidics system of a flow cytometer
Cells must pass uniformly through the center of focused laser beams to accurately measure optical properties of cells in any flow cytometer. The purpose of the fluidic system is to move the cells one by one through the lasers beam and throughout the instrument. Fluidics in a flow cytometer with cell sorting capabilities also use the stream to carry sorted cells into collection tubes or wells.

Hydrodynamic focusing
For precise positioning of cells in a liquid jet, hydrodynamic focusing is used in most cytometers. The cells in suspension enter into the instrument enclosed by an outer sheath fluid. The sample core is maintained in the center of the sheath fluid. The sample input rate or how fast the cells flow through to the laser interrogation can be controlled by the pressure of the sheath fluid on the sample core. Under optimal conditions, the central fluid stream and sheath fluid do not mix.

Acoustic-assisted hydrodynamic focusing
Acoustic focusing technology is used in some flow cytometers to support hydrodynamic focusing. Acoustic waves (>2 MHz) pre-focus the sample before introduction to sheath fluid. The pre-focused sample is then injected into the hydrodynamic core and flowed through the instrument. This may help with increasing data accuracy under high sample input rates.

Optical filters
Light emitted from fluorophores are in a spectrum of wavelengths, so combining multiple fluorophores may cause overlap. To add specificity, optical filters and dichroic mirrors are used to filter and move light to the detectors such as photomultiplier tubes (PMTs) or avalanche photodiodes (APD). Optical filters are designed as band pass (BP), long pass (LP), or short pass (SP) filters. Most flow cytometers uses dichroic mirrors and band pass filters to select specific bands of the optical spectrum.

Prisms, gratings, and spectral flow cytometry
Spectral flow cytometry uses prisms or diffraction gratings to disperse the emitted light of a marker across a detector array. This allows for the full spectra from each particle to be measured. The measured spectra from single cells are subsequently unmixed by using reference spectra of all used dyes and the autofluorescence spectrum. This may allow for a wider panel design and the application of new biological markers.

Imaging flow cytometry
Imaging flow cytometry (IFC) captures multichannel images of cells. Detectors used in imaging platforms can be equipped with charge-coupled device (CCD) or complementary metal–oxide–semiconductor (CMOS) to capture images of individual cells.

Compensation
Each fluorochrome has a broad fluorescence spectrum. When more than one fluorochrome is used, the overlap between fluorochromes can occur. This situation is called spectrum overlap. This situation needs to be overcome. For example, the emission spectrum for FITC and PE is that the light emitted by the fluorescein overlaps the same wavelength as it passes through the filter used for PE. This spectral overlap is corrected by removing a portion of the FITC signal from the PE signals or vice versa. This process is called color compensation, which calculates a fluorochrome as a percentage to measure itself.

Compensation is the mathematical process by which spectral overlap of multiparameter flow cytometric data is corrected. Since fluorochromes can have wide-ranging spectrum, they can overlap, causing the undesirable result of confusion during the analysis of data. This overlap, known as spillover and quantified in the spillover coefficient, is usually caused by detectors for a certain fluorochrome measuring a significant peak in wavelength from a different fluorochrome. Linear algebra is most often used to make this correction.

In general, when graphs of one or more parameters are displayed, it is to show that the other parameters do not contribute to the distribution shown. Especially when using the parameters which are more than double, this problem is more severe. Currently, no tools have been discovered to efficiently display multidimensional parameters. Compensation is very important to see the distinction between cells.

Gating
The data generated by flow cytometers can be plotted in a single dimension, to produce a histogram, or in two-dimensional dot plots, or even in three dimensions. The regions on these plots can be sequentially separated, based on fluorescence intensity, by creating a series of subset extractions, termed "gates." Specific gating protocols exist for diagnostic and clinical purposes, especially in relation to hematology. Individual single cells are often distinguished from cell doublets or higher aggregates by their "time-of-flight" (denoted also as a "pulse-width") through the narrowly focused laser beam

The plots are often made on logarithmic scales. Because different fluorescent dyes' emission spectra overlap, signals at the detectors have to be compensated electronically as well as computationally. Data accumulated using the flow cytometer can be analyzed using software. Once the data is collected, there is no need to stay connected to the flow cytometer and analysis is most often performed on a separate computer. This is especially necessary in core facilities where usage of these machines is in high demand.

Computational analysis
Recent progress on automated population identification using computational methods has offered an alternative to traditional gating strategies. Automated identification systems could potentially help findings of rare and hidden populations. Representative automated methods include FLOCK in Immunology Database and Analysis Portal (ImmPort), SamSPECTRAL and flowClust  in Bioconductor, and FLAME in GenePattern. T-Distributed Stochastic Neighbor Embedding (tSNE) is an algorithm designed to perform dimensionality reduction, to allow visualization of complex multi-dimensional data in a two-dimensional "map". Collaborative efforts have resulted in an open project called FlowCAP (Flow Cytometry: Critical Assessment of Population Identification Methods, ) to provide an objective way to compare and evaluate the flow cytometry data clustering methods, and also to establish guidance about appropriate use and application of these methods.

FMO controls
Fluorescence minus one (FMO) controls are important for data interpretation when building multi-color panels – in which a cell is stained with multiple fluorochromes simultaneously. FMO controls provide a measure of fluorescence spillover in a given channel and allow for compensation. To generate a FMO control, a sample is stained with all the fluorochromes except the one that is being tested – meaning if you are using 4 different fluorochromes your FMO control must contain only 3 of them (example: fluorochromes – A, B, C, D; FMOs – ABC_, AB_D, A_CD, _BCD).

Cell sorting by flow cytometry
Cell sorting is a method to purify cell populations based on the presence or absence of specific physical characteristics. In flow cytometers with sorting capabilities, the instrument detects cells using parameters including cell size, morphology, and protein expression, and then droplet technology to sort cells and recover the subsets for post-experimental use.

The first prototype sorter was built at the Los Alamos National Laboratory (LANL) in 1965 by physicist Mack J. Fulwyler by joining a Coulter volume sensor with the newly invented ink jet printer. Live cell cell sorter or fluorescence-activated cell sorter (FACS) was generated by Len Herzenberg, who subsequently won the Kyoto Prize in 2006 for his seminal work.

Flow cytometry cell sorters have a collection system unlike flow cytometry analyzers. The collection process starts when a sample is injected into a stream of sheath fluid that passes through the flow cell and laser intercepts. The stream then carries the cell through a vibrating nozzle which generates droplets with most containing either one cell or no cells. An electrical charging ring is placed just at the point where the stream breaks into droplets and a charge is placed on the ring based immediately prior to fluorescence intensity being measured; the opposite charge is trapped on the droplet as it breaks from the stream and the droplets are therefore charged. The charged droplets then fall through an electrostatic deflection system that diverts droplets into containers based on their charge. In some systems, the charge is applied directly to the stream, and the droplet breaking off retains charge of the same sign as the stream. The stream is then returned to neutral after the droplet breaks off. After collecting, these cells can be further cultured, manipulated, and studied.

Labels
Flow cytometry uses the light properties scattered from cells or particles for identification or quantitative measurement of physical properties. Labels, dyes, and stains can be used for multi-parametric analysis (understand more properties about a cell). Immunophenotyping is the analysis of heterogeneous populations of cells using labeled antibodies and other fluorophore containing reagents such as dyes and stains.

Fluorescent labels
A wide range of fluorophores can be used as labels in flow cytometry. Fluorophores, or simply "fluors", are typically attached to an antibody that recognizes a target feature on or in the cell; they may also be attached to a chemical entity with affinity for the cell membrane or another cellular structure. Each fluorophore has a characteristic peak excitation and emission wavelength, and the emission spectra often overlap. Consequently, the combination of labels which can be used depends on the wavelength of the lamp(s) or laser(s) used to excite the fluorochromes and on the detectors available. Flow cytometry uses fluorescence as a quantitative tool; the utmost sensitivity of flow cytometry is unmatched by other fluorescent detection platforms such as confocal microscopy. Absolute fluorescence sensitivity is generally lower in confocal microscopy because out-of-focus signals are rejected by the confocal optical system and because the image is built up serially from individual measurements at every location across the cell, reducing the amount of time available to collect signal.

Quantum dots
Quantum dots are sometimes used in place of traditional fluorophores because of their narrower emission peaks.

Isotope labeling
Mass cytometry overcomes the fluorescent labeling limit by utilizing lanthanide isotopes attached to antibodies. This method could theoretically allow the use of 40 to 60 distinguishable labels and has been demonstrated for 30 labels. Mass cytometry is fundamentally different from flow cytometry: cells are introduced into a plasma, ionized, and associated isotopes are quantified via time-of-flight mass spectrometry. Although this method permits the use of a large number of labels, it currently has lower throughput capacity than flow cytometry. It also destroys the analysed cells, precluding their recovery by sorting.

Cytometric bead array
In addition to the ability to label and identify individual cells via fluorescent antibodies, cellular products such as cytokines, proteins, and other factors may be measured as well. Similar to ELISA sandwich assays, cytometric bead array (CBA) assays use multiple bead populations typically differentiated by size and different levels of fluorescence intensity to distinguish multiple analytes in a single assay. The amount of the analyte captured is detected via a biotinylated antibody against a secondary epitope of the protein, followed by a streptavidin-R-phycoerythrin treatment. The fluorescent intensity of R-phycoerythrin on the beads is quantified on a flow cytometer equipped with a 488 nm excitation source. Concentrations of a protein of interest in the samples can be obtained by comparing the fluorescent signals to those of a standard curve generated from a serial dilution of a known concentration of the analyte. Commonly also referred to as cytokine bead array (CBA).

Impedance flow cytometry
Impedance-based single cell analysis systems are commonly known as Coulter counters. They represent a well-established method for counting and sizing virtually any kind of cells and particles. The label-free technology has recently been enhanced by a "lab-on-a-chip" based approach and by applying high frequency alternating current (AC) in the radio frequency range (from 100 kHz to 30 MHz) instead of a static direct current (DC) or low frequency AC field. This patented technology allows a highly accurate cell analysis and provides additional information like membrane capacitance and viability. The relatively small size and robustness allow battery powered on-site use in the field.

Measurable parameters
Measurable factors in cellular analysis and flow cytometry include a diverse set of traits and indicators that provide important insights into cell biology and function. Flow cytometry techniques can quantify and evaluate these factors, allowing researchers to investigate and analyze various aspects of cells. Here are some important quantifiable parameters that are frequently investigated:
 * Apoptosis: Apoptosis can be quantified by flow cytometry by measuring DNA destruction, mitochondrial membrane potential, permeability alterations, and caspase activity. These measurements reveal important details about planned cell death.
 * Cell adherence: Flow cytometry can be used to investigate cell adherence, such as pathogen-host cell adherence. Researchers can quantify and analyze cell adhesion events by utilizing particular markers or fluorescent tags.
 * Cell pigments: Chlorophyll and phycoerythrin are pigments found in certain cells. The presence and amounts of these pigments can be measured by flow cytometry, providing information about cellular metabolism and physiological states.
 * Cell surface antigens: Flow cytometry is often used to identify and quantify cell surface antigens, also known as Cluster of Differentiation (CD) markers. Researchers can classify cell populations based on surface antigen expression by labeling cells with specific antibodies.
 * Cell viability: Flow cytometry can be used as a cell viability assay by utilizing fluorescent dyes or markers that distinguish between live and dead cells. This parameter is critical in determining cell health and response to experimental or therapeutic settings. Viability of the cells in flow cytometry should be around 95% but not less than 90%.
 * Circulating tumor cells: Flow cytometry is essential for isolating and purifying circulating tumor cells (CTCs) from blood samples. CTCs can be discovered and examined by targeting certain markers or features, assisting in cancer diagnosis, prognosis, and treatment monitoring.
 * Characterizing multidrug resistance (MDR): Flow cytometry can be used to characterize multidrug resistance (MDR) in cancer cells by assessing the efflux of fluorescent dyes or particular markers associated with drug resistance mechanisms. This knowledge aids in the understanding and battling of medication resistance in cancer treatment.
 * Chromosome analysis and sorting: Flow cytometry can help with chromosome analysis and sorting, allowing for the creation of libraries and the identification of specific chromosomes or chromosomal abnormalities.
 * DNA copy number variation: DNA copy number variation can be measured using flow cytometry techniques such as Flow-FISH or BACs-on-Beads technology. These technologies shed light on genetic changes linked to diseases such as cancer.
 * Protein expression and modifications: Using fluorescently labeled antibodies or probes, flow cytometry makes it possible to assess protein expression levels and alterations such as phosphorylation. This parameter contributes to a better understanding of protein function and signaling networks.
 * Membrane fluidity: Flow cytometry can detect membrane fluidity by utilizing fluorescent probes that are sensitive to membrane characteristics. This parameter gives information on the dynamics and function of cell membranes.
 * Total DNA and RNA content: Flow cytometry can measure total DNA and RNA content in cells. This data is useful for cell cycle analyses, proliferation investigations, and determining gene expression changes.
 * Monitoring intracellular parameters: Intracellular factors can be measured by flow cytometry, including pH, intracellular ionized calcium and magnesium levels, membrane potential, glutathione levels, and oxidative burst. These data provide information about cellular metabolism, signaling, and oxidative stress.
 * Light scattering: Forward scatter (FSC) and side scatter (SSC) measures are used in flow cytometry to assess cell volume and morphological complexity, respectively. These metrics describe the size, granularity, and shape of cells.
 * Transgenic products: Flow cytometry is useful for assessing transgenic products in vivo, particularly fluorescent proteins like as green fluorescent protein (GFP) or similar variations. This enables scientists to investigate gene expression, protein localisation, and cellular dynamics.
 * Various combinations: Flow cytometry allows you to integrate many measurable data, such as DNA/surface antigens, to acquire a comprehensive understanding of biological features and functions.

Applications
The technology has applications in a number of fields, including molecular biology, pathology, immunology, virology, plant biology and marine biology. It has broad application in medicine especially in transplantation, hematology, tumor immunology and chemotherapy, prenatal diagnosis, genetics and sperm sorting for sex preselection. Flow cytometry is widely applied to detect sperm cells abnormality associated with DNA fragmentation in male fertility assays. Also, it is extensively used in research for the detection of DNA damage, caspase cleavage and apoptosis. Photoacoustic flow cytometry is used in the study of multi-drug-resistant bacteria (most commonly MRSA) to detect, differentiate, and quantify bacteria in the blood marked with dyed bacteriophages. In neuroscience, co-expression of cell surface and intracellular antigens can also be analyzed. In microbiology, it can be used to screen and sort transposon mutant libraries constructed with a GFP-encoding transposon (TnMHA), or to assess viability. In protein engineering, flow cytometry is used in conjunction with yeast display and bacterial display to identify cell surface-displayed protein variants with desired properties. The main advantages of flow cytometry over histology and IHC is the possibility to precisely measure the quantities of antigens and the possibility to stain each cell with multiple antibodies-fluorophores, in current laboratories around 10 antibodies can be bound to each cell. This is much less than mass cytometer where up to 40 can be currently measured, but at a higher price and a slower pace.

Aquatic research
In aquatic systems, flow cytometry is used for the analysis of autofluorescing cells or cells that are fluorescently-labeled with added stains. This research started in 1981 when Clarice Yentsch used flow cytometry to measure the fluorescence in a red tide producing dinoflagellate. The next year researchers published flow cytometric measurements of multiple algal species which could be distinguished based on their fluorescence characteristics. By 1983, marine researchers were assembling their own flow cytometers or using commercially available flow cytometers on seawater samples collected off Bermuda to demonstrate that phytoplankton cells could be distinguished from non-living material and that cyanobacteria could be sorted from a mixed community and subsequently cultured in the lab. Flow cytometry also allowed marine researchers to distinguish between dimly-fluorescing Prochlorococcus and heterotrophic microorganisms, a distinction that is difficult with microscopy-based assessments. Advances in technology now allow aquatic scientists to use flow cytometers continuously during research cruises and flow cytometers are used to provide images of individual phytoplankton cells. Marine scientists use the sorting ability of flow cytometers to make discrete measurements of cellular activity and diversity, to conduct investigations into the mutualistic relationships between microorganisms that live in close proximity, and to measure biogeochemical rates of multiple processes in the ocean.

Cell proliferation assay
Cell proliferation is the major function in the immune system. Often it is required to analyse the proliferative nature of the cells in order to make some conclusions. One such assay to determine the cell proliferation is the tracking dye carboxyfluorescein diacetate succinimidyl ester (CFSE). It helps to monitor proliferative cells. This assay gives quantitative as well as qualitative data during time-series experiments. This dye binds covalently with the long-lived molecules present inside the cell. When the cells divide, the molecules divide too and, the daughter cells possess half the dye than the parent population. This decrease in the intensity can be visualized by flow cytometry. In literature, this powerful technique of flow cytometry and CFSE has been used to find the efficiency of T-cells in killing the target cells in cancer such as leukemia. In order to visualize the target cell death, both rapid and slow, scientists have used CFSE labelling with antibody staining of certain kinds of cells and fluorescently labelled microbeads. This also gave information regarding the proliferation of the target cells upon the treatment of certain cytokines.

Measuring genome size
Flow cytometry has been used to measure genome sizes, or more precisely: the amount of DNA in a cell or nucleus. Although genomes can be analyzed with more precision by genome sequencing, this is often difficult due to a high fraction of micro-chromosomes or repetitive sequences which may be missed by sequencing (or which get filtered out during the analysis step when they cannot be assigned to chromosomes). However, flow cytometry is not perfect either. The resulting genome sizes may differ based on the dye used. An analysis of fish genomes resulted in significantly different genome sizes when propidium iodide (PI) and DAPI were used, respectively. For instance, the genome of Anguilla japonica was found to contain 1.09 pg of DNA with PI vs. 1.25 pg with DAPI. Similarly, the genome of Myxocyprinus asiaticus was found to contain 2.75 pg of DNA (PI) vs. 3.08 pg (DAPI). That is, the differences were on the order of 12–14%.

license: Creative Commons - Attribution Share-Alike - https://creativecommons.org/licenses/by-sa/4.0/

title: Flow cytometry


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Talk:List of television shows set in London

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Hi. What is keen eddie - we don't have it here. Secretlondon 21:49, Mar 2, 2004 (UTC)

 * I started the list on the principle that London is at least as important as New York, L.A., or D.C., particularly to Londoners. However, from where I sit in New York I could think of very few shows (not that I didn't watch Absolutely Fabulous fairly regularly, it just slipped my mind for some reason). Anyway, Keen Eddie is an original (and I use the term loosely) show on Bravo. It's American, best I can tell, so it's peopled more by stereotypes than actual Londoners.

 * So you're not missing much. --Charles A. L. 04:21, Mar 3, 2004 (UTC)


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Talk:Multiple agent system

Multiple agent system and Multi-agent system are about the same topic. They should be merged and cleaned up.

How do I add keywords so when I search for MAS or for Multiple Agent System (not Systems) I still land on this page?

What's with this weird pseudocode? How are we supposed to interpret it?

title: Talk:Multiple agent system


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Talk:Slow play

Article rewrite
I boldly rewrote the article. I'm still not particularly happy with it, but at least it has more of an encyclopedic tone about it now. Can anyone find a reference for fishing for overcalls? I'm not sure the topic deserves inclusion in the article. Opinions? Also, I think the term "slow play" is far more common today than "sandbag". Any consensus on renaming the article to "Slow playing (poker)"?--Toms2866 18:40, 6 May 2006 (UTC)

 * Seems like a good idea. Slow playing and slow play is far more common.  However there are existing redirects from Slowplay and Slowplaying, so perhaps one of them should be chosen. Scrunching the words together is done.  On the other hand, Slow playing (poker) uses the words separately and is a normal structure so I'd support the move if you want to do that -- also setting up a redirect from Slow play (poker). 2005 19:36, 6 May 2006 (UTC)

 * I moved the article from "Sandbagging (poker)" to "Slow play (poker)". I picked Slow play instead of Slow playing to stay consistent with the other poker articles, which tend not to use the "ing" ending: Draw (poker), Check-raise (poker), Isoloation (poker), etc. The page's edit history and talk page moved over fine. I fixed the double redirects.--Toms2866 21:09, 6 May 2006 (UTC)

Check raising as a slow play
Sandbagging is NOT the same as slowplaying, one is not a "special case" of the other. This is made clear on the link provided to Sklansky. He says they are not identical. In fact, it's worse than that -- they're not even similar. A slowplay is an attempt to KEEP people in a pot, when you have what you think is a very strong hand that will almost certainly win. A check-raise is most often used to KNOCK PLAYERS OUT of a pot, when you think you have a strong hand, but not a VERY strong hand. This goal is exactly the opposite goal of a slowplay. I think this really needs to be cleared up (Sklansky's book is a good place to start). Revolver 04:27, 3 Mar 2004 (UTC)

 * I agree that Sklansky differentiates between check-raising and slowplaying as you described. There's a separate article for sandbagging (poker), so I'm inclined to mention the difference between slow-playing and check-raising and reference the check-raise article.--Toms2866 21:20, 30 April 2006 (UTC)

title: Talk:Slow play