question: Why is it that when looking at microscopic things, they always look two dimensional
comment: because the gap between in focus and out of focus is a really thin 2d plane, and you can only clearly see things in that area
passages: In static image acquisition only one field of view image is captured at a time. If the user wishes to image other portions of the same sample on the slide, they can use the X-Y positioning hardware (typically composed of two linear stages on the microscope to move to a different area of the slide. Care must be taken to insure that two images do not overlap so as not to count and measure the same particles more than once.
   Most modern instruments provide simple solutions for micro-photography and image recording electronically. However such capabilities are not always present and the more experienced microscopist will, in many cases, still prefer a hand drawn image to a photograph. This is because a microscopist with knowledge of the subject can accurately convert a three-dimensional image into a precise two-dimensional drawing. In a photograph or other image capture system however, only one thin plane is ever in good focus.
   Section::::Analysis.
  
  Analysis of images will vary considerably according to application. Typical analysis includes determining where the edges of an object are, counting similar objects, calculating the area, perimeter length and other useful measurements of each object. A common approach is to create an image mask which only includes pixels that match certain criteria, then perform simpler scanning operations on the resulting mask. It is also possible to label objects and track their motion over a series of frames in a video sequence.
  
  Section::::See also.
  
  BULLET::::- Image processing
  
  Section::::References.
  
  BULLET::::- Jan-Mark Geusebroek, Color and Geometrical Structure in Images, Applications in Microscopy,
   BULLET::::- "Dehydration" – or replacement of water with organic solvents such as ethanol or acetone, followed by critical point drying or infiltration with embedding resins. Also freeze drying.
   Section::::Microstructure Characterizations.
  
  To quantify microstructural features, both morphological and material property must be characterized. Image processing is a robust technique for determination of morphological features such as volume fraction, inclusion morphology, void and crystal orientations. To acquire micrographs, optical as well as electron microscopy are commonly used. 
   The large working distance at low magnification is useful in examining large solid objects such as fracture surfaces, especially using fibre-optic illumination as discussed below. Such samples can also be manipulated easily so as to determine the points of interest. There are severe limitations on sample size in scanning electron microscopy, as well as ease of manipulation in the specimen chamber.
  
  Section::::Magnification.
   There are several overall categories of methods used to characterize nanoparticles. Microscopy methods generate images of individual nanoparticles to characterize their shape, size, and location. Electron microscopy and scanning probe microscopy are the dominant methods. Because nanoparticles have a size below the diffraction limit of visible light, conventional optical microscopy is not useful. Electron microscopes can be coupled to spectroscopic methods that can perform elemental analysis. Microscopy methods are destructive, and can be prone to undesirable artifacts from sample preparation, or from probe tip geometry in the case of scanning probe microscopy. Additionally, microscopy is based on single-particle measurements, meaning that large numbers of individual particles must be characterized to estimate their bulk properties.
   Moreover, one must also consider the temporal resolution requirements of the application. A lower resolution detector will often have a significantly higher acquisition rate, permitting the observation of faster events. Conversely, if the observed object is motionless, one may wish to acquire images at the highest possible spatial resolution without regard to the time required to acquire a single image.
  
  Section::::2D image techniques.
   The ultrastructure of nanocellulose derived from various sources has been extensively studied. Techniques such as transmission electron microscopy (TEM), scanning electron microscopy (SEM), atomic force microscopy (AFM), wide angle X-ray scattering (WAXS), small incidence angle X-ray diffraction and solid state C cross-polarization magic angle spinning (CP/MAS), nuclear magnetic resonance (NMR) and spectroscopy have been used to characterize typically dried nanocellulose morphology.
   A macroscopic view of a ball is just that: a ball. A microscopic view could reveal a thick round skin seemingly composed entirely of puckered cracks and fissures (as viewed through a microscope) or, further down in scale, a collection of molecules in a roughly spherical shape. An example of a physical theory that takes a deliberately macroscopic viewpoint is thermodynamics. An example of a topic that extends from macroscopic to microscopic viewpoints is histology.
   Section::::History.
   (By contrast, traditional optical microscopy, which is not appropriate for the "direct" observation of dislocations, typically offers magnifications up to a maximum of only around 2000 times).
   BULLET::::- "Replication" – A surface shadowed with metal (e.g. platinum, or a mixture of carbon and platinum) at an angle is coated with pure carbon evaporated from carbon electrodes at right angles to the surface. This is followed by removal of the specimen material (e.g. in an acid bath, using enzymes or by mechanical separation) to produce a surface replica that records the surface ultrastructure and can be examined using transmission electron microscopy.
   Section::::Characterization of 2D materials.
  
  Microscopy techniques such as transmission electron microscopy, 3D electron diffraction, scanning probe microscopy, scanning tunneling microscopy, and atomic force microscopy are used to characterize the thickness and size of the 2D materials. Electrical properties and structural properties such as composition and defects are characterized by raman spectroscopy, x-ray diffraction, and x-ray photoelectron spectroscopy.
  
  Section::::Applications.
   By convention, the microscopic scale also includes classes of objects that are most commonly too small to see but of which some members are large enough to be observed with the eye. Such groups include the "Cladocera", planktonic green algae of which "Volvox" is readily observable, and the protozoa of which "stentor" can be easily seen without aid.
  
  The submicroscopic scale similarly includes objects that are too small to see with an optical microscope.
  
  Section::::Thermodynamics.
   Section::::Image acquisition methods.:Static imaging particle analysis.
  
  Static image acquisition is the most common form. Almost all microscopes can be easily adapted to accept a digital camera via a C mount adaptor. This type of set-up is often referred to as a digital microscope, although many systems using that name are used only for displaying an image on a monitor.
   The achievable thickness of the focal plane is defined mostly by the wavelength of the used light divided by the numerical aperture of the objective lens, but also by the optical properties of the specimen. The thin optical sectioning possible makes these types of microscopes particularly good at 3D imaging and surface profiling of samples.
  
  Successive slices make up a 'z-stack', which can either be processed to create a 3D image, or it is merged into a 2D stack (predominately the maximum pixel intensity is taken, other common methods include using the standard deviation or summing the pixels).
   Given the above, the primary method for imaging particle analysis is using optical microscopy. While optical microscopes have been around and used for particle analysis since the 1600s, the "analysis" in the past has been accomplished by humans using the human visual system. As such, much of this analysis is subjective, or qualitative in nature. Even when some sort of qualitative tools are available, such as a measuring reticle in the microscope, it has still required a human to determine and record those measurements.
   Although similar physics guides the focusing process, Scanning probe microscopes and scanning electron microscopes are not typically discussed in the context of optical sectioning as these microscopes only interact with the surface of the sample. 
  
  Total internal reflection microscopy is a fluorescent microscopy technique, which intentionally restricts observation to either the top or bottom surfaces of a sample, but with extremely high depth resolution.
  
  3D imaging using a combination of focal sectioning and tilting has been demonstrated theoretically and experimentally in order to provide exceptional 3D resolution over large fields of view.
  
  Section::::Alternatives.
   The creation of careful and accurate micrographs requires a microscopical technique using a monocular eyepiece. It is essential that both eyes are open and that the eye that is not observing down the microscope is instead concentrated on a sheet of paper on the bench besides the microscope. With practice, and without moving the head or eyes, it is possible to accurately record the observed details by tracing round the observed shapes by simultaneously "seeing" the pencil point in the microscopical image.
   Another common requirement is to take a series of images at a fixed position, but at different focal depths. Since most microscopic samples are essentially transparent, and the depth of field of the focused sample is exceptionally narrow, it is possible to capture images "through" a three-dimensional object using 2D equipment like confocal microscopes. Software is then able to reconstruct a 3D model of the original sample which may be manipulated appropriately. The processing turns a 2D instrument into a 3D instrument, which would not otherwise exist. In recent times this technique has led to a number of scientific discoveries in cell biology.
   The visualization of single molecules, single cells, biological tissues and nanomaterials is an important and attractive approach in analytical science. Also, hybridization with other traditional analytical tools is revolutionizing analytical science. Microscopy can be categorized into three different fields: optical microscopy, electron microscopy, and scanning probe microscopy. Recently, this field is rapidly progressing because of the rapid development of the computer and camera industries.
  
  Section::::Instrumental methods.:Lab-on-a-chip.
   Small, stable specimens such as carbon nanotubes, diatom frustules and small mineral crystals (asbestos fibres, for example) require no special treatment before being examined in the electron microscope. Samples of hydrated materials, including almost all biological specimens have to be prepared in various ways to stabilize them, reduce their thickness (ultrathin sectioning) and increase their electron optical contrast (staining). These processes may result in "artifacts", but these can usually be identified by comparing the results obtained by using radically different specimen preparation methods. Since the 1980s, analysis of cryofixed, vitrified specimens has also become increasingly used by scientists, further confirming the validity of this technique.
   Section::::Commonly Used Methods.:Squashes.
  
  Squashes are methods, in which objects are crushed with force. This method is suitable for preparing both transparent and tender tissues. When preparing squashes slides, specimens are supposed to be thin and transparent so that objects can be observed clearly under microscopes.
   Section::::Career.:Electron microscopy.
  