Scanning electron microscope image ofpollen

Microscopic examination in a biochemicallaboratory

MicroscopyMicroscopy is the technical field of using microscopes to viewobjects and areas of objects that cannot be seen with the nakedeye (objects that are not within the resolution range of thenormal eye).[1] There are three well-known branches ofmicroscopy: optical, electron, and scanning probe microscopy,along with the emerging field of X-ray microscopy.

Optical microscopy and electron microscopy involve thediffraction, reflection, or refraction of electromagneticradiation/electron beams interacting with the specimen, and thecollection of the scattered radiation or another signal in order tocreate an image. This process may be carried out by wide-fieldirradiation of the sample (for example standard lightmicroscopy and transmission electron microscopy) or byscanning a fine beam over the sample (for example confocallaser scanning microscopy and scanning electron microscopy).Scanning probe microscopy involves the interaction of ascanning probe with the surface of the object of interest. Thedevelopment of microscopy revolutionized biology, gave rise tothe field of histology and so remains an essential technique inthe life and physical sciences. X-ray microscopy is three-dimensional and non-destructive, allowing for repeated imagingof the same sample for in situ or 4D studies, and providing theability to "see inside" the sample being studied beforesacrificing it to higher resolution techniques. A 3D X-raymicroscope uses the technique of computed tomography(microCT), rotating the sample 360 degrees and reconstructingthe images. CT is typically carried out with a flat panel display.A 3D X-ray microscope employs a range of objectives, e.g.,from 4X to 40X, and can also include a flat panel.

HistoryOptical microscopy

LimitationsTechniques

Bright fieldOblique illuminationDark fieldDispersion stainingPhase contrastDifferential interference contrastInterference reflection

Contents

FluorescenceConfocalSingle plane illumination microscopy and light sheet fluorescence microscopyWide-field multiphoton microscopyDeconvolution

Sub-diffraction techniquesSerial time-encoded amplified microscopyExtensionsOther enhancementsX-ray

Electron microscopyScanning probe microscopy

Ultrasonic force

Ultraviolet microscopyInfrared microscopyDigital holographic microscopyDigital pathology (virtual microscopy)Laser microscopyPhotoacoustic microscopyAmateur microscopyApplication in forensic scienceSee alsoReferencesFurther readingExternal links

The field of microscopy (optical microscopy) dates back to at least the 17th-century. Earlier microscopes,single lens magnifying glasses with limited magnification, date at least as far back as the wide spread use oflenses in eyeglasses in the 13th century[3] but more advanced compound microscopes first appeared inEurope around 1620[4][5] The earliest practitioners of microscopy include Galileo Galilei, who found in1610 that he could close focus his telescope to view small objects close up[6][7] and Cornelis Drebbel, whomay have invented the compound microscope around 1620[8][9] Antonie van Leeuwenhoek developed avery high magnification simple microscope in the 1670s and is often considered to be the firstacknowledged microscopist and microbiologist.[2][10]

Optical or light microscopy involves passing visible light transmitted through or reflected from the samplethrough a single lens or multiple lenses to allow a magnified view of the sample.[11] The resulting image canbe detected directly by the eye, imaged on a photographic plate, or captured digitally. The single lens withits attachments, or the system of lenses and imaging equipment, along with the appropriate lighting

History

Optical microscopy

Often considered to be the firstacknowledged microscopist andmicrobiologist, Antonie van Leeuwenhoekis best known for his pioneering work inthe field of microscopy and for hiscontributions toward the establishment ofmicrobiology as a scientific discipline.[2]

Stereo microscope

equipment, sample stage, and support, makes up the basic lightmicroscope. The most recent development is the digitalmicroscope, which uses a CCD camera to focus on the exhibitof interest. The image is shown on a computer screen, so eye-pieces are unnecessary.

Limitations of standard optical microscopy (bright fieldmicroscopy) lie in three areas;

This technique can only image dark or stronglyrefracting objects effectively.Diffraction limits resolution to approximately0.2 micrometres (see: microscope). This limits thepractical magnification limit to ~1500x.Out-of-focus light from points outside the focal planereduces image clarity.

Live cells in particular generally lack sufficient contrast to bestudied successfully, since the internal structures of the cell arecolorless and transparent. The most common way to increasecontrast is to stain the different structures with selective dyes,but this often involves killing and fixing the sample. Stainingmay also introduce artifacts, which are apparent structuraldetails that are caused by the processing of the specimen andare thus not legitimate features of the specimen. In general,these techniques make use of differences in the refractive indexof cell structures. Bright field microscopy is comparable tolooking through a glass window: one sees not the glass butmerely the dirt on the glass. There is a difference, as glass is adenser material, and this creates a difference in phase of thelight passing through. The human eye is not sensitive to thisdifference in phase, but clever optical solutions have beendevised to change this difference in phase into a difference inamplitude (light intensity).

In order to improve specimen contrast or highlight certainstructures in a sample, special techniques must be used. A hugeselection of microscopy techniques are available to increasecontrast or label a sample.

Four examples of transillumination techniques usedto generate contrast in a sample of tissue paper.

1.559 μm/pixel.

Limitations

Techniques

Bright fieldillumination, samplecontrast comes fromabsorbance of lightin the sample.

Cross-polarized lightillumination, samplecontrast comes fromrotation of polarizedlight through thesample.

Dark fieldillumination, samplecontrast comes fromlight scattered bythe sample.

Phase contrastillumination, samplecontrast comes frominterference ofdifferent pathlengths of lightthrough the sample.

Bright field microscopy is the simplest of all the light microscopy techniques. Sample illumination is viatransmitted white light, i.e. illuminated from below and observed from above. Limitations include lowcontrast of most biological samples and low apparent resolution due to the blur of out-of-focus material. Thesimplicity of the technique and the minimal sample preparation required are significant advantages.

The use of oblique (from the side) illumination gives the image a three-dimensional (3D) appearance andcan highlight otherwise invisible features. A more recent technique based on this method is Hoffmann'smodulation contrast, a system found on inverted microscopes for use in cell culture. Oblique illuminationsuffers from the same limitations as bright field microscopy (low contrast of many biological samples; lowapparent resolution due to out of focus objects).

Dark field microscopy is a technique for improving the contrast of unstained, transparent specimens.[12]

Dark field illumination uses a carefully aligned light source to minimize the quantity of directly transmitted(unscattered) light entering the image plane, collecting only the light scattered by the sample. Dark field candramatically improve image contrast – especially of transparent objects – while requiring little equipmentsetup or sample preparation. However, the technique suffers from low light intensity in final image of manybiological samples and continues to be affected by low apparent resolution.

Rheinberg illumination is a special variant of dark field illumination in which transparent, colored filters areinserted just before the condenser so that light rays at high aperture are differently colored than those at lowaperture (i.e., the background to the specimen may be blue while the object appears self-luminous red).Other color combinations are possible, but their effectiveness is quite variable.[13]

Bright field

Oblique illumination

Dark field

Dispersion staining

A diatom under Rheinberg illumination

Phase-contrast light micrograph ofundecalcified hyaline cartilage showingchondrocytes and organelles, lacunae andextracellular matrix.

Dispersion staining is an optical technique that results in acolored image of a colorless object. This is an optical stainingtechnique and requires no stains or dyes to produce a coloreffect. There are five different microscope configurations usedin the broader technique of dispersion staining. They includebrightfield Becke line, oblique, darkfield, phase contrast, andobjective stop dispersion staining.

In electron microscopy: Phase-contrast imaging

More sophisticated techniques will show proportionaldifferences in optical density. Phase contrast is a widely usedtechnique that shows differences in refractive index asdifference in contrast. It was developed by the Dutch physicistFrits Zernike in the 1930s (for which he was awarded the NobelPrize in 1953). The nucleus in a cell for example will show updarkly against the surrounding cytoplasm. Contrast is excellent;however it is not for use with thick objects. Frequently, a halo isformed even around small objects, which obscures detail. Thesystem consists of a circular annulus in the condenser, whichproduces a cone of light. This cone is superimposed on a similarsized ring within the phase-objective. Every objective has adifferent size ring, so for every objective another condensersetting has to be chosen. The ring in the objective has specialoptical properties: it, first of all, reduces the direct light inintensity, but more importantly, it creates an artificial phasedifference of about a quarter wavelength. As the physicalproperties of this direct light have changed, interference withthe diffracted light occurs, resulting in the phase contrast image.One disadvantage of phase-contrast microscopy is haloformation (halo-light ring).

Superior and much more expensive is the use of interference contrast. Differences in optical density willshow up as differences in relief. A nucleus within a cell will actually show up as a globule in the most oftenused differential interference contrast system according to Georges Nomarski. However, it has to be keptin mind that this is an optical effect, and the relief does not necessarily resemble the true shape. Contrast isvery good and the condenser aperture can be used fully open, thereby reducing the depth of field andmaximizing resolution.

The system consists of a special prism (Nomarski prism, Wollaston prism) in the condenser that splits lightin an ordinary and an extraordinary beam. The spatial difference between the two beams is minimal (lessthan the maximum resolution of the objective). After passage through the specimen, the beams are reunitedby a similar prism in the objective.

In a homogeneous specimen, there is no difference between the two beams, and no contrast is beinggenerated. However, near a refractive boundary (say a nucleus within the cytoplasm), the difference betweenthe ordinary and the extraordinary beam will generate a relief in the image. Differential interference contrast

Phase contrast

Differential interference contrast

Images may also contain artifacts. This isa confocal laser scanning fluorescencemicrograph of thale cress anther (part ofstamen). The picture shows among otherthings a nice red flowing collar-likestructure just below the anther. However,an intact thale cress stamen does nothave such collar, this is a fixation artifact:the stamen has been cut below the pictureframe, and epidermis (upper layer of cells)of stamen stalk has peeled off, forming anon-characteristic structure. Photo: HeitiPaves from Tallinn University ofTechnology.

requires a polarized light source to function; two polarizing filters have to be fitted in the light path, onebelow the condenser (the polarizer), and the other above the objective (the analyzer).

Note: In cases where the optical design of a microscope produces an appreciable lateral separation of thetwo beams we have the case of classical interference microscopy, which does not result in relief images, butcan nevertheless be used for the quantitative determination of mass-thicknesses of microscopic objects.

An additional technique using interference is interference reflection microscopy (also known as reflectedinterference contrast, or RIC). It relies on cell adhesion to the slide to produce an interference signal. If thereis no cell attached to the glass, there will be no interference.

Interference reflection microscopy can be obtained by using the same elements used by DIC, but without theprisms. Also, the light that is being detected is reflected and not transmitted as it is when DIC is employed.

When certain compounds are illuminated with high energylight, they emit light of a lower frequency. This effect is knownas fluorescence. Often specimens show their characteristicautofluorescence image, based on their chemical makeup.

This method is of critical importance in the modern lifesciences, as it can be extremely sensitive, allowing the detectionof single molecules. Many different fluorescent dyes can beused to stain different structures or chemical compounds. Oneparticularly powerful method is the combination of antibodiescoupled to a fluorophore as in immunostaining. Examples ofcommonly used fluorophores are fluorescein or rhodamine.

The antibodies can be tailor-made for a chemical compound.For example, one strategy often in use is the artificialproduction of proteins, based on the genetic code (DNA). Theseproteins can then be used to immunize rabbits, formingantibodies which bind to the protein. The antibodies are thencoupled chemically to a fluorophore and used to trace theproteins in the cells under study.

Highly efficient fluorescent proteins such as the greenfluorescent protein (GFP) have been developed using themolecular biology technique of gene fusion, a process that linksthe expression of the fluorescent compound to that of the targetprotein. This combined fluorescent protein is, in general, non-toxic to the organism and rarely interfereswith the function of the protein under study. Genetically modified cells or organisms directly express thefluorescently tagged proteins, which enables the study of the function of the original protein in vivo.

Growth of protein crystals results in both protein and salt crystals. Both are colorless and microscopic.Recovery of the protein crystals requires imaging which can be done by the intrinsic fluorescence of theprotein or by using transmission microscopy. Both methods require an ultraviolet microscope as proteinabsorbs light at 280 nm. Protein will also fluorescence at approximately 353 nm when excited with 280 nmlight.[14]

Interference reflection

Fluorescence

Since fluorescence emission differs in wavelength (color) from the excitation light, an ideal fluorescentimage shows only the structure of interest that was labeled with the fluorescent dye. This high specificity ledto the widespread use of fluorescence light microscopy in biomedical research. Different fluorescent dyescan be used to stain different biological structures, which can then be detected simultaneously, while stillbeing specific due to the individual color of the dye.

To block the excitation light from reaching the observer or the detector, filter sets of high quality are needed.These typically consist of an excitation filter selecting the range of excitation wavelengths, a dichroicmirror, and an emission filter blocking the excitation light. Most fluorescence microscopes are operated inthe Epi-illumination mode (illumination and detection from one side of the sample) to further decrease theamount of excitation light entering the detector.

An example of fluorescence microscopy today is two-photon or multi-photon imaging. Two photon imagingallows imaging of living tissues up to a very high depth by enabling greater excitation light penetration andreduced background emission signal.

See also: total internal reflection fluorescence microscope Neuroscience

Confocal microscopy uses a scanning point of light and a pinhole to prevent out of focus light from reachingthe detector. Compared to full sample illumination, confocal microscopy gives slightly higher resolution,and significantly improves optical sectioning. Confocal microscopy is, therefore, commonly used where 3Dstructure is important.

Using a plane of light formed by focusing light through a cylindrical lens at a narrow angle or by scanning aline of light in a plane perpendicular to the axis of objective, high resolution optical sections can betaken.[15][16][17] Single plane illumination, or light sheet illumination, is also accomplished using beamshaping techniques incorporating multiple-prism beam expanders.[18][19] The images are captured by CCDs.These variants allow very fast and high signal to noise ratio image capture.

Wide-field multiphoton microscopy[20][21][22][23] refers to an optical non-linear imaging technique tailoredfor ultrafast imaging in which a large area of the object is illuminated and imaged without the need forscanning. High intensities are required to induce non-linear optical processes such as two-photonfluorescence or second harmonic generation. In scanning multiphoton microscopes the high intensities areachieved by tightly focusing the light, and the image is obtained by stage- or beam-scanning the sample. Inwide-field multiphoton microscopy the high intensities are best achieved using an optically amplifiedpulsed laser source to attain a large field of view (~100 µm).[20][21][22] The image in this case is obtained asa single frame with a CCD without the need of scanning, making the technique particularly useful tovisualize dynamic processes simultaneously across the object of interest. With wide-field multiphotonmicroscopy the frame rate can be increased up to a 1000-fold compared to multiphoton scanningmicroscopy.[21]

Confocal

Single plane illumination microscopy and light sheet fluorescence microscopy

Wide-field multiphoton microscopy

Deconvolution

Mathematically modeled Point SpreadFunction of a pulsed THz laser imagingsystem.[24]

Fluorescence microscopy is a powerful technique to show specifically labeled structures within a complexenvironment and to provide three-dimensional information of biological structures. However, thisinformation is blurred by the fact that, upon illumination, all fluorescently labeled structures emit light,irrespective of whether they are in focus or not. So an image of a certain structure is always blurred by thecontribution of light from structures that are out of focus. This phenomenon results in a loss of contrastespecially when using objectives with a high resolving power, typically oil immersion objectives with a highnumerical aperture.

However, blurring is not caused by random processes, such aslight scattering, but can be well defined by the optical propertiesof the image formation in the microscope imaging system. If oneconsiders a small fluorescent light source (essentially a brightspot), light coming from this spot spreads out further from ourperspective as the spot becomes more out of focus. Under idealconditions, this produces an "hourglass" shape of this pointsource in the third (axial) dimension. This shape is called thepoint spread function (PSF) of the microscope imaging system.Since any fluorescence image is made up of a large number ofsuch small fluorescent light sources, the image is said to be"convolved by the point spread function". The mathematicallymodeled PSF of a terahertz laser pulsed imaging system isshown on the right.

The output of an imaging system can be described using the equation:

Where n is the additive noise.[25] Knowing this point spread function[26] means that it is possible to reversethis process to a certain extent by computer-based methods commonly known as deconvolutionmicroscopy.[27] There are various algorithms available for 2D or 3D deconvolution. They can be roughlyclassified in nonrestorative and restorative methods. While the nonrestorative methods can improve contrastby removing out-of-focus light from focal planes, only the restorative methods can actually reassign light toits proper place of origin. Processing fluorescent images in this manner can be an advantage over directlyacquiring images without out-of-focus light, such as images from confocal microscopy, because light signalsotherwise eliminated become useful information. For 3D deconvolution, one typically provides a series ofimages taken from different focal planes (called a Z-stack) plus the knowledge of the PSF, which can bederived either experimentally or theoretically from knowing all contributing parameters of the microscope.

A multitude of super-resolution microscopy techniques have been developed in recent times whichcircumvent the diffraction barrier.

This is mostly achieved by imaging a sufficiently static sample multiple times and either modifying theexcitation light or observing stochastic changes in the image. The deconvolution methods described in theprevious section, which removes the PSF induced blur and assigns a mathematically 'correct' origin of light,are used, albeit with slightly different understanding of what the value of a pixel mean. Assuming most ofthe time, one single fluorophore contributes to one single blob on one single taken image, the blobs in theimages can be replaced with their calculated position, vastly improving resolution to well below thediffraction limit.

Sub-diffraction techniques

Example of super-resolution microscopy. Image of Her3 and Her2, target of the breast cancer drugTrastuzumab, within a cancer cell.

Torealizesuch

assumption, Knowledge of and chemical control over fluorophore photophysics is at the core of thesetechniques, by which resolutions of ~20 nanometers are regularly obtained.[28][29]

Serial time encoded amplified microscopy (STEAM) is an imaging method that provides ultrafast shutterspeed and frame rate, by using optical image amplification to circumvent the fundamental trade-off betweensensitivity and speed, and a single-pixel photodetector to eliminate the need for a detector array and readouttime limitations[30] The method is at least 1000 times faster than the state-of-the-art CCD and CMOScameras. Consequently, it is potentially useful for a broad range of scientific, industrial, and biomedicalapplications that require high image acquisition rates, including real-time diagnosis and evaluation ofshockwaves, microfluidics, MEMS, and laser surgery.[31]

Most modern instruments provide simple solutions for micro-photography and image recordingelectronically. 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 withknowledge of the subject can accurately convert a three-dimensional image into a precise two-dimensionaldrawing. In a photograph or other image capture system however, only one thin plane is ever in good focus.

The creation of careful and accurate micrographs requires a microscopical technique using a monoculareyepiece. It is essential that both eyes are open and that the eye that is not observing down the microscope isinstead concentrated on a sheet of paper on the bench besides the microscope. With practice, and withoutmoving the head or eyes, it is possible to accurately record the observed details by tracing round theobserved shapes by simultaneously "seeing" the pencil point in the microscopical image.

Practicing this technique also establishes good general microscopical technique. It is always less tiring toobserve with the microscope focused so that the image is seen at infinity and with both eyes open at alltimes.

Microspectroscopy:spectroscopy with a microscope

Serial time-encoded amplified microscopy

Extensions

Other enhancements

As resolution depends on the wavelength of the light. Electron microscopy has been developed since the1930s that use electron beams instead of light. Because of the much smaller wavelength of the electronbeam, resolution is far higher.

Though less common, X-ray microscopy has also been developed since the late 1940s. The resolution of X-ray microscopy lies between that of light microscopy and electron microscopy.

Until the invention of sub-diffraction microscopy, the wavelength of the light limited the resolution oftraditional microscopy to around 0.2 micrometers. In order to gain higher resolution, the use of an electronbeam with a far smaller wavelength is used in electron microscopes.

Transmission electron microscopy (TEM) is quite similar to the compound light microscope, bysending an electron beam through a very thin slice of the specimen. The resolution limit in2005 was around 0.05 nanometer and has not increased appreciably since that time.Scanning electron microscopy (SEM) visualizes details on the surfaces of specimens andgives a very nice 3D view. It gives results much like those of the stereo light microscope. Thebest resolution for SEM in 2011 was 0.4 nanometer.

Electron microscopes equipped for X-ray spectroscopy can provide qualitative and quantitative elementalanalysis. This type of electron microscope, also known as analytical electron microscope, can be a verypowerful characterisation tool for investigation of nanomaterials.[32]

This is a sub-diffraction technique. Examples of scanning probe microscopes are the atomic forcemicroscope (AFM), the Scanning tunneling microscope, the photonic force microscope and the recurrencetracking microscope. All such methods use the physical contact of a solid probe tip to scan the surface of anobject, which is supposed to be almost flat.

Ultrasonic force microscopy (UFM) has been developed in order to improve the details and image contraston "flat" areas of interest where AFM images are limited in contrast. The combination of AFM-UFM allowsa near field acoustic microscopic image to be generated. The AFM tip is used to detect the ultrasonic wavesand overcomes the limitation of wavelength that occurs in acoustic microscopy. By using the elastic changesunder the AFM tip, an image of much greater detail than the AFM topography can be generated.

Ultrasonic force microscopy allows the local mapping of elasticity in atomic force microscopy by theapplication of ultrasonic vibration to the cantilever or sample. In an attempt to analyze the results ofultrasonic force microscopy in a quantitative fashion, a force-distance curve measurement is done withultrasonic vibration applied to the cantilever base, and the results are compared with a model of thecantilever dynamics and tip-sample interaction based on the finite-difference technique.

X-ray

Electron microscopy

Scanning probe microscopy

Ultrasonic force

Ultraviolet microscopy

Human cells imaged by DHM phase shift(left) and phase contrast microscopy(right).

Ultraviolet microscopes have two main purposes. The first is to utilize the shorter wavelength of ultravioletelectromagnetic energy to improve the image resolution beyond that of the diffraction limit of standardoptical microscopes. This technique is used for non-destructive inspection of devices with very smallfeatures such as those found in modern semiconductors. The second application for UV microscopes iscontrast enhancement where the response of individual samples is enhanced, relative to their surrounding,due to the interaction of light with the molecules within the sample itself. One example is in the growth ofprotein crystals. Protein crystals are formed in salt solutions. As salt and protein crystals are both formed inthe growth process, and both are commonly transparent to the human eye, they cannot be differentiated witha standard optical microscope. As the tryptophan of protein absorbs light at 280 nm, imaging with a UVmicroscope with 280 nm bandpass filters makes it simple to differentiate between the two types of crystals.The protein crystals appear dark while the salt crystals are transparent.

The term infrared microscopy refers to microscopy performed at infrared wavelengths. In the typicalinstrument configuration, a Fourier Transform Infrared Spectrometer (FTIR) is combined with an opticalmicroscope and an infrared detector. The infrared detector can be a single point detector, a linear array or a2D focal plane array. The FTIR provides the ability to perform chemical analysis via infrared spectroscopyand the microscope and point or array detector enable this chemical analysis to be spatially resolved, i.e.performed at different regions of the sample. As such, the technique is also called infraredmicrospectroscopy[33][34] (an alternative architecture involves the combination of a tuneable infrared lightsource and single point detector on a flying objective). This technique is frequently used for infraredchemical imaging, where the image contrast is determined by the response of individual sample regions toparticular IR wavelengths selected by the user, usually specific IR absorption bands and associatedmolecular resonances . A key limitation of conventional infrared microspectroscopy is that the spatialresolution is diffraction-limited. Specifically the spatial resolution is limited to a figure related to thewavelength of the light. For practical IR microscopes, the spatial resolution is limited to 1-3X thewavelength, depending on the specific technique and instrument used. For mid-IR wavelengths, this sets apractical spatial resolution limit of ~3-30 μm.

IR versions of sub-diffraction microscopy (see above) also exist.[33][34] These include IR NSOM,[35]

photothermal microspectroscopy, and atomic force microscope based infrared spectroscopy (AFM-IR).

In digital holographic microscopy (DHM), interfering wavefronts from a coherent (monochromatic) light-source arerecorded on a sensor. The image is digitally reconstructed by acomputer from the recorded hologram. Besides the ordinarybright field image, a phase shift image is created.

DHM can operate both in reflection and transmission mode. Inreflection mode, the phase shift image provides a relativedistance measurement and thus represents a topography map ofthe reflecting surface. In transmission mode, the phase shiftimage provides a label-free quantitative measurement of theoptical thickness of the specimen. Phase shift images ofbiological cells are very similar to images of stained cells andhave successfully been analyzed by high content analysissoftware.

Infrared microscopy

Digital holographic microscopy

A unique feature of DHM is the ability to adjust focus after the image is recorded, since all focus planes arerecorded simultaneously by the hologram. This feature makes it possible to image moving particles in avolume or to rapidly scan a surface. Another attractive feature is DHM's ability to use low cost optics bycorrecting optical aberrations by software.

Digital pathology is an image-based information environment enabled by computer technology that allowsfor the management of information generated from a digital slide. Digital pathology is enabled in part byvirtual microscopy, which is the practice of converting glass slides into digital slides that can be viewed,managed, and analyzed.

Laser microscopy is a rapidly growing field that uses laser illumination sources in various forms ofmicroscopy.[36] For instance, laser microscopy focused on biological applications uses ultrashort pulselasers, in a number of techniques labeled as nonlinear microscopy, saturation microscopy, and two-photonexcitation microscopy.[37]

High-intensity, short-pulse laboratory x-ray lasers have been under development for several years. When thistechnology comes to fruition, it will be possible to obtain magnified three-dimensional images of elementarybiological structures in the living state at a precisely defined instant. For optimum contrast between waterand protein and for best sensitivity and resolution, the laser should be tuned near the nitrogen line at about0.3 nanometers. Resolution will be limited mainly by the hydrodynamic expansion that occurs while thenecessary number of photons is being registered.[38] Thus, while the specimen is destroyed by the exposure,its configuration can be captured before it explodes.[39][40][41][42][43][44]

Scientists have been working on practical designs and prototypes for x-ray holographic microscopes, despitethe prolonged development of the appropriate laser.[45][46][47][48][49][50][51][52]

A microscopy technique relying on the photoacoustic effect,[53] i.e. the generation of (ultra)sound caused bylight absorption. A focused and intensity modulated laser beam is raster scanned over a sample. Thegenerated (ultra)sound is detected via an ultrasound transducer. Commonly, piezoelectric ultrasoundtransducers are employed.[54]

The image contrast is related to the sample's absorption coefficient . This is in contrast to bright or darkfield microscopy, where the image contrast is due to transmittance or scattering. In principle, the contrast offluorescence microscopy is proportional to the sample's absorption too. However, in fluorescencemicroscopy the fluorescence quantum yield needs to be unequal to zero in order that a signal can bedetected. In photoacoustic microscopy, however, every absorbing substance gives a photoacoustic signal

which is proportional to

Here is the Grüneisen coefficient, is the laser's photon energy and is the sample's band gapenergy. Therefore, photoacoustic microscopy seems well suited as a complementary technique tofluorescence microscopy, as a high fluorescence quantum yield leads to high fluorescence signals and a lowfluorescence quantum yield leads to high photoacoustic signals.

Digital pathology (virtual microscopy)

Laser microscopy

Photoacoustic microscopy

Neglecting non-linear effects, the lateral resolution dx is limited by the Abbe diffraction limit:

where is the wavelength of the excitation laser and NA is the numerical aperture of the objective lens. TheAbbe diffraction limit holds if the incoming wave front is parallel. In reality, however, the laser beam profileis Gaussian. Therefore, in order to the calculate the achievable resolution, formulas for truncated Gaussianbeams have to be used.[55]

Amateur Microscopy is the investigation and observation of biological and non-biological specimens forrecreational purposes. Collectors of minerals, insects, seashells, and plants may use microscopes as tools touncover features that help them classify their collected items. Other amateurs may be interested in observingthe life found in pond water and of other samples. Microscopes may also prove useful for the water qualityassessment for people that keep a home aquarium. Photographic documentation and drawing of themicroscopic images are additional tasks that augment the spectrum of tasks of the amateur. There are evencompetitions for photomicrograph art. Participants of this pastime may either use commercially preparedmicroscopic slides or engage in the task of specimen preparation.

While microscopy is a central tool in the documentation of biological specimens, it is, in general,insufficient to justify the description of a new species based on microscopic investigations alone. Oftengenetic and biochemical tests are necessary to confirm the discovery of a new species. A laboratory andaccess to academic literature is a necessity, which is specialized and, in general, not available to amateurs.There is, however, one huge advantage that amateurs have above professionals: time to explore theirsurroundings. Often, advanced amateurs team up with professionals to validate their findings and (possibly)describe new species.

In the late 1800s, amateur microscopy became a popular hobby in the United States and Europe. Several'professional amateurs' were being paid for their sampling trips and microscopic explorations byphilanthropists, to keep them amused on the Sunday afternoon (e.g., the diatom specialist A. Grunow, beingpaid by (among others) a Belgian industrialist). Professor John Phin published "Practical Hints on theSelection and Use of the Microscope (Second Edition, 1878)," and was also the editor of the "AmericanJournal of Microscopy."

Examples of amateur microscopy images:

"house bee" Mouth100X

Rice Stem cs 400X

Rabbit Testis 100X

Fern Prothallium400X

Amateur microscopy

Application in forensic science

Microscopy has many applications in the forensic sciences; it provides precision, quality, accuracy, andreproducibility of results.[56] These applications are almost limitless. This is due to the ability of microscopeto detect, resolve and image the smallest items of evidence, often without any alteration or destruction. Themicroscope is used to identify and compare fibers, hairs, soils, and dust...etc.

The aim of any microscope is to magnify images or photos of a small object and to see fine details. Inforensic; the type of specimen, the information one wishes to obtain from it and the type of microscopechosen for the task will determine if the sample preparation is required. For example, ink lines, blood stainsor bullets, no treatment is required and the evidence shows directly from appropriate microscope withoutany form of sample preparation, but for traces of particular matter, the sample preparation must be donebefore microscopical examination occurs.

A variety of microscopes are used in forensic science laboratory. The light microscopes are the most use inforensic and these microscopes use photons to form images4, these microscopes which are most applicablefor examining forensic specimens as mentioned before are as follows:[57]

1. The compound microscope

2. The comparison microscope

3. The stereoscopic microscope

4. The polarizing microscope

5. The micro spectrophotometer

This diversity of the types of microscopes in forensic applications comes mainly from their magnificationranges, which are (1- 1200X), (50 -30,000X) and (500- 250,000X) for the optical microscopy, SEM andTEM respectively.[57]

Acronyms in microscopyDigital microscopeDigital pathologyImaging cycler microscopyInfinity correctionInterferometric microscopyKöhler illuminationList of microscopistsMicrodensitometerTimeline of microscope technologyTwo-photon excitation microscopyUSB microscope

1. The University of Edinburgh (March 6, 2018). "What is Microscopy?" (https://www.ed.ac.uk/clinical-sciences/edinburgh-imaging/for-patients-study-participants/tell-me-more-about-my-scan/what-is-microscopy). The University of Edinburgh. Retrieved April 9, 2018.

See also

References

2. Ford, Brian J. (1992). "From Dilettante to Diligent Experimenter: a Reappraisal ofLeeuwenhoek as microscopist and investigator" (http://www.brianjford.com/a-avl01.htm).Biology History. 5 (3).

3. Atti Della Fondazione Giorgio Ronchi E Contributi Dell'Istituto Nazionale Di Ottica, Volume 30,La Fondazione-1975, page 554

4. Albert Van Helden; Sven Dupré; Rob van Gent (2010). The Origins of the Telescope (https://books.google.com/books?id=XguxYlYd-9EC&pg=PA24). Amsterdam University Press. p. 24.ISBN 978-90-6984-615-6. Archived (https://web.archive.org/web/20170215200146/https://books.google.com/books?id=XguxYlYd-9EC&pg=PA24) from the original on 15 February 2017.

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General

Microscopy glossary (http://www.3dham.com/microgallery/glossary.htm), Common terms usedin amateur light microscopy.Nikon MicroscopyU (http://www.microscopyu.com/) Extensive information on light microscopyOlympus Microscopy (http://www.olympusmicro.com/index.html) Microscopy Resource centerCarl Zeiss "Microscopy from the very beginning" (http://www.zeiss.de/C1256B5E0047FF3F?Open), a step by step tutorial into the basics of microscopy.Microscopy in Detail (https://web.archive.org/web/20011214072652/http://www.biologie.uni-hamburg.de/b-online/e03/03.htm) - A resource with many illustrations elaborating the mostcommon microscopy techniquesManawatu Microscopy (http://confocal-manawatu.pbworks.com/) - first known collaborationenvironment for Microscopy and Image Analysis.Audio microscope glossary (http://www.histology-world.com/microscope/audiomicroscope/audiomicroscope.htm)

Techniques

55. Langer, G.; Buchegger, B.; Jacak, J.; Klar, T. A.; Berer, T. (2016). "Frequency domainphotoacoustic and fluorescence microscopy" (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4948622). Biomedical Optics Express. 7 (7): 2692–702. doi:10.1364/BOE.7.002692 (https://doi.org/10.1364%2FBOE.7.002692). PMC 4948622 (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4948622). PMID 27446698 (https://pubmed.ncbi.nlm.nih.gov/27446698).

56. Kotrly M (August 2015). "New Possibilities of Using Micrscopic Techniques in Forensic Field".Proceedings of Microscopy & Microanallysis. 21 (S3): 1365–1366.Bibcode:2015MiMic..21S1365K (https://ui.adsabs.harvard.edu/abs/2015MiMic..21S1365K).doi:10.1017/S1431927615007618 (https://doi.org/10.1017%2FS1431927615007618).

57. Basu S, Millette JR (1986). Electron Microscopy in Forensic Occupational and EnvironmentalHealth Sciences. New York: Plenum Press.

Further reading

External links

Ratio-metric Imaging Applications For Microscopes (https://web.archive.org/web/20081121044712/http://www.pti-nj.com/EasyRatio/EasyRatioPro-Applications.html) Examples of RatiometricImaging Work on a MicroscopeInteractive Fluorescence Dye and Filter Database (https://www.micro-shop.zeiss.com/?s=2525647761b33&l=en&p=us&f=f) Carl Zeiss Interactive Fluorescence Dye and Filter Database.New approaches to microscopy (http://spie.org/x112606.xml) Eric Betzig: Beyond the NobelPrize—New approaches to microscopy.

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