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69-732: John Thomas Quekett (11 August 1815 – 20 August 1861) was an English microscopist and histologist . Quekett studied medicine at the London Hospital in 1831. He became a licentiate of the Apothecaries' Company and a member of the Royal College of Surgeons . In 1839, along with his brother Edwin John Quekett ) co-founded the Royal Microscopical Society . Quekett served as the society's secretary from 1841 to 1860. In 1843 he

138-618: A pulsed infrared laser is used for excitation. Only in the tiny focus of the laser is the intensity high enough to generate fluorescence by two-photon excitation , which means that no out-of-focus fluorescence is generated, and no pinhole is necessary to clean up the image. This allows imaging deep in scattering tissue, where a confocal microscope would not be able to collect photons efficiently. Two-photon microscopes with wide-field detection are frequently used for functional imaging, e.g. calcium imaging , in brain tissue. They are marketed as Multiphoton microscopes by several companies, although

207-522: A 1000-fold compared to multiphoton scanning microscopy . In scattering tissue, however, image quality rapidly degrades with increasing depth. Fluorescence microscopy is a powerful technique to show specifically labeled structures within a complex environment and to provide three-dimensional information of biological structures. However, this information 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

276-499: A Z-stack) plus the knowledge of the PSF, which can be derived 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 which circumvent the diffraction limit . This is mostly achieved by imaging a sufficiently static sample multiple times and either modifying the excitation light or observing stochastic changes in

345-432: A cell will actually show up as a globule in the most often used differential interference contrast system according to Georges Nomarski . However, it has to be kept in mind that this is an optical effect , and the relief does not necessarily resemble the true shape. Contrast is very good and the condenser aperture can be used fully open, thereby reducing the depth of field and maximizing resolution. The system consists of

414-467: A central locality, at an annual charge to cover incidental expenses". The name agreed was "The Quekett Microscopical Club", 'club' was chosen instead of 'society' to reflect the aims of the association. The first President was Edwin Lankester . The Club is named after the famous Victorian microscopist Professor John Thomas Quekett , and is the second oldest organisation in the world dedicated to microscopy;

483-414: A certain structure is always blurred by the contribution of light from structures that are out of focus. This phenomenon results in a loss of contrast especially when using objectives with a high resolving power, typically oil immersion objectives with a high numerical aperture. However, blurring is not caused by random processes, such as light scattering, but can be well defined by the optical properties of

552-444: A circular annulus in the condenser, which produces a cone of light. This cone is superimposed on a similar sized ring within the phase-objective. Every objective has a different size ring, so for every objective another condenser setting has to be chosen. The ring in the objective has special optical properties: it, first of all, reduces the direct light in intensity, but more importantly, it creates an artificial phase difference of about

621-642: A few meetings in other parts of the United Kingdom. During the warmer months, the Club arranges excursions where members can collect specimens and examine them using their own microscopes. The Club holds an annual exhibition in the Natural History Museum each autumn. Reports of meetings are published in the Club’s Bulletin and on its website. The Club’s publications include the amateur-friendly Bulletin of

690-644: A focused laser beam (e.g. 488 nm) that is scanned across the sample to excite fluorescence in a point-by-point fashion. The emitted light is directed through a pinhole to prevent out-of-focus light from reaching the detector, typically a photomultiplier tube . The image is constructed in a computer, plotting the measured fluorescence intensities according to the position of the excitation laser. Compared to full sample illumination, confocal microscopy gives slightly higher lateral resolution and significantly improves optical sectioning (axial resolution). Confocal microscopy is, therefore, commonly used where 3D structure

759-448: A glass window: one sees not the glass but merely the dirt on the glass. There is a difference, as glass is a denser material, and this creates a difference in phase of the light passing through. The human eye is not sensitive to this difference in phase, but clever optical solutions have been devised to change this difference in phase into a difference in amplitude (light intensity). To improve specimen contrast or highlight structures in

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828-404: A large area of the object is illuminated and imaged without the need for scanning. High intensities are required to induce non-linear optical processes such as two-photon fluorescence or second harmonic generation . In scanning multiphoton microscopes the high intensities are achieved by tightly focusing the light, and the image is obtained by beam scanning. In wide-field multiphoton microscopy

897-404: A lower frequency. This effect is known as fluorescence . Often specimens show their characteristic autofluorescence image, based on their chemical makeup. This method is of critical importance in the modern life sciences, as it can be extremely sensitive, allowing the detection of single molecules. Many fluorescent dyes can be used to stain structures or chemical compounds. One powerful method

966-424: 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 trace the observed shapes by simultaneously "seeing" the pencil point in the microscopical image. It is always less tiring to observe with

1035-617: A most extensive and valuable collection of microscopic preparations, injected by himself, illustrating the tissues of plants and animals in health and in disease, and showing the results and uses of microscopic investigation. In November 1843 he was appointed by the College of Surgeons assistant conservator of the Hunterian Museum , under Professor (afterwards Sir) Richard Owen, and in 1844 he was appointed demonstrator of minute anatomy. In 1846 his collection of two thousand five hundred preparations

1104-627: A parasol, and a few pieces of brass purchased at a neighbouring marine-store shop. On leaving school he was apprenticed, first to a surgeon in Langport, and afterwards to his brother Edwin John Quekett , entering King's College, London , and the London Hospital medical school. In 1840 he qualified at Apothecaries' Hall, and at the Royal College of Surgeons won the three-years studentship in human and comparative anatomy, then first instituted. He formed

1173-413: A quarter wavelength. As the physical properties of this direct light have changed, interference with the diffracted light occurs, resulting in the phase contrast image. One disadvantage of phase-contrast microscopy is halo formation (halo-light ring). Superior and much more expensive is the use of interference contrast . Differences in optical density will show up as differences in relief. A nucleus within

1242-420: A sample, special techniques must be used. A huge selection of microscopy techniques are available to increase contrast or label a sample. Bright field microscopy is the simplest of all the light microscopy techniques. Sample illumination is via transmitted white light, i.e. illuminated from below and observed from above. Limitations include low contrast of most biological samples and low apparent resolution due to

1311-603: A single-pixel photodetector to eliminate the need for a detector array and readout time limitations The method is at least 1000 times faster than the state-of-the-art CCD and CMOS cameras. Consequently, it is potentially useful for scientific, industrial, and biomedical applications that require high image acquisition rates, including real-time diagnosis and evaluation of shockwaves, microfluidics , MEMS , and laser surgery . Most modern instruments provide simple solutions for micro-photography and image recording electronically. However such capabilities are not always present and

1380-426: A special prism ( Nomarski prism , Wollaston prism ) in the condenser that splits light in an ordinary and an extraordinary beam. The spatial difference between the two beams is minimal (less than the maximum resolution of the objective). After passage through the specimen, the beams are reunited by a similar prism in the objective. In a homogeneous specimen, there is no difference between the two beams, and no contrast

1449-494: A very powerful tool for investigation of nanomaterials . This is a sub-diffraction technique. Examples of scanning probe microscopes are the atomic force microscope (AFM), the scanning tunneling microscope , the photonic force microscope and the recurrence tracking microscope . All such methods use the physical contact of a solid probe tip to scan the surface of an object, which is supposed to be almost flat. Quekett Microscopical Club The Quekett Microscopical Club

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1518-564: Is a learned society for the promotion of microscopy . Its members come from all over the world, and include both amateur and professional microscopists. It is a registered charity and not-for-profit publisher, with the stated aims of promoting the understanding and use of all aspects of the microscope. The Club was founded in 1865 as a result of a letter from W. Gibson published in Science Gossip in May 1865 suggesting that "some association among

1587-405: Is a variant of dark field illumination in which transparent, colored filters are inserted just before the condenser so that light rays at high aperture are differently colored than those at low aperture (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. Dispersion staining

1656-581: Is a widely used technique that shows differences in refractive index as difference in contrast. It was developed by the Dutch physicist Frits Zernike in the 1930s (for which he was awarded the Nobel Prize in 1953). The nucleus in a cell for example will show up darkly against the surrounding cytoplasm. Contrast is excellent; however it is not for use with thick objects. Frequently, a halo is formed even around small objects, which obscures detail. The system consists of

1725-495: Is an optical technique that results in a colored image of a colorless object. This is an optical staining technique and requires no stains or dyes to produce a color effect. There are five different microscope configurations used in the broader technique of dispersion staining. They include brightfield Becke line, oblique, darkfield, phase contrast, and objective stop dispersion staining. More sophisticated techniques will show proportional differences in optical density. Phase contrast

1794-451: Is being generated. However, near a refractive boundary (say a nucleus within the cytoplasm), the difference between the ordinary and the extraordinary beam will generate a relief in the image. Differential interference contrast requires a polarized light source to function; two polarizing filters have to be fitted in the light path, one below the condenser (the polarizer), and the other above the objective (the analyzer). Note: In cases where

1863-601: Is commemorated by the Quekett Microscopical Club , which was established in 1865, under the presidency of Edwin Lankester . Quekett's chief publications were: Twenty-two papers by him are also enumerated in the Royal Society's Catalogue of Scientific Papers (v. 53–4), mostly contributed to the Microscopical Society's Transactions, and dealing with animal histology. One of the most important of these

1932-428: Is important. A subclass of confocal microscopes are spinning disc microscopes which are able to scan multiple points simultaneously across the sample. A corresponding disc with pinholes rejects out-of-focus light. The light detector in a spinning disc microscope is a digital camera, typically EM-CCD or sCMOS . A two-photon microscope is also a laser-scanning microscope, but instead of UV, blue or green laser light,

2001-608: Is that on the Intimate Structure of Bones in the four great Classes, Mammals, Birds, Reptiles, and Fishes, with Remarks on the Value of the Knowledge in determining minute Organic Remains, Microscopical Society's Transactions, vol. ii. 1846, pp. 46–58. Microscopy Optical microscopy and electron microscopy involve the diffraction , reflection , or refraction of electromagnetic radiation /electron beams interacting with

2070-456: Is the additive noise. Knowing this point spread function means that it is possible to reverse this process to a certain extent by computer-based methods commonly known as deconvolution microscopy. There are various algorithms available for 2D or 3D deconvolution. They can be roughly classified in nonrestorative and restorative methods. While the nonrestorative methods can improve contrast by removing out-of-focus light from focal planes, only

2139-417: Is the combination of antibodies coupled to a fluorophore as in immunostaining . Examples of commonly 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 artificial production of proteins, based on the genetic code (DNA). These proteins can then be used to immunize rabbits, forming antibodies which bind to

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2208-568: The conchologist John Frederick Whitlie Quekett . There is a lithographic portrait of Quekett in Maguire's Ipswich series of 1849, and a coloured one by W. Lens Aldous . Upon Quekett's death, Joseph Henry Green , Thomas Wormald , George Gulliver and several other members of the Council of the Royal College of Surgeons strongly supported the college's granting of a pension to the widow; Wormald and James Moncrieff Arnott each contributed £100 in addition to

2277-426: The specimen , and the collection of the scattered radiation or another signal in order to create an image. This process may be carried out by wide-field irradiation of the sample (for example standard light microscopy and transmission electron microscopy ) or by scanning a fine beam over the sample (for example confocal laser scanning microscopy and scanning electron microscopy ). Scanning probe microscopy involves

2346-531: The 17th-century. Earlier microscopes, single lens magnifying glasses with limited magnification, date at least as far back as the wide spread use of lenses in eyeglasses in the 13th century but more advanced compound microscopes first appeared in Europe around 1620 The earliest practitioners of microscopy include Galileo Galilei , who found in 1610 that he could close focus his telescope to view small objects close up and Cornelis Drebbel , who may have invented

2415-852: The Club, including Edwin Lankester (1865–66), Peter le Neve Foster (1869), Lionel Smith Beale (1870–71), Robert Braithwaite (bryologist) (1872–1873), Henry Lee (1875–77), Thomas Henry Huxley (1877–1879), Thomas Spencer Cobbold (1879–80), Mordecai Cubitt Cooke (1881–1883), William Benjamin Carpenter (1883–1885), William Dallinger (1889–1892), George Edward Massee (1899–1903), Edward Alfred Minchin (1908–1912) Arthur Dendy (1912–1916), Alfred Barton Rendle (1916–1921), Sir David Prain (1924–1926), William Thomas Calman (1926–1928), John Ramsbottom (1928–1931) and Hamilton Hartridge (1951–1954). Members include amateurs, professionals, beginners and experts with an interest in microscopes, microscopy or microscope slides. Members receive two issues of

2484-484: The ability to "see inside" the sample being studied before sacrificing it to higher resolution techniques. A 3D X-ray microscope uses the technique of computed tomography ( microCT ), rotating the sample 360 degrees and reconstructing the 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. The field of microscopy ( optical microscopy ) dates back to at least

2553-421: The amateur microscopists of London is desirable". The suggestion was taken up by Mordecai Cubitt Cooke , Thomas Ketteringham and Witham Bywater, and they met on 14 June 1865 and agreed a provisional committee. About sixty people attended the first meeting of the Club on Friday 7 July 1865 for the purpose of establishing the Club to "give amateurs the opportunity of assisting each other, holding monthly meetings in

2622-612: The benefit of his health, on 20 Aug. 1861. In 1841 Quekett succeeded Arthur Farre as secretary of the Microscopical Society, a post which he retained until 1860, when he was elected president, but was unable to attend any meetings during his year of office. He was elected a fellow of the Linnean Society in 1857, and of the Royal Society in 1860. In 1846 Quekett married Isabella Mary Anne (d. 1872), daughter of Robert Scott, Bengal Civil Service, by whom he had four children, including

2691-534: The blur of out-of-focus material. The simplicity 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 appearance and can highlight otherwise invisible features. A more recent technique based on this method is Hoffmann's modulation contrast , a system found on inverted microscopes for use in cell culture. Oblique illumination enhances contrast even in clear specimens; however, because light enters off-axis,

2760-407: The compound microscope around 1620. Antonie van Leeuwenhoek developed a very high magnification simple microscope in the 1670s and is often considered to be the first acknowledged microscopist and microbiologist . Optical or light microscopy involves passing visible light transmitted through or reflected from the sample through a single lens or multiple lenses to allow a magnified view of

2829-445: The diffraction limit. To realize such assumption, Knowledge of and chemical control over fluorophore photophysics is at the core of these techniques, by which resolutions of ~20 nanometers are obtained. Serial time encoded amplified microscopy (STEAM) is an imaging method that provides ultrafast shutter speed and frame rate, by using optical image amplification to circumvent the fundamental trade-off between sensitivity and speed, and

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2898-407: The exhibit of interest. The image is shown on a computer screen, so eye-pieces are unnecessary. Limitations of standard optical microscopy ( bright field microscopy ) lie in three areas; Live cells in particular generally lack sufficient contrast to be studied successfully, since the internal structures of the cell are colorless and transparent. The most common way to increase contrast is to stain

2967-666: The gains of using 3-photon instead of 2-photon excitation are marginal. Using a plane of light formed by focusing light through a cylindrical lens at a narrow angle or by scanning a line of light in a plane perpendicular to the axis of objective, high resolution optical sections can be taken. Single plane illumination, or light sheet illumination, is also accomplished using beam shaping techniques incorporating multiple-prism beam expanders . The images are captured by CCDs. These variants allow very fast and high signal to noise ratio image capture. Wide-field multiphoton microscopy refers to an optical non-linear imaging technique in which

3036-436: The high intensities are best achieved using an optically amplified pulsed laser source to attain a large field of view (~100 μm). The image in this case is obtained as a single frame with a CCD camera without the need of scanning, making the technique particularly useful to visualize dynamic processes simultaneously across the object of interest. With wide-field multiphoton microscopy the frame rate can be increased up to

3105-412: The image formation in the microscope imaging system. If one considers a small fluorescent light source (essentially a bright spot), light coming from this spot spreads out further from our perspective as the spot becomes more out of focus. Under ideal conditions, this produces an "hourglass" shape of this point source in the third (axial) dimension. This shape is called the point spread function (PSF) of

3174-403: The image plane, collecting only the light scattered by the sample. Dark field can dramatically improve image contrast – especially of transparent objects – while requiring little equipment setup or sample preparation. However, the technique suffers from low light intensity in the final image of many biological samples and continues to be affected by low apparent resolution. Rheinberg illumination

3243-476: The image. The deconvolution methods described in the previous 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 of the time , one single fluorophore contributes to one single blob on one single taken image, the blobs in the images can be replaced with their calculated position, vastly improving resolution to well below

3312-401: The interaction of a scanning probe with the surface of the object of interest. The development of microscopy revolutionized biology , gave rise to the field of histology and so remains an essential technique in the life and physical sciences . X-ray microscopy is three-dimensional and non-destructive, allowing for repeated imaging of the same sample for in situ or 4D studies, and providing

3381-425: The intrinsic fluorescence of the protein or by using transmission microscopy. Both methods require an ultraviolet microscope as proteins absorbs light at 280 nm. Protein will also fluorescence at approximately 353 nm when excited with 280 nm light. Since fluorescence emission differs in wavelength (color) from the excitation light, an ideal fluorescent image shows only the structure of interest that

3450-603: 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 of traditional microscopy to around 0.2 micrometers. In order to gain higher resolution, the use of an electron beam with a far smaller wavelength is used in electron microscopes. Electron microscopes equipped for X-ray spectroscopy can provide qualitative and quantitative elemental analysis. This type of electron microscope, also known as analytical electron microscope, can be

3519-466: The microscope focused so that the image is seen at infinity and with both eyes open at all times. Microspectroscopy:spectroscopy with a microscope As resolution depends on the wavelength of the light. Electron microscopy has been developed since the 1930s that use electron beams instead of light. Because of the much smaller wavelength of the electron beam, resolution is far higher. Though less common, X-ray microscopy has also been developed since

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3588-570: The microscope imaging system. Since any fluorescence image is made up of a large number of such small fluorescent light sources, the image is said to be "convolved by the point spread function". The mathematically modeled PSF of a terahertz laser pulsed imaging system is shown on the right. The output of an imaging system can be described using the equation: s ( x , y ) = P S F ( x , y ) ∗ o ( x , y ) + n {\displaystyle s(x,y)=PSF(x,y)*o(x,y)+n} Where n

3657-409: The more experienced microscopist may 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. The creation of accurate micrographs requires a microscopical technique using

3726-523: The oldest is the Royal Microscopical Society . Some of the traditions of the Club’s Victorian founders are continued, but the Quekett is now very much a friendly club for today’s microscopists and covers all aspects of the subject ranging from the history of the microscope and slide collecting to the latest advances in digital imaging with the microscope. Several eminent scientists have been presidents of

3795-471: The optical design of a microscope produces an appreciable lateral separation of the two beams we have the case of classical interference microscopy , which does not result in relief images, but can 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 reflected interference contrast, or RIC). It relies on cell adhesion to

3864-428: The organism and rarely interferes with the function of the protein under study. Genetically modified cells or organisms directly express the fluorescently 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

3933-506: The pension. Quekett's work as an histologist was remarkable for its originality and for its influence upon the anatomical studies of the medical profession in this country. His Practical Treatise on the Use of the Microscope (1848, 8vo) did much also to promote the study among medical men and amateurs, and among those who came to him for instruction was the prince consort. His work in this direction

4002-422: The position of an object will appear to shift as the focus is changed. This limitation makes techniques like optical sectioning or accurate measurement on the z-axis impossible. Dark field microscopy is a technique for improving the contrast of unstained, transparent specimens. Dark field illumination uses a carefully aligned light source to minimize the quantity of directly transmitted (unscattered) light entering

4071-455: The protein. The antibodies are then coupled chemically to a fluorophore and used to trace the proteins in the cells under study. Highly efficient fluorescent proteins such as the green fluorescent protein (GFP) have been developed using the molecular biology technique of gene fusion , a process that links the expression of the fluorescent compound to that of the target protein. This combined fluorescent protein is, in general, non-toxic to

4140-500: The range of excitation wavelengths , a dichroic mirror, and an emission filter blocking the excitation light. Most fluorescence microscopes are operated in the Epi-illumination mode (illumination and detection from one side of the sample) to further decrease the amount of excitation light entering the detector. See also: total internal reflection fluorescence microscope Neuroscience Confocal laser scanning microscopy uses

4209-425: The restorative methods can actually reassign light to its proper place of origin. Processing fluorescent images in this manner can be an advantage over directly acquiring images without out-of-focus light, such as images from confocal microscopy , because light signals otherwise eliminated become useful information. For 3D deconvolution, one typically provides a series of images taken from different focal planes (called

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4278-419: The sample. The resulting image can be detected directly by the eye, imaged on a photographic plate , or captured digitally . The single lens with its attachments, or the system of lenses and imaging equipment, along with the appropriate lighting equipment, sample stage, and support, makes up the basic light microscope. The most recent development is the digital microscope , which uses a CCD camera to focus on

4347-649: The scholarly Quekett Journal of Microscopy and two issues of the informal Bulletin of the Quekett Microscopical Club each year. Members have access to a private area of the Club’s website that includes meeting reports, videos of lectures, and galleries of entries from slide and photograph competitions. The Club holds monthly meetings in London for its members, normally in the Natural History Museum , and

4416-422: The slide to produce an interference signal. If there is 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 the prisms. 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 energy light, they emit light of

4485-419: The structures with selective dyes, but this often involves killing and fixing the sample. Staining may also introduce artifacts , which are apparent structural details that are caused by the processing of the specimen and are thus not features of the specimen. In general, these techniques make use of differences in the refractive index of cell structures. Bright-field microscopy is comparable to looking through

4554-438: Was appointed assistant conservator of the Hunterian Museum , and in 1856 conservator of the museum and professor of histology on the retirement of professor Richard Owen . Quekett, born at Langport , Somerset, on 11 August 1815, was the youngest son of William Quekett and Mary, daughter of John Bartlett. The father was at Cockermouth grammar school with William and Christopher Wordsworth , and from 1790 till his death in 1842

4623-511: Was labeled with the fluorescent dye. This high specificity led to the widespread use of fluorescence light microscopy in biomedical research. Different fluorescent dyes can be used to stain different biological structures, which can then be detected simultaneously, while still being 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

4692-399: Was master of Langport grammar school. He educated his sons at home, and each of them was encouraged to collect specimens in some branch of natural history. The eldest brother, William Quekett , was a rector and author. When only sixteen John gave a course of lectures on microscopic subjects, illustrated by original diagrams and by a microscope which he had himself made out of a roasting-jack,

4761-538: Was purchased by the college, and he was directed to prepare a descriptive illustrated catalogue of the whole histological collection belonging to the college, of which they constituted the chief part. In 1852 the title of his demonstratorship was changed to that of professor of histology; and on Owen's obtaining permission to reside at Richmond, Quekett was appointed resident conservator, finally succeeding Owen as conservator in 1856. His health, however, soon failed, and he died at Pangbourne, Berkshire , whither he had gone for

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