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Between 1838 and 1839 two German scientists, Mathias Schleiden (1804–81) and Theodor Schwann (1810–82) proposed that cells were the building blocks for plant and animal life.

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Stereomicroscope

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The light from the microscope source passes through the specimen/slide and continues through the air (or an immersion medium) as a cone of light between the cover glass and the objective front lens. The ‘angular aperture’ refers to the maximum angle of the edges of this image-forming cone of light which can be collected by the objective front lens when the specimen is in focus. In addition to an increasing NA, image brightness and image detail (resolution) are also related to the angular aperture.

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And technological innovations in digital technology improved techniques such as microsurgery, which combines surgery and microscopy to allow detailed and precise manipulations inside the body.

Hassall’s investigative work with the microscope showed how laboratory science could be used to gather evidence from the field about health and disease.

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His second book, ‘A microscopical examination of the water supplied to the inhabitants of London and the suburban districts’ (1850), became an influential work in promoting the cause of water reform.

The NA of an objective is the simply the ability of the lenses to collect light at a fixed distance from the sample which you are viewing. When light passes through and leaves a specimen, it enters the front lens of an objective as an inverted cone. However, a percentage of this image-forming light is refracted and reflected. Objectives which have a high NA allow for increasingly oblique light waves to be collected by the front lens which will in turn form a final image which is not only relatively brighter, but contains more information and detail and is highly resolved.

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Arthur Hill Hassall (1817– 1894) was a British physician, and a pioneer in the use of the microscope as a tool in medicine and public health. In 1846 he published a two-volume study, ‘The Microscopic Anatomy of the Human Body in Health and Disease’, the first English textbook on the subject.

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Leeuwenhoek observed animal and plant tissue, human sperm and blood cells, minerals, fossils, and many other things that had never been seen before on a microscopic scale.

He made use of the latest developments in microscopy such as the use of microtomes to cut very thin slices of tissue and the development of stains to highlight the parts of a cell.

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Snell’s Law describes the relationship between the angles of incidence and refraction of light as it travels through the boundary of two different medium (e.g. from air to glass). The Law states that the ratio of the sines of the angles of incident and refracted light are equivalent to the reciprocal of the ratio of the refractive indices through which the light passes.

Hooke published the ‘Micrographia’ (1665), an astonishing collection of copper-plate illustrations of objects he had observed with his own compound microscope.

Microscope

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Therefore, the refractive index of air is a limiting factor in achieving the highest possible NA of an objective. As a result, objectives with NA values greater than one are the immersion objectives where the air gap is replaced by a medium such as water or oil. An angular aperture of 1800 is physically unachievable- the widest angle of light which can be collected by an objective is around 1440. Consequently, the maximum achievable NA of a non-immersion objective is approximately 0.95 (which is equal to the sine of 72).

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Etched onto the barrel of each objective on a microscope, you will find a variety of information. In addition to the magnification and the optical correction (see my article published entitled ‘Looking Down and Looking Through: The Optics of a Microscope 2: The Objectives’ for more information on aberrations and corrections), you will find a number without units. This is the Numerical Aperture (or ‘NA’) of the objective.

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Metaphorically, think of it this way: if you are standing in front of a door with a key hole which leads into another room, then when you are at a distance, you will only be able to see a little of the light and objects within the room. If you press your eye against the key hole, you will then see more of the detail and light in the room as you have, in theory, increased the angular aperture of your eye.

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Like the scanning electron microscope, the transmission electron microscope (TEM) uses electrons to create a magnified image, and samples are scanned in a vacuum so they must be specially prepared.

In the 20th century, new instruments such as the electron microscope increased magnification and offered new insights into the body and disease, allowing scientists to see organisms such as viruses for the first time.

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Many researchers refused to use the early microscopes because they could not trust what they were seeing. Aberrations and impurities in the lenses caused distortions, which led to errors in observations.

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Where ‘n’ is the refractive index of the medium between the cover glass and the objective front lens (e.g. air, water or oil).

Where ‘θ’ is half of the angle of the cone of light which is collected by the front lens lens (i.e., the angular aperture).

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His theory was taken up by another German researcher, Rudolf Virchow (1821–1902), possibly the most influential teacher of pathology in the 1800s.

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Electronmicroscope

Refraction is described in a formula known as ‘Snell’s Law’. Refraction was first described in the year 984 by a Persian physicist and mathematician called Ibn Sahl. In 984, he presented a manuscript in which he described how mirrors and curved lenses focused and bent light. Snell’s Law is actually named after a Dutch mathematician and astronomer called Willebrord Snellius (1580-1626). Although he was credited for mathematically describing refraction, it is more accurate to say that he ‘rediscovered’ diffraction after the work of Ibn Sahl.

The NA of an objective is an important aspect as it relates to the final image formation seen when looking down through the eyepieces (which will be covered in full in a forthcoming blog article). Briefly, resolution relates to the amount of detail which can be seen in the final formation of an image. An objective with a high magnification would be unable to resolve detail in your sample without a similarly high NA.

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To help to understand NA, it is useful to also have some understanding of refraction. In microscopy and optics, refraction refers to the change in direction of light waves which results from a change in the medium though which light passes (for example, glass, air, oil or water).

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From the 1830s, cells and cell theory became the focus of medical and biological research, thanks to the central role of the microscope in laboratory science. Researchers were able to describe the body at the microscopic level more consistently and with greater confidence in what they saw.

There is an inverse relationship between the angular aperture and the working distance of an objective. I have covered working distance in my article entitled ‘Looking Down and Through: Microscope Optics 3: Oil Immersion Objectives’, but to briefly recap, the working distance is the actual distance (in millimetres or microns) between the objective front lens and the surface of the cover slip when the object is in sharp focus. Objectives with short working distances will consequently have a greater ability to gather more oblique light rays from a specimen compared to longer working distance objectives. Angular aperture is usually determined by the optics within the objective and each objective lens will have an optimal focal length and working distance- it can’t simply be increased by moving the objective closer to the slide!

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Not much changed in basic microscope design over the next 200 years, but improvements in lens manufacture (such as the use of purer glass) helped to solve problems like colour distortion and poor image resolution. Mirrors were added to compound microscopes to add more light and improve the image.

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Unlike stereo and compound microscopes, which use regular light for image formation, the confocal microscope uses a laser light to scan samples that have been dyed. These samples are prepared on slides, which the device then converts into a magnified digital image. Operators can also create 3D images by assembling multiple scans. They are commonly used in cell biology and medical applications.

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The simple microscope combines a convex lens with a holder for specimens. Magnifying between 200 and 300 times, it is essentially a magnifying glass.

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In the 1660s, another Dutchman, Antonie van Leeuwenhoek (1632-1723) made microscopes by grinding his own lenses. His simple microscopes were more like magnifying glasses, with only one lens.

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To simplify this further, as light travels through one medium to another, it changes speed (e.g. when passing from air to water, light slows down). When light passes across the boundary of two different medium at an angle other than 900, this results in a change of direction. Although the frequency of light doesn’t change, the resultant wavelength will be determined by nature of the medium.

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History ofmicroscope

He presented his findings to the Royal Society in London, where Robert Hooke was also making remarkable discoveries with a microscope.

He was the first person to use the term ‘cell’ to describe what would later be recognised as the building blocks of all living organisms, plant and animal.

How to usemicroscope

The scanning electron microscope, or SEM, uses electrons rather than light for image formation. The sample is dehydrated and coated with a thin layer of a conductive material, such as gold. Samples are placed in an evacuated chamber to be scanned. The SEM produces a black-and-white digital image. SEMs are used by researchers to examine a range of specimens from insects to bones.

This image is a transmission electron micrograph showing four views of the Herpes simplex virus, computer-coloured mauve.

Schwann had a medical training and proposed that understanding cellular behaviour was the key to understanding the body in health and illness.

Compound microscope designed by Robert Hooke, 1671–1700, and thought to have been made by Christopher Cock of Covent Garden, London.

But at the start of the 1800s century, the pioneering French pathologist Xavier Bichat, who carried out many investigations into tissue samples and organs, still refused to use a microscope.

And in the early 1850s he also studied food adulteration, publishing his findings in medical journal The Lancet. The journal's campaign on food adulteration led directly to the 1860 Food Adulteration Act.

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Compoundmicroscope

The microscope was at the centre of Virchow’s work on disease processes, he would urge his students to 'learn to see microscopically'. Much of Virchow’s work involved investigating tissue and cells in the laboratory and then relating his findings back to clinical changes in his patients.

Numerical aperture is proportional to refractive index. For example, air has a refractive index of 1.00, water has a refractive index of 1.33, whereas many of the immersion oils have refractive indexes around 1.52.

In summary, without a correspondingly high NA, a high magnification objective will have low resolution. Most microscope companies offer objectives which have high NA values for use with immersion medium. If you are in the lucky position of buying a custom microscope, or buying new objectives for your existing instrument, you should always consider buying objectives which offer the highest NA value which you can afford.

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Two main problems hindered lens manufacture: image blurring (spherical aberration) and colour separation (chromatic aberration). Around 1830, Joseph Jackson Lister, in collaboration with instrument maker William Tulley, made one of the first microscopes that corrected for both these faults.

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microscope中文

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Compound microscopes have two lenses: the second lens magnifies the image enlarged by the first lens. Modern compound microscopes can provide a magnification of 1,000 times. They are still the most commonly used general purpose microscopes, found everywhere from research labs to school biology laboratories.

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It’s not clear who invented the first microscope, but the Dutch spectacle maker Zacharias Janssen (b.1585) is credited with making one of the earliest compound microscopes (ones that used two lenses) around 1600. The earliest microscopes could magnify an object up to 20 or 30 times its normal size.

The theoretical maximum angular aperture of light entering the front lens of an objective is 1800. This would give a θ value of 900 (half of the angle of the light cone). As a result, the theoretical maximum NA of an objective would be one (which is equal to the sine of 90). The refractive index of air is also one, therefore the maximum (theoretical) NA of an objective with an air gap between the front lens and the specimen would only equal one.

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