Working with Microscopes

A display of items drawn from the Scientific Instrument Collection of the Macleay Museum

Accessories

The compound microscope - what is normally thought of as a microscope - having an eyepiece and an objective separated by a tube and used for examining small objects was invented about 1600. A number of investigators made significant discoveries with compound microscopes in the late 17th century. It was not until the early decades of the 19th century that various technical problems were significantly diminished that the microscope became a valuable instrument of scientific research.

The problems were especially those of optics. Although achromatic lenses had been developed for telescopes in the mid 18th century, the small lenses used in microscopes presented difficulties of manufacture. The improvement of objectives, eyepieces and illumination between 1830 and 1880 brought the light microscope to the limits of resolution. In the latter years of the 19th century many of the micro-organisms responsible for infectious diseases were discovered. These discoveries and consequent improvements to public health could not have occurred without the dramatic improvements to the microscope.

This display looks at various accessories used with microscopes. Many of these were developed in the 19th century and contributed to the scientific use of microscopes.

Optical Glass

The lenses of microscopes and other optical instruments use special sorts of optical glass. Individual lenses with spherically polished surfaces produce spherical and chromatic aberration. Lines of light passing through the lens at various distances from the centre come to a focus at different distances from the lens. Also a wedge of the lens is like a prism dispersing the different wavelengths of white light into the colours of the rainbow. White light passing through a spherical lens is slightly separated into colours which also come to a focus at different distances from the lens.

Early telescopes and microscopes consisted of a number of individual lenses. The combination exaggerated the two forms of aberration, leading to indistinct images. This problem was further compounded by variations in the optical density of the glass available in earlier centuries. These problems were partially overcome in the 18th century with the development of special optical glass. It was found that combinations of lenses with different dispersive properties could reduce the aberrations significantly. Because of the smallness of the lenses, achromatic microscopes were not produced until the 19th century.

Objectives

At the start of the 19th century microscopes were individually crafted and objectives made for one microscope would not necessarily fit another. In the 1820s and 1830s objectives were beginning to be produced with lens combinations that largely corrected the problems of chromatic and spherical aberration. With the rapid improvement of microscope optics a standardised screw thread was introduced about 1860 by the Microscopical Society of London so that new objectives would fit an existing microscope body.

With an increased understanding of geometrical optics and improved lens construction other improvements followed. The use of a glass cover slip to sandwich a specimen on to a slide affected the rays of light passing from the specimen into the objective. To compensate for this Andrew Ross introduced a cover slip adjustment into his objectives in 1837. The last major improvement to objectives came with the introduction of a droplet connecting the top of the cover slip with the bottom surface of the objective, water in the 1860s and then oil in the 1880s. This significantly increased the aperture of the lens and brought the resolution close to the theoretical limits for light. The next major advance came with the development of the electron microscope half a century later.

Testing Objectives

The idea that the detailed structure of natural objects could be used to test the power of resolution of objectives was introduced by Dr R.C. Goring in the 1820s. These test objects were diatoms, the scales of butterfly wings and other small, highly structured objects. The introduction of test objects provided the means to compare one objective with another. This was a powerful stimulus for the production of achromatic objectives with larger apertures in the following years.

Natural objects, such as a particular species of diatom, show a certain amount of variation from one specimen to another. A more consistent way of testing objectives was introduced by F.A. Nobert (1806-1881) who made very fine rulings on glass from the 1840s. These rulings were produced in a series of bands. This was a further stimulus to improve objectives. When the finest band had been resolved Nobert produced a new test plate with bands of finer rulings. Eventually Nobert produced rulings finer than the limits of resolution with light microscopes at about one tenth of a micron.

Test Objects

Abbe test plate, Carl Zeiss, Jena, c. 1900 (96/017)
This tests for the appropriate thickness of cover slip to use with a particular objective. The six circles are cover slips of different thickness. Under them are rough diffraction gratings. Light passing through the gratings and the cover slip produces a diffraction pattern when viewed down the microscope with the eyepiece removed.

Diatoms

Test diatoms, Ernst Leitz, Wetzlar, c. 1890 (98/027)

This slide bears numerous examples of the same species of diatom, Pleurosigma angulatum. The photograph shows a detail of this slide. The fine internal structure of a diatom, not visible in the photograph, was used to test the resolution of an objective.


Grayson ruling, about 1900 (Private Collection)
Following in the footsteps of Nobert, H.J. Grayson in Melbourne produced rulings that gained a world-wide reputation. This test ruling has 12 bands ranging from 10,000 to 120,000 lines per inch.

Illumination

17th century illustrations show microscopes being held up to the light to illuminate specimens. It was generally found more satisfactory to have the microscope fixed on a firm base. This led to the use of artificial light and large condensing lens such as a spherical glass vessel filled with water. Such an arrangement was illustrated by Robert Hooke in 1665. In the 18th century several improvements were made to the mechanical design of microscopes including a substantial stage for mounting the specimen. A mirror mounted below the stage for reflecting light through the specimen then became a standard feature.

In due course various devices were introduced below the stage to control the light passing through the specimen. These ranged from a wheel of stops, a series of holes of various sizes that could be rotated into position, to a complex series of lenses making up a substage condenser. With the improvement of objectives in the 19th century, precise control of illumination passing through the specimen became critical. Mirrors and substage condensers can be seen on the microscopes in the case behind you. Other devices for illumination are shown here.

Opaque Illumination

The microscope is especially suited to examining translucent objects illuminated from below. Other techniques are required to illuminate opaque objects so that sufficient light passes into the objective. These involve side illumination and various kinds of reflectors. The Lieberkühn reflector is a collar with a silvered parabolic flange. The collar fitted over the end of the objective with the silvered surface reflecting light on to the object. This was introduced in the 18th century and was much used in the 19th century. Two other forms of reflector are shown here.

Side reflector and cover

Side reflector and cover, c. 1900 (91/030)
With the microscope lamp placed close to one side of the stage, the parabolic side reflector was mounted close to the specimen and objective on the opposite side so that the opaque object would be well illuminated from both sides. The reflector has an arm to fix it to the microscope and a cover to protect the silvered surface when not in use.

Vertical illuminator

Vertical illuminator, [R. & J. Beck] c. 1900 (91/030)
This is placed between the bottom of the body and the objective. Light entering from the hole on the side is reflected by a thin glass disc (in fact a cover slip) down through the objective on to the object.

Polarisation

Polarised light can bring out otherwise invisible details in various objects, especially translucent objects. William Nicol (c. 1771-1851) announced the use of crystals of Iceland spar to polarise light in 1829 but it was not until the second half of the 19th century that Nicol prisms were widely adopted. A pair of prisms is required, the polariser mounted below the stage and the analyser mounted between the objective and the eyepiece. One or both can be rotated. By 1900 microscopes designed for petrological work (examining thin slices of rock) incorporated polarising prisms as a built-in feature.

Objects examined in polarised light display rich colours. With the rapid improvement of the compound microscope in the middle decades of the 19th century a world of miniature things was discovered. Polarisation also enabled familiar things to be seen in a new light. Even such a commonplace object as a finger nail presents a rich splash of colour.

Illustration

Representing what was seen through the microscope presented difficulties to early microscopists. For the most part what was seen through telescopes were distant but familiar things enlarged - buildings, mountains, ships and so on. Much of what was seen through microscopes was completely unfamiliar. This presented greater problems of representation. The first substantial series of microscopic illustrations was published by Robert Hooke in 1665 in his Micrographia. Hooke studied his objects under many different lighting conditions before sketching what he saw.

The adaptation of the camera lucida to the microscope provided a significantly improved means of drawing microscopic objects. Invented by W.H. Wollaston at the beginning of the 19th century, the camera lucida enables a scene and the drawing paper to be brought into view together. Several designs were invented for use with the microscope.

Hooke, Micrographia

Recording Microscopic Objects in 1665

"… of these kind of Objects there is much more difficulty to discover the true shape, then of those visible to the naked eye, the same Object seeming quite differing, in one position to the Light, from what it really is, and may be discover’d in another. And therefore I never began to make any draught before by many examinations in several lights, and in several positions of those lights, I had discover’d the true form. For it is exceeding difficult in some Objects, to distinguish between a prominency and a depression, between a shadow and a black stain, or a reflection and a whiteness in the colour. Besides, the transparency of most Objects renders them much more difficult then if they were opacous."

Robert Hooke, Micrographia, 1665

Abbe camera lucida

Aids to Drawing

Abbe camera lucida, unsigned, c. 1890 (89/017)
This mounts above the eyepiece of the microscope in such a way that the large mirror and a small prism bring the object and paper into the same view. This camera lucida belonged to W.A. Haswell (1854-1925) who was the first Challis Professor of Zoology, 1890-1917. His initials can be seen on the wooden case.

Camera lucida, unsigned, c. 1900 (86/018/3)
This more compact version of the Abbe camera lucida has a small prism in place of the silvered mirror.

Photomicrography

The introduction of photography in 1839 provided a new means of recording visual evidence. The technique was soon adapted to the microscope, especially after the introduction of collodion wet-plate negatives in the early 1850s. Photographs taken through microscopes became an essential technique for illustrating scientific publications by the end of the 19th century.

Common wood ant, lantern slide, c. 1900
This photomicrograph has been commercially reproduced as a lantern slide for classroom use. It comes from a large collection of lantern slides used by Professor Edgeworth David in the Department of Geology early this century.
Historic Photograph Collection

Microphotography

The Manchester optical instrument maker, John Benjamin Dancer (1812-1887), was the first to produce microscopic photographs. He had experimented with producing photographs to be examined under the microscope soon after the daguerreotype process appeared in England. These were not successful. Scott Archer’s wet collodion process enabled photographs to be produced on glass. Using this process Dancer produced the first successful microphotographs in 1852. He subsequently supplied an extensive selection of microphotographs commercially. A number of other people produced microphotographs privately and commercially from the mid 1850s.

The growth of an amateur market for microscopes in the second half of the 19th century created an extensive demand for microphotographs and a very wide range of subjects was produced from photographs of the Royal Family to reproductions of famous pictures. The recreational market for microphotographs faded out around 1900 but by then the value of miniaturisation of documents had been demonstrated.

Measurement

Measurement is a basic scientific activity. As microscopy became an increasingly important scientific practice in the 19th century methods were developed to measure microscopic objects. A simple technique involved comparing an object directly with a scale. First the microscopic object was projected on to a piece of paper, then without adjusting the settings on the microscope the object was replaced by a scale. The object, then the scale, could be traced on to the piece of paper.

A more involved technique uses a known scale on the stage and another, often arbitrary scale in the eyepiece. The eyepiece scale then provides the point of comparison between the object and stage scale.

Stage micrometer, Carl Zeiss, Jena, c. 1890 (97/033)
Within the small black circle is a scale 1 mm long divided into 100 parts.

Screw-micrometer eyepiece, c. 1930 (86/018/1)
Originally developed in the 17th century for use with telescopes, the screw-micrometer eyepiece has a graduated drum on the side to indicate the separation of two index threads within the eyepiece by which the length of the image is measured. One thread is fixed, the other movable.

Jackson eyepiece micrometer

Jackson eyepiece micrometer, c. 1910 (99/001)
The micrometer is mounted in a small slider that can be inserted into a slot in the eyepiece.

Eyepiece micrometer, c. 1930 (96/034)
The glass disc is placed on the diaphragm within the eyepiece. The scale is 10 mm long and is divided into 100.

Dr Dollar

Dr Dollar’s Integrating Micrometer, Unicam, c. 1940 (86/018/10)
"This instrument is a microscope stage-micrometer for the quantitative estimation of one to six different kinds of constituents in a substance, by the Delesse-Rosiwal method. It is intended primarily for linear micrometric analysis in petrography, but has similar uses in mineralogy, chemistry and metallography. Economic applications include the volumetric evaluation of components in building stones, road-metals, solid fuels, refractories, and slags." The integrating micrometer was invented by A.J.T. Dollar in 1936.
Transferred from Geology and Geophysics

Slide Preparation

Early Slides

In the 18th century microscopes were often supplied with sliders bearing a series of four or five specimens. The sliders were made of wood or bone. By the 1830s glass slides were coming into use. These were of various sizes, often 1½ x ½ inch. When the Microscopical Society of London was founded in 1839 one of its first decisions was to standardise slides to 3 x 1 inch and 3 x 1½ inch. While in England the former size was generally adopted by commercial preparators European preparators continued to use a variety sizes until late in the 19th century. The 3 x 1 inch slide is now used universally except where the specimen requires a larger slide. The standardisation of slides was a great benefit for the design of microscope accessories such as mechanical stages as well as for the storage and transmission of slides.

Wooden sliders, about 1840 (82/013)
Microscopes provided as recreational toys in the 18th and 19th centuries often included sliders made of bone or wood, each with a number of specimens. These four wooden sliders each contain four objects. Two of the sliders contain opaque objects in wells, the other two transparent objects sandwiched between clear mica. These were supplied with the drum microscope behind.

French slide, about 1860 (89/049)
The specimen is the tongue of a terrestrial snail and is mounted between two pieces of glass.

Cutting Thin Slices

Many biological substances are best examined under the microscope when cut into very thin slices. Robert Hooke described the use of a very sharp knife to cut cork by hand in the 1660s. The first mechanical microtomes were not invented until about 1770. Substantial mechanical microtomes became a standard tool in the later decades of the 19th century.

Hand microtome, Nachet, Paris, c. 1890 (89/061)
This is the simplest form of microtome with the embedded specimen in the central well raised by a screw and cut with a hand-held knife.

Microtome knife, John Heiffor, Sheffield, c. 1950 (90/008)

Diamantine powder, Olivier Mathey, Geneva, c. 1900 (98/016)
This fine powder was used for sharpening steel microtome knives.

Strop, Cambridge Instrument Co., c. 1960 (97/025)
It is important that microtome knives have a sharp and smooth edge for cutting fine and even sections. A rough edge would produce sections of uneven thickness and be liable to tear the specimen.

Fossil wood

Fossil Wood from Coal, Andrew Pritchard (?), c. 1840 (89/049)

The sections of fossil wood from Derbyshire have been prepared in transverse, tangential, radial sections. Sections of fossil wood were first prepared in this manner in the 1830s. This slide with diamond-engraved inscriptions is in the style of Andrew Pritchard.

Stem of Pinus, possibly F.W. Hall, c. 1890 (98/027)
Transverse, Longitudinal and Tangential Sections This slide come from a collection which includes several slides by F.W. Hall dating from the 1880s. Frederick William Hall (d.1933) was a Sydney doctor.

Serial sections: Transverse sections of Trout embryo (89/049)

Clearing and Mounting

Specimens often go through several stages of preparation before they are ready for viewing. Substances used in one step may need to be removed in the next. A specimen is mounted in paraffin wax for cutting by the microtome. The wax is dissolved out by alcohol. This has a low refractive index and therefore needs to be removed to improve the visibility of the specimen. The substance used for this, such a cedarwood oil, is known as a clearing agent.

Cedarwood oil bottle

Cedarwood oil bottle, May and Baker, London, c. 1910 (89/010)
Cedarwood oil has a long history of use for clearing animal tissues and was also used as an immersion oil with immersion objectives.

Canada balsam bottles
Glass bottle with wooden lid and applicator, c. 1930 (97/036)
Metal canister with applicator, c. 1960 (98/032)
This canister mimics an objective box and so could be easily slipped into an objective fitting in a microscope case for easy transport. The applicators made it easy to deposit small quantities of mountant or clearing agent on the specimen before sealing under a cover slip.

Staining

Stains are used to bring out detail in biological specimens. Different stains react on different structures within cells and in tissues and so various combinations of stains can considerably enhance specimens, including transparent specimens, where detail would otherwise be difficult to distinguish.

Although Leeuwenhoek used saffron as a stain at the beginning of the 18th century it was not until the mid 19th century that microscopists began to use stains with any regularity. With the improvements in microscope optics and the capacity to resolve finer detail stains became an essential tool of research by the 1880s. The development of aniline dyes greatly increased the range of useful stains.

Slide staining trough, c. 1960 (96/074)
The glass tank has ten slots for placing 20 slides back to back into the tank for staining the specimens.

Staining pallets, c. 1930 (79/003/57)
Made by R. Fowler Ltd in Marrickville, these pallets were used in the University for preparing alizarin stains. Sometimes specimens were mounted on the cover slip and stained before mounting on the slide. These pallets were used for such staining.

Eosin stains, c. 1950 (97/034)
These two packets contain tiny glass phials with very small quantities of dry stains. These would have been added to methyl alcohol for use.

Stained sections, 4 March 1905 (98/027)
The slide shows three sections of the spleen of a fowl stained with haematoxylin and eosin. The slide is one of an extensive series prepared by Hugh Poate while a medical student at the University of Sydney. He went on to a distinguished career as a surgeon.

Mounting

In the 19th century a number of substances were applied to specimens to preserve them and improve their visibility. Canada balsam, a resinous liquid, was commonly used. The balsam was dissolved in a volatile solvent such as xylol so that it would dry out sufficiently to secure the specimen under a cover slip.

Sira mountant, Stafford, Allen & Sons, c. 1930 (98/016)
'Sira' was a proprietary mountant suitable for inorganic substances.

Cover slips
Circular cover slips in box, c. 1920 (93/028)
Square cover slips in box, c. 1960 (97/041)
Cover slips secure a specimen on to the slide. With improved objectives, especially the introduction of immersion techniques, the thickness of cover slips became important. Gauges were produced for measuring this thickness and objectives could be adjusted accordingly.

Various microscope slide mounts

Cells and Ringing

Cell mounts, c. 1910 (98/027)
The first slide has a manufactured cell mounted on it ready to take a thick specimen. This would then have been covered with a cover slip and sealed. The second slide shows such a preparation. Here a whole fly has been mounted and sealed. The blue and gold lines have been applied on a turntable.

Microscope turntable stage

Turntable, R. & J. Beck, London, c. 1880 (89/061)
Turntables were used to ring mounts on slides. The specimen was covered with a circular cover slip and the edge was then sealed. Sometimes amateur microscopists had a particular set of colours which acted like a signature.

Three-dimensional Specimens

A number of techniques were developed for viewing larger objects. Fish plates (or frog plates) were cradles that could be mounted on to the microscope stage for observing the circulation of the blood through a thin membrane such as the tail of a live fish. Live boxes could constrain small animals within the field of view of the microscope. Specimens were also preserved wet or dry in deep cells mounted on a slide.

Wet Cell, Lung of Fowl, outside, Norman Preparer, c. 1880 (98/027)
John Thomas Norman (c. 1814-1893) began selling microscopical preparations in 1846. His son continued the business until the 1930s. Slides by ‘Norman’ from throughout this period are noted for their quality. This wet cell is still in fine condition after more than a century.

Live Box, c.1930 (93/028)
Viewing live insects and other small creatures through the microscope was facilitated by the use of a live box from an early period. Such live boxes were a standard accessory supplied with microscopes in the 18th century.

Microscopic Traditions

Techniques of slide mounting evolved over a long period. Sometimes connections can be traced from one commercial preparator to the next. This is illustrated with these three slides.

C.M. Topping, Tongue of scorpion fly, c. 1860 (94/003)
Charles Morgan Topping (1800-1874) began preparing slides commercially about 1840. He gained a high reputation for his mounts of the mouth parts of flies.

Edmund Wheeler, diatomaceous dredgings, 1875 (89/049)
Wheeler (d. 1885) gained a high reputation for the mounts he sold including Webb’s diamond engravings. This slide was prepared from dredgings taken in Antarctic waters on the scientific exploration voyage of HMSS Challenger.

W. Watson & Sons, Wheeler mount, section of mica, 1884 (89/049)
William Watson began his optical business in London in 1837 and began manufacturing microscopes in the 1870s. The firm acquired Wheeler’s stock and business in 1884 and remained major suppliers of microscopical mounts until the late 1930s.

Slide Preparation

H.G. Wells provided a vignette of commercial slide preparation in London in 1888:

"I made my sketches under the Bloomsbury Dome and enlarged them as diagrams in a small laboratory Jennings shared with a microscopist named Martin Cole in 27 Chancery Lane. Cole, at the window, prepared, stained and mounted the microscope slides he sold, while I sprawled on a table behind him and worked at my diagram painting. Cole’s slides were sold chiefly to medical students and neatly arranged upon his shelves were innumerable bottles containing scraps of human lung, liver, kidney and so forth, diseased or healthy, obtained more or less surreptitiously from post mortems and similar occasions."

H.G. Wells, Experiment in Autobiography, 1934

Martin Cole: Prepared Slide, c. 1890 (89/049)
This preparation of a transverse section of human oesophagus was one of a series produced for medical students and well illustrates Wells’ recollection.

H.W.H. Darlaston

Herbert William Hutton Darlaston (1867-1949) operated as a commercial mounter in Birmingham from about 1900 until the late 1930s. These examples show his superb mounting of insects. It in not surprising that he made flea mounts for the well known entomologist, Miriam Rothschild.

Dragon Fly; Scorpion Fly; Forest Hopper (98/027)
Snipe Fly (94/003)

Foreign Mounters

While most of the commercial microscope preparations sold in Australia before 1940 were English, a small number of commercially produced slides from other countries are represented in the Macleay Museum.

Charles Bourgogne: France, c. 1870 (89/049)
The Bourgogne family were active as slide preparators in Paris from the 1850s. This slide was retailed in Dublin by Yeates & Son.

Dr Ek



Dr Ek: Germany, late 19th century (89/049)

Germany played a leading role in the development of biology in the 19th century. The use of the microscope was an essential part of this. A considerable number of makers of microscopes were active in Germany from mid century of whom Carl Zeiss and Ernst Leitz are the best known. A photograph of the spicules from the stomach of a sea urchin on this slide is shown at the left.

M. & KATERA: Japan, c. 1930 (89/049)
The Optical Workshop of M. & KATERA was established in 1915 by three partners, F. Matsumoto, Y. Kato and S. Terada. The firm, which made microscopes, reflects the beginning of the scientific industry in Japan which became so significant after the Second World War. The name was discontinued in 1934.

Australian Mounters

Very little is known about early Australian microscopists. A number of slides with printed labels of Australian microscopists are held in the Museum. It is possible that some of these operated commercially though a keen amateur preparing numerous slides would have found it convenient to have labels with their names printed. Microscopists often exchanged slides.

W. Bäuerlen: Bandicoot hairs, c. 1890 (89/049)
William Bäuerlen was primarily a botanist. Little is known of his life. Born in 1845, he spent much of his life in Australia. He was a member of the New Guinea Exploring Expedition of 1885.

Monkman section of Pampas grass



Noel Monkman: Transverse section - leaf of Pampas grass, c. 1950 (89/049)

Monkman (1896-1969) gave up a career as a cellist to be a full-time naturalist. He worked on the Great Barrier Reef from about 1930 and was a pioneer of cine-micrography. Monkman was elected a Fellow of the Royal Microscopical Society in 1948.

T. Steel: Spicules from siliceous sponge, March 1885 (99/002)
Born in Glasgow, Thomas Steel (1858-1925), came to Sydney in 1882 to work for the Colonial Sugar Refining Co. as an industrial chemist. He was also an active naturalist throughout his life and was president of the Linnean Society of NSW, 1905-07.

W.H. Wooster: Rasp of shellfish, Cronulla (98/027)
Wooster was a Victorian microscopist active in the 1880s.

Photomicrographs for this display were taken by Malcolm Ricketts, School of Biological Sciences