Monthly Archives: February 2015

Glass Chamber and Hanging Droplet

The development of the “hanging droplet method” was a crucial step towards establishing the micromanipulator as a useful tool with which to study the living cell. Developed around 1904  by Marshall A. Barber, a professor of bacteriology at the University of Kansas, this method involved placing a sample in a droplet of liquid (usually water) adhering to the bottom surface of a large cover slip that formed the roof of a glass chamber.

This “moist chamber” was placed on the microscope stage such that the user observed the sample in the droplet through the flat surface of the cover slip. The chamber was open on at least one end to permit the entry of microtools whose tips operated upward into the droplet. While these tool tips could be moved independently relative to the sample using a micromanipulator, the sample itself could be moved relative to the tips by clasping the chamber in a mechanical stage as shown in the photo below.

Glass chamber mounted

A glass chamber in place on a microscope stage with microtools (one microscalpel, one microsyringe) extending into it. The chamber is held in a mechanical stage which can position it along the x and y axes. The micromanipulator mechanisms can move the microtools along three axes relative to the chamber. In this case, the microscope incorporates a microscope stage that can be rotated. [Chambers 1929, 63]

Chambers 1921

In this illustration, a hooked microscalpel is used to cut through the soft membrane of a sea-urchin egg. The surface tension of the droplet is firm enough to hold the egg in place as the scalpel is drawn downward through it. [Chambers 1921b, 327.]

While a droplet of liquid might seem a weak enclosure, it could usefully hold a variety of microscopic objects. Individual bacteria or other microorganisms could be recovered from droplets of growth medium adhering to the cover glass. Larger microorganisms, such as amoebas or paramecia, could be caged within  a liquid droplet for the purpose of vivisection or the injection of colour indicating dyes to study cell metabolism. Fresh tissues and cell samples could also be studied in this way.

The hanging droplet method addressed several problems with previous methods. The glass enclosure provided a moist atmosphere that prevented the liquid from evaporating. This was maintained by pooling water in a shallow well on the floor of the chamber or by lining its sides with damp blotting paper. The sample could be preserved in the chamber by closing the open end around the shafts of the microtools. A simple seal could be made using a cardboard frame containing cotton fibres and Vaseline. [Chambers and Kopac 1950, 511-512]

An inverted microscope developed around 1933 for use with the Chambers' micromanipulator. It would be interesting to discover exactly what role the hanging droplet method played in the development of the inverted microscope as a laboratory instrument.

An inverted microscope developed for use with the Chambers’ micromanipulator. It would be interesting to discover exactly what role the hanging droplet method played in the development of the inverted microscope.

Whereas earlier methods had required the observer to  look through the meniscus of a pool of liquid, which introduced optical distortions when the tool tips entered the liquid,  the hanging droplet method placed the sample beneath the flat surface of a cover slip. In addition, previous methods had placed the microtool tips between the microscope objective and the sample. When the tools were located beneath the droplet, the microscope objective could be  moved closer to the sample. This made room for more powerful objectives with shorter focal lengths.

The system was easier to use with an inverted microscope. One such instrument was developed by the Leitz company for the highly-effective Chambers’ micromanipulator in 1933. In this case, the droplet sat on the floor of the chamber and the position of the tools and objective were reversed.

Evolution and elaboration

From the invention of the hanging droplet technique in 1904 to the mid-to-late nineteen thirties, when the technique had become widely used, the technology evolved considerably. The chamber was initially made locally, most likely by laboratory glass blowers. As it was relatively easy to make, this practice no doubt continued—instructions for making it were provided in the 1950s and possibly later as well. [Chambers and Kopac 1950, 511-512]

Moist chambers did become available commercially, notably a version—again manufactured by Leitz—that was used with  a dark field condenser created specifically for micrurgical work. Other specialized equipment such as an electrically heated jacket for the chamber was devised by researchers. [Howland and Belkin 1931, 16-18]

As the system began to reach its potential, shorter chambers were used. This is because the height of the chamber was determined by two factors: the need to manoeuvre the delicate glass tool tips within the glass enclosure without breaking them, and the need to properly illuminate the sample using the microscope condenser.

The microscope condenser is an optical system located beneath the microscope stage that focuses light onto the sample. Since samples are usually placed on glass slides between 1 – 1.2mm  thick, standard microscope condensers are designed to focus light within a narrow range just above the microscope stage.

A particularly tall moist chamber set up for isolating bacteria. The cross marked on the top of the cover glass is to help locate bacteria.

A particularly tall moist chamber set up for isolating bacteria. The cross marked on the cover glass is to help orient the user to the location of bacteria colonies. [Kahn 1922, 346]

The hanging droplet method located the sample substantially beyond the normal operating range of the unmodified microscope condenser. This was not a problem initially since the hanging droplet method was first used to isolate microorganisms, a process that did not require especially high magnification. This type of operation would always use a larger chamber for convenience since it involved the frequent removal and reinsertion of micropipettes into the chamber in order to gather multiple samples.

As the micromanipulator was increasingly used with more powerful objectives to discern the structures within living cells,  stouter chambers were required in order to achieve proper illumination from the condenser. This also required condensers that could focus light well above the microscope stage. Specialized condensers were sold for this purpose, though many conventional research microscopes featured condensers that could be easily modified. [Chambers 1922a, 339-340]

Sealing the chamber, isolating the droplet

chambre a huile_small

The hanging droplet method, as originally devised, had several limitations. Beyond the difficulty of illuminating the sample, the open enclosure, heated unevenly by the microscope illuminator, permitted moisture to evaporate from the droplet and recondense on the walls of the chamber. [Fonbrune 1949, 130]

The “oil chamber” (chambre à huile), developed in the early 1930s by French researchers Pierre de Fonbrune (1901-1963) and Jean Comandon at the Pasteur Institute, addressed a number of these limitations. [Fonbrune 1949,  130-131] This was a very shallow chamber (1-3 mm high) open at both ends.

Fonbrune_nematode_138

Biological investigation in the oil chamber could involve blood, living tissue or microorganisms. In the upper two photos, a nematode caught in the ring-shaped traps of the carnivorous Dactylaria brochopaga fungus. The bottom two photos show a trap, open in the first photo, closed in the second after having been touched by the tip of a microneedle. [Fonbrune 1949, 138]

The sample in an oil chamber was contained, as before, in a liquid droplet suspended from a coverslip. However, this droplet was surrounded by paraffin oil which effectively created a microenvironment isolated from the atmosphere. The shallow space between the coverslip and the glass floor held the oil in place by capillary action. This prevented the loss of moisture to the atmosphere and increased the survival time of living samples .

The oil chamber was likely made possible by developments in the technology of micrurgy that had accrued by the 1930. This would have included instruments for making microtools that made it possible to produce the very short tool tips necessary to operate within the confines of the shallow oil chamber.

There were other reasons for sealing the chamber. Over the mid 1920s, Cambridge biologists Joseph  (1900-1995) and Dorothy Needham (1896-1987) experimented on cell metabolism using a modified Chambers’ micromanipulator. Some of these chemical operations—for instance those involving anaerobic bacteria or the injection of highly reduced dyes for testing the oxidation potential of microorganisms—required an anaerobic environment within the chamber. This was achieved by passing the the microneedles through a shallow trough of mercury while the chamber was filled with a stream of nitrogen. This anaerobic chamber—notably the bent needle and mercury trough arrangement—was first suggested by  Marshall A. Barber in 1918 but it was the Needhams who first explored it. [Needham and Needham 1926, 385]

Needham and Needham's anaerobic chamber. Specially formed glass microneedles (L) pass into the chamber through a trough of mercury (G). Nitrogen flows through the chamber, entering through tube D and exiting through C. The chamber is roofed over by a large cover slip (R) to which a smaller "flying" cover slip adheres by a film of water. The drop (T) us suspended from this second cover slip. [Needham and Needham 1926 , 386]

Needham and Needham’s anaerobic chamber. Specially formed glass microneedles (L) pass into the chamber through a trough of mercury (G). Nitrogen flows through the chamber, entering through tube D and exiting through C. The chamber is roofed over by a large cover slip (R) to which a smaller “flying” cover slip adheres by a film of water. The drop (T) is suspended from this second cover slip. [Needham and Needham 1926 , 386]

As a laboratory technology, the hanging droplet technique was highly adaptable and hence widely adopted. It is still in use. One also finds variations of this technique beyond the biological realm, for instance in chemistry where the  moist chamber provided a kind of miniature laboratory bench upon which capillary vessels were used to perform experiments at the microgram and nanogram level.

(Left) Top and side view of a moist chamber used in chemical experiments that was developed in the early 1750s. The chamber dimensions are around 6 x 6 cm. (Right) A simple rack and pinion micromanipulator holding a micrometer syringe for use in chemical operations. [El Badry 1963, 102]

(Left) Top and side view of a moist chamber used in chemical experiments that was developed in the early 1950s. The chamber dimensions are around 6 x 6 cm. (Right) A simple rack and pinion micromanipulator holding a micrometer syringe for use in chemical operations. [El Badry 1963, 102]

 One form of chamber in particular—a common type that was made by hand locally—is relevant to the project to recreate parts missing from the U of T’s Chambers’ Micromanipulator. The next post will describe the process of remaking that in order to produce a version that is both cosmetically similar to the original and functional.