No.2 - Method of Micro-measurement.
Many times it is desirable to obtain the measurement of various objects met in the course of routine observations through the microscope. Approximate overall dimensions of such objects may be readily obtained by means of a calibrated micrometer eyepiece with a ruled scale located at its inner diaphragm.
When more accurate measurements are required, they may be obtained by means of a carefully calibrated filar eyepiece micro- meter, with a traversing spider-line actuated by a micrometer screw. This latter method calls for considerable skill in manipulation, and I have found its operation to be very tiring on the eyes, particularly when measuring the intervals of closely spaced structure, such as the markings of the finer diatoms.
A third method, prefered by many microscopists, is to make a photomicrograph of the object to be measured, and knowing the magnification at which the picture was taken, the size of an object on the print, may be measured with a ruler and its actual size calculated.
A variation of this method is to make a separate photomicrograph of the rulings of a stage micrometer under identical conditions of magnification, and use the print showing the enlarged rulings as a scale to measure directly the true size of the object.
Another and more accurate variation of the photographic method, that I prefer to any of the methods already described is to make a single photomicrographic negative showing not only the object to be measured, but also showing a narrow scale along the edge of the same negative indicating some unit of measure, say 10 microns, at that magnification.The final measurement of any part of the object shown on the photomicrograph may then be readily determined by making a direct comparison with the known intervals of the superimposed scale.
To obtain such a negative showing the object and the magnified scale simultaneously, I use an open backed plate holder, into which is first placed a clear plate of selected glass bearing a row of opaque markings along one side of its inner face. These markings were cut into the glass, spaced by direct comparison with the image of the rulings of a stage micrometer projected onto the glass, and accurately indicating actual units of measurement at that magnification.
The plate holder is loaded, in the dark room, with a piece of cut film, emulsion side down and in direct contact with the markings on the glass plate. Both plate and film are then held in position by the clamping action of the back of the plate holder.
The exposure is made in the conventional manner, except that it is made through the glass pieta held in direct contact with the photographic emulsion. The resulting negative and all subsequent prints will then show the enlarged image of the object superimposed on a scale indicating its true size.
A differently marked glass plate will be required for each different magnification used, as produced by various combinations of objectives, tube lengths, eyepieces, and camera bellows draw. However, unless unusually versatile in his work, the average microscopist will find that not more than two or three such plates will suffice for all requirements. (It should be noted that if an objective with a correction collar is being used, different magnifications result at different settings of the collar.)
By this method, as described, we eliminate any possible errors due to the unpredictable shrinkage of either the photographic film or paper during processing, both of which are considerable in actual practice, This method also permits the enlargement of the final print to any convenient size and magnification without affecting the relative size of the scale and the object, which will remain constant during any enlargement or reduction.
With reasonable care to maintain conditions identical with those under which the markings on the glass plates were originally prepared, the accuracy of the final results by this method will compare very favorably with those obtained by the expert use of a filar eyepiece micrometer. The convenience of making the final measurements of closely spaced structure by this method, as compared with the eye racking observationthrough a filar will be obvious to all who have subjected themselves to that ordeal. The described method provides the added advantage that a permanent record is provided, available at any future time.