I found the following correspondence in some old issues of The Microscope, and thought that it might be of interest to modern-day readers. Personally, rather than risk damaging my objective lenses, I use the glass plate method referred to in the text and insert a circular coverslip of the correct diameter, previously marked with a soot stripe. My Watson 40x parachromat has a ledge just above the lens and this seems to be an ideal place for a home-made phase plate.
FROM The Microscope Vol 9, No. 6, March April 1953, p148:
A NEW METHOD OF LIGHT MICROSCOPY
UNDER the above title, in a letter to the Editor of Nature, 1953, 171, 353, A. Wilska, Institute of Physiology, University of Helsinki, describes how he obtained enhanced contrast images of refractile bodies somewhat resembling conventional phase contrast images, by depositing soot from a candle flame upon the objective lens surface nearest to the image of the substage condenser until the transparency was reduced to one-half: then mounting in a watchmakers' lathe, and using a sharp tip of soft material, removing a narrow ring-shaped area of the soot, of diameter more than half the exit pupil of the objective. A corresponding disc was also prepared for use below the condenser. By sooting until a transparency of 8 to 9 per cent. was reached and then removing all the soot except for an area covering the image of the condenser ring aperture, " the background of the microscope image appears in an agreeable sepia tone, often giving the illusion of sky-illuminated objects lying on the sand floor of a shallow sea. Many features which are generally invisible with the normal use of the microscope are rendered visible; for example, influenza virus on red cell ghosts, bacterial flagella, sperm tail pencil, and delicate membranous margins in living thrombocytes.... Apparently the method can be adapted to ultra-violet microscopy and mirror optics as well." No explanations of the phenomena are offered.
C. M. WEBB, Toronto, Canada
IN connection with the excerpt from Nature entitled, ` A New Method of Light Microscopy,' published in the March-April, 1953 issue of The Microscope, (9,148) I think that a re-reading of the original will show that it has been wrongly interpreted in your extract and that it is actually a phase contrast system.
Some two years ago, after experimenting with discs and etching grooves, etc., without much success, I arrived at the method of smoking one of the elements of the objective itself and then removing most of the soot, except for a phase pattern. Taking an objective apart and sooting the lenses seems a somewhat drastic procedure at first, but if ordinary common sense is used, it is not attended by any danger or difficulty. In fact it is a very simple and expense-free method of converting ordinary objectives to phase contrast with the added advantage of having no glass discs to make or introduce aberrations into the optical system.
Positive contrast, in my opinion, cannot be successfully managed by this method, as the direct light cannot be attenuated effectively in relation to the diffracted beam. Negative contrast, however, offers most effective and beautiful results, the object appearing in ` life-like' contrast against a warm sepia background, the effect being almost stereoscopic, even with the highest objectives. Resolution is equal, and in some respects, superior to ordinary methods of illumination.
I have tried various phase contrast patterns including the circle and the cross, but I have come to the conclusion that the simple diametrical stripe is the best. This is the easiest to apply and affords sharper definition and perhaps assists in the `stereoscopic' effect.
The procedure followed is to first of all select the element of the objective which is to receive the phase pattern, that is, the surface nearest the back focal plane. In 6-mm. and 4-mm. lenses the inside surface of the rear element works very well. In the case of objectives of four combinations or more such as oil immersions and some dry fluorites, etc., the lower surface of the second element from the rear is the place for the pattern.
Separating the objective sometimes requires a little care and study. The mounts are differently arranged in the various makes and in the older type screw-together mounts, the accurate finish usually makes the actual parting lines on the exterior practically invisible. However, a close inspection will generally indicate the necessary action. With the lens separated, the selected surface is passed through the flame of a candle or cigarette lighter several times until an even coat of soot is deposited, taking care not to overheat in the process. The thickness of this coating controls the degree of contrast, a medium contrast gives the best all round results. This is perhaps best found by experiment. My experience is that the coating is at about the best thickness when with the sooted surface held under a light, the reflection from the glass just shows through.
All the soot is then removed from the lens with the exception of a narrow diametrical stripe, of which the width is about is the diameter of the lens. The handiest tool for this process is a sliver of hardwood, pointed and slightly charred at one end: it will not scratch the glass.
With a little practice this can be done, quickly and accurately, freehand. The soot comes away cleanly, and the cleaning may be finished with lens paper wrapped around the sliver.
A condenser diaphragm is then made with an aperture to coincide with the pattern on the objective, in the usual phase contrast manner. This can be either a slit aperture in opaque material, or a heavily smoked glass disc with a clear stripe; the slight transmission of the smoked area of the latter will modify the contrast somewhat.
This mask is placed in a position as near as is practical to the rear focal plane of the condenser. The condenser filter carrier will do but it is not ideal.
The stripe should be orientated in a vertical position in relation to the observer, and, of course, when the microscope is in focus no direct light should appear that is not covered by the stripe on the objective. No auxiliary telescope is necessary for the adjustment of the illumination. With the eyepiece removed, and a clear portion of a slide in focus, both the dark objective stripe and the bright condenser aperture are clearly visible, and only need to be superimposed. A corrected condenser, even of moderate aperture, it to be preferred to Abbe types. Clean slides and cover glasses are needed. Smears or particles of dust on the top surface of the cover glass or underside of the slide will spoil the definition.
The photomicrographs that were published in The Microscope volume 9, number 5 with the letter' Phase contrast and Resolution,' were made using the above method.
Readers who are following the discussion of Prof. Wilska's method of illumination will be interested to read Dr. R. Barer's contribution in Nature (171, 697; 1953) in which he shows that the effects in question are mainly due to " amplitude contrast," with some degree of phase contrast as well, should the partially absorbing layer introduce any phase change. Any such secondary phase change would result in a heightening of the effect due to the amplitude contrast. The Editor acknowledges a separate from Dr. Barer of the Letter referred to above.