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Surface reflexion interference microscopy of bullspermatozoa

By SIMON IVERSEN

(From the Cancer Research Department, Royal Beatson Memorial Hospital, Glasgow)

With 2 plates (figs, i and 2)

SummaryThis type of microscopy gives absolute measurements of the vertical dimension andconfiguration of an object. As an example bull spermatozoa were used and measure-ments showed the vertical dimension and thus the volume to be considerably smallerthan obtained by other methods.

Method and resultsThe surface reflexion interference microscope makes it possible to measure

the thickness of fixed, stained or unstained, biological objects to within afraction of the wavelength of the illuminating light.

The principle of this type of microscopy is that of Newton's rings. Theinstrument is therefore self-calibrating, the measurements are absolute andtheir interpretation is not based upon any assumptions. There are only 2limitations for applying this type of microscopy to biological material. Thefirst is that the object must be dry, which necessitates fixation of the object.For this investigation all smears were fixed in a fluid containing 3 partsethyl alcohol to 1 part glacial acetic acid. The other is that the objective issuitable for the type of reflectivity shown by the object. For this investigationa Baker surface finish interference microscope was used with objectivessuitable for objects with such low reflectivity, as for example ceramics.

When a plane surface is viewed in this microscope the field is seen traversedby parallel, equally spaced lines or fringes. Both the width of these fringesand their linear separation depend upon the degree of tilt of the observedplane in relation to the optical axis. When the angle between the observedsurface and the optical axis increases both the width of the fringes and thelinear separation will decrease. As the linear separation between 2 adjacentfringes invariably represents a vertical distance of A/2, we have for mercurylight of wavelength 0-546/4 that linear separation = cot F x 0-273 /*> whereV is the angle between the observed plane and the horizontal.

If the surface has irregularities a deformation of the fringes will take place.The pattern of this deformation will depend on the shape of the irregularityand its orientation with respect to the optical axis.

The fringe pattern is thus in certain respects comparable to the contourlines in cartography, but it is important to realize that the extent of thefringe deformation only gives an absolute measurement of the difference in

[Quart. J. micr. Sci., Vol. 105, pt. 2, pp. 245-6, 1964.]

246 Iversen—Surface reflexion microscopy of sperm

vertical direction between the adjacent surface and the measured point. Thusa solid and a hollow sphere of identical diameter will give rise to identicalfringe deformation pattern.

The determination of the thickness of an object can be carried out bydensitometry, which has the advantage of giving information of the thicknessat any point of the object, or by simply measuring the extent of the fringedeformation. The latter way of measuring is illustrated in fig. 1, A, whichshows bull spermatozoa from a fixed, unstained smear preparation. A mereinspection of the fringe pattern shows that the thickness of the head is lessthan 1 fringe, i.e. less than 0-273 /• except for a small circumscribed region ofd o-6 fj. where the thickness is about 1-5 fringe. The thickness at point a ofhead I is xyjxz = o-8i fringe = 0-221 JU., and at point a of head II is 0-82fringe = 0-224 ("-• The thickness at a point just lateral to point p of head IIis o-8i fringe and at pointy 1-5 fringe = 0-41 /x. The figure also shows thatthe tail is approximately 1 fringe over the greater part of its length and taper-ing to a thickness of approximately o-i fringe at the end.

Fig. 1, B shows the appearance of the field when observed under suchsmall tilt that the near 'fluffed out' position is reached. This demonstratesthat the height variation per unit length is very slight over the acrosomalregion of the head, increasing over the nuclear part and practically nil overthe greater part of the middle piece and tail.

Fig. 2, A to D show preparations from different bulls and demonstrate thatthe bull spermatozoon is convex upwards with its vertex corresponding to thesagittal axis. It appears from these figures that the degree of convexity isinversely related to the head area: a more slender head is more convex thana wider head, which seems to indicate a constant head volume. Taking theaverage head area to be 44 JU,2 (van Duijn, i960) the volume of the averagehead is 11 to 13 fj.3 and the volume of the tail, when calculated as a cylinder,at most 3 to 4 /x3.

This work was carried out with the aid of a full-time grant from theBritish Empire Cancer Campaign.

ReferenceDuijn, C. van (Jnr.), (i960). Mikroskopie, 14, 265.

FIG. 1 (plate), A and B, fixed, unstained smear preparations of bull spermatozoa.

FIG. 2 (plate). All fixed, unstained smear preparations of spermatozoa. A, B, and c,from Ayrshire bulls; D, from Friesian bull.

FIG. I

S. IVERSEN

FIG. 2

S. IVERSEN