HIGH SPEED LIMEAE PHOTOGRAPRY

E. NEWTON HARVEY AND F. J. M. SICHEL Physiological Labwatories, Prtnceton Unecerszty, New Jersey nnd

Univus t ty of Vermont Medrcal Col lege ,

TWO FIGURES

For liigh speed photography of objects cliflering in different directions or liaviiig components of niotioii in various directions, a fairly coni- plicated mechaiiism is necessary. Either the film must stop mornen- tarily while the exposure is made, or, if the film moves contiiiuously, the exposure time must be very sliort, or some optical and mechanical device must be interposed to make the image move at the same rate arid iii the same direction as the film during an exposure period.

Whenever tlie pattern of an object is uniform in one direction and it is important to record structural changes or movements which are limited to one direction at riglit angles to tlie patterii, the simplest type of high speed photography call be used. There is necessary only a light source, a cylindrical lens and a pliotograpliic film moving rapidly and continuously iii a direction perpendicular to the direction of the movement studied. This arraiigemeiit is familiar to all who have re- corded the deflections of a string galvanometer.

Tlie method can be ixsecl for. minute movements invisible without the microscope, and is admirably adapted for studying the changes in width of bands in single striated muscle fibers during a contraction. We oh- taiiied a number of such photographs just before the United States entered the war and an abstract was publislied (Harvey and Sichel, '42), but various duties have preveiited continuation of the experiments. The following description is given at this time in the hope that details of the method may prove useful to others.

A single striated muscle fiber from the sartorius muscle of a bull- frog is removed by the usual procedure (Sicliel, '34) and mounted on a cover slip 14 mm. wide, by folding the muscle fiber ends around tlic edge of the glass. The ends are allowed to dry, thereby holding the fiber with a slight initial stretch and so that a contraction will be as iso- metric as possible. Tlie rest of the fiber is kept moist and in good func- tional condition by bathing in Ringer 'R solution. The cover slip wit11 fiber attached is then placed 011 supports on a microscope slide so that

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176 E. NEWTON HARVEY AND F. J. M. SICHEL

the space between cover slip and slide, filled with Ringer's solution, is equal to the diameter of the muscle fiber, about 0.1 mm. Fine platinum electrodes placed near the fiber serve for stimulation.

The slide with mounted muscle fiber and electrodes is then placed on the mechanical stage of a microscope, used in the horizontal position so that tlie two dimensional microscope image can be projected through a cylindrical lens on the General Radio moving film camera. The cylin- drical lens has its axis horizontal and parallel to the muscle fiber and the film moves vertically. The cylindrical lens forms a one dimensional image, focussed on the plane of the film, and a t right angles to the dircctioii of' the film motion. The striations of the muscle fiber are rep- resented in this linear image by alternate dark and light segments of the line. Tlie boundary between dark and light segments will be sharply focussed on the film only if the cross-striations are accurately oriented so that their images as formed by the microscope are a t right angles to the axis of the cyliridrical lens. Any change in width of these will be recorded on the moving film as a function of time.

The light source is a D. C. carbon arc using 15 to 20 amperes. The light is collimated, passes a water filter to remove some heat, and strikes tlie regular optical system of tlie microscope - condenser, high power dry apocliromatic objective ( X 62 magnification) arid compensating ocular ( x 20 magnification). The cyliridrical lens of short focus is placed between ocular and camera, wliich has no lens of its own but merely a slit parall(11 to the image of the rriuscle fiber and at right angles to the image of the cross-striation and the direction of movement of the film. The cylindrical lens merely concentrates the light of the image on the slit. If the slit width is 1.0 mm. and the film moves 1000 mm. a second, the temporal resolution is the equivalent of 1000 pictures per second. We have found that an adequate exposure on super XX panchro- matic film can be obtained a t this speed. Greater light intensities could be attained with the type A-H6 water-cooled mercury arc and hence greater equivalent speed in photography. While focussing, the light intensity from tlie carbon arc should be reduced by a filter in order to prevent injury to the muscle fiber. The optical arrangement is dia- grammed in figure l.

The muscle fiber stimulus was a condenser discharge, 1 . 0 p f at 22.5 volts, passing tbi*ough the resistance of the muscle chamber and a 100- ohm resistor in deries. To record moment of stimulation the voltage drop BCYOSS the resistor was amplified and the output applied to a mercury vapor discharge tube ( a small stelailamp) placed near the slit in front of the moving film and as nearly in the optical axis as possible to re-

HIGH SPEED LINEAR PHOTOGRAPHY 177

duce parallax. Each stimulus was thereby recorded by a flash from the lamp producing an outline of the slit on the film.

Although the muscle fiber is mounted so that no great movemelit caii occur, a small movemeiit is apparent from the records. Such movement is important only so fa r a s it changes the orientation of the fiber image with respect to tlie cylindrical lens and slit, since this would interfere with the focus, arid with the apparent interstriae distance on the record. For accurate recording it is therefore important to maintain the muscle fiber image parallel to the slit. Otherwise any movement of the muscle will result in apparent and not real changes in width of muscle stria- tions. The effect can be visualized by tying two pencils together, placing

Fig. 1 Diagram of the optical systeni for high speed photography in one dimension. A, a rc light. I,, condensing lens. W, heat absorbing water filter. D, diaphragm under microscope (Mic). C, condenser. 8, microscope slide with muscle, M, in cross section and cover slip, C S , Cyl., cylindrical lens in cross section, throwing image on slit just before film, F, moving on reels R and R’.

their points on a sheet of paper and then moving the paper in one direction arid the pencils in a direction a t right angles to the paper movement. If the line connecting the two pencil points is at right angles to the dii*ection of paper movemeiit the pencil lines on the paper will always be equidistant as measured at right angles to the direction of paper travel, but if the line connecting the pencil points makes a slight angle with the direction of paper movement, then the pencil lines will no longer be spaced at this distance and distortion will appear.

Figure 2 is repi-odueed to sliow the type of record which is obtainecl by this method of photography, but more work will be necessary to make certain of changes in the striations, The moment of stimulation is clearly visible as well as the light and dark bands. Interstriae dis- tance is about 2.5 micra. By enlarging prints of the films this distance can be increased to 12 mm., an enlargement of 5000 diameters. The time

178 E. NEWTON HARVEY AND F. J. M. SICHEL

axis is recorded by a 100-cycle electric tuning fork whose shadow shows in the upper par t of each figure. A reflected spot of light from the fork is also evident as a trace in the top record. We recommend this method of recording as the simplest possible where data a re needed in one direction only.

Fig. 2 Top. A print of the record (B, Oct. 20, 1941) of a muscle contraction as recorded on 35-mm. moving picture film. The highly magnified striations of the single muscle fiber are clearly visible a t left, before the vertical streak of light which marks the time of stimulation. Time scale on record is 0.01 seconds and distance scale a t right is 0.01 inin. between horizontal lines. The contraction in this record probably took place in a region of the fiber out of the field of view, so tha t only passive displacement of the fiber is recorded. Middle, a similar record of another single muscle fiber (Al, Oct. 20, 1941). Bottom, The A,, Oct 20, 1941, record enlarged t o show details of the change in early par t of contraction. The time and distance scales ( a t right) are correspondingly enlarged.

SUMMARY

A simple method of recording the change in width of living muscle fiber striations during contraction is described. It can be used with high powers of the microscope and is the equivalent of taking 1000 or more

H I G H SPEED LINEAR PHOTOGRAPHY 179

pictures a second. The method is adaptable for recording any changes in position of structures which take place in only one direction.

LITERATURE CITED

HARVEY, E. NEWTON AND F. J. M. SICHEL 1942

SICHEL, FDRDINAND J. M.

A method of recording the dimensions

The elasticity of isolat,ed resting skeletal muscle fibers. J. of muscle fiber striations during contraction. Fed. Proc., vol. 1, p. 38.

Cell. and Comp. Physiol., vol. 5, pp. 21-42. 1934