chemical microscopg ewmpoelunr
Color Photomicrography in the laboratory R . P. LO\-ELAND, Eastman Kodak Company, Rochester, N . Y . placed directly. If the illumination characteristic in this plane were completely known, the photographs would be satisfactory. Methods for the quantitative and qualitative analysis of illumination are described and criteria of optimum exposure are given. Utilizing a photoelectric photometer, whose spectral response is adjusted to that of the color film, greatly simplifies illumination analysis for photography and allows a trichroic ratio method of qualitative analysis to be used that has proved practical.
An apparatus andmethod for simplifiedphotomicrography in color are described. Use of a roll-film camera and flash bulbs makes darlcroom and processing facilities unnecessary. No compensation is required because of variation in intensity or time of exposure, but a reasonably sensitive photometer is needed. Visual and photoelectric photometers are discussed. It is very advantageous for color photomicrography to have an image plane that is open for inspection and analysis, and in which the film is
T
it can be mailed to a service center or processed by the microscopist.
0 T H E laboratory worker for whom the microscope is a valuable tool, photomicrography may be either an essential technique or merely a means of making occasional records. The records are often desired in color. Hornever, the facilities and the time available for color photomicrography vary in different laboratories, and probably most workers would merely like to make an occasional photographic exposure of a field. RIicroscopists )Tho have neither photographic inclinations nor experience and lack darkroom facilities may employ a simplified method of black-and-white photomicrography ( 7 ) utilizing rollfilm cameras. Alternatively, there may be sufficient time or personnel available for making photomicrographs, and also a special photomiclographic stand for the work. The more elaborate method is to be preferred to the simplified one, but a chemist’s needs may be met satisfactorily by the latter. Usually, time is saved with adequate apparatus. Sowhere is this better exemplified than by the use of an adequate photometer properly calibrated by photographic teats for the determination of exposure.
For color Fork, a view box or camera lucida with an open image plane, that can be swung alternatively above the microscope in place of the camera, should be constructed. This may be supported on a large wooden shelf which can be held by a laboratory shelf clamp. The author’s view box was easily made from a cylindrical waxed cardboard container of the type used in packaging Ice cream; the inside was painted black and a 2 5-em. (1-inch) hole was cut in the cap. This cap was held on with tape and could be slipped on and off, as desired. Thus, the lens could be rested on this horizontal surface surrounding the hole and held in place by wax. The bottom of a 4 X 5 inch plate box having an appropriate hole cut in it is placed on top of the shelf, surrounding the cylinder. Different tops, each containing a piece of clear or ground glass, can be fitted on the view box for measuring the light or viewing the image. The lens of the viewing box should have the same focal length as that of the camera. .4s neither the focusing nor the photography will be done with it, a spectacle lens may be used. There is some advantage in substituting, for a single lens with strong curvatures, a combination of several lovxr-power lenses in a pile, whose diopter power adds up to that of the camera lens. For instance, if there is a 100-mm. lens in the camera, as is common, then instead of using a 10-diopter lens, it would be preferable to use two 5-diopter lenses. There is also some advantage in using a meniscus lens with the concave side toward the microscope. Because the camera lens and the viewing-box lens have the same focal length, the lens is focused best in the box by making the magnification on the glass of the view box the same as that in the camera, for which purpose the camera should be temporarily opened and a piece of matte film placed in it.
SIJIPLIFIED METHOD OF COLOR PHOTOMICROGRAPHY
In describing the simplified method, it is assumed that visual microscopy is the routine procedure, and hence to be interrupted as little as possible. The method consists, in general, of first independently focusing the microscope visually, then placing above the ocular a roll-film camera which has previously been focused at infinity or some other predetermined distance. .lily hand camera will do; in fact, a simple lens, such as that in a Brownie camera, is actually preferable to a more elaborate one having many optical surfaces. B miniature camera with a focaiplane shutter, which protects the film so that the lens may be removed, can temporarily be excluded from consideration. Definition in the picture equivalent to that from a large bench is obtained by the simple expedient of focusing the microscope through a telescope. The camera is preferably held independently at the point where the eye point of the microscope lies in the front surface of the lens. This method has a few disadvantages, none serious enough to make the method unsuitable for record purposes: The picture taken covers the whole visual field of the microscope, which is rarely in focus a t one setting, especially with apochromatic objectives; a flare spot is frequently formed in the center of the picture; and any dust particles or marks on the camera lens mill show on the photograph. This method may be adapted to color work by the use of 35mm. roll film, Kodachrome film, Type A, for miniature-film cameras, or Ektachrome roll film, for daylight, for KO.620-size film cameras. In metropolitan areas at least, processing service ,for Ektachrome film is usually available; in other locations,
Whereas the arrangement described can also be used with a 35-mm. film camera, if the lens is kept in the camera as in a Kodak 35, many commercial devices are available for photomicrography with the 35-mm. cameras with a removable lens. The best types for this purpose employ an alternative groundglass viewing box. Illumination for Daylight Film. Selecting a method of ‘illumination suitable for simplified photomicrography with daylight film was a problem, because the incandescent tungsten lamps used for visual ivork are definitely not suitable for this purpose. The carbon arc can be made satisfactory for this purpose, but for the average microscopist it cannot be included in a simplified method. The solution seems to be the use of the G.E. KO.5B-type flash bulbs which are coated with the proper filter layer. This method has given excellent results. The microscope may be set up for visual work as usual, but if some projection lamp is used, one with a relatively long-focus lens (such as the common 60-mm. aspheric lens) is of considerable advantage. Then a bayonet socket, to hold a flash bulb, is fastened to the end of a rod and held a short distance before the camera by means of a swivel clamp. A No. 111 opal-glass enlarger lamp bulb is about the same size as the No. 5B and fits 467
468
ANALYTICAL CHEMISTRY
obtain the exact value desired. Changes of light intensity can be obtained by using evaporated Inconel Neutral Densities, such BS those supplied by the Bausch & Lomb Optical Company. The present series have a density range of 0.3 to 1.2, by 8. difference of 0.3. An additional density of 0.15 would be desirable; otherwise, - ..__some use of the iris diaphragm for light control is required. light across the IlO-Golt side; the alternite circuit is from a For speed and simplicity of operation, a photometer is needed. flashlight case which requires a separate push an a button to ignite the flash bulb. Obviously i t is the illumination from the alternate No. 111 enThe procedure is simple. When in the course of visual examine larger lamp that must be measured, but this measurement has tion a field to be recorded has been found, the visual lamp is proved satisfactory. The photometer must he sensitive because turned off and the No. 111opal bulb is swung down into position the illumination in the image plane amounts to only approxiby means of the swivel clamp; the 32-mm. objective is substituted for the lamp, along with a law-power eyepiece, the lamp is cenmately 0.01 foot-candle. However, with a good instrument, sdtered, and the condenser is focused. The original objective and justment of the illumination requires only a minute or two. ocular are then replaced. or a hisher-Dower ocular is used with a A practical visual type of photometer can be made inexpensively. This type demands a standard lamp for compasison. Actually, the visual type of photometer is the most sensitive and ~~. ~. . . .. _..”_ have as diaphragm pieces of sheet metal, with holes of different can handle the greatest range of illumination within one instrusizes and backed by a piece of verv finely amund alass. Such a ment. (A description of the visual photometer shown in Figure 2 will be sent on request.) The details of construction of a variable distance type that is clumsier to use but easier to construct have . .~ = . It is necessary to protect the optical system from the stray light been described (6). of the flash. A lens hood is useful, but a sheet of cardboard Before the color film is exposed to a specimen, the illuminance should dso be placed in the proper position to cut off direct illufrom the No. 111 enlarger lamp must be calibrated by the premination from bulb to camera lens. liminary exposure of a roll of film. Moreover, beesuse the ilThe exposure is made by open flash-Le., with the camera luminance is read from a different lamp from that actually used shutter set on “bulb.” In this case, the time of exposure is confor photography, and the brightness varies appreciably from one stant; therefore, the illuminance must be brought to a constant No. 111 bulb to another, i t is wise t o have a number of spare value. The word “illuminance” is the term recommended by the Committee on Colorimetry of the Optical Society of America (8) calibrated bulbs. The calibration is most simply done by determining the ratio between the brightnesses of the bulbs with one for the quantitative aspeck of illumination. The problem is to as a standard. When these bulbs are burned at their rated 115 obtain this illuminance value by changing the illumination withvolts, they have high brightness but a relatively short life. It is out affecting its quality. Use of the iris diaphragm for this adworth while, therefore, to include a resistance-e.g., 35 ohmsjust.ment. is highly nndesirahle, except for slight variations to ~~
~
~~
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~~
Figure 1.
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~
~~
Apparatus Assembly for Simplified Photomicrography
V O L U M E 21, NO. 4, A P R I L 1 9 4 9
469
ocular (7). This can be overcome by merely choosing a higher ocular for the photography. However, the illuminance in the film plane decreases with the square of the magnification. The illuminance is read with the sensitive photometer and an Inconel density inserted into the beam to reduce the reading to the prescribed value. The aperture of the substage diaphragm may be manipulated slightly to give the desired readingexactly. Photomicrographs have been made of crystals and crystal structure by polarized light using this method, some of which were shown as color slides during presentation a t St. Louis. For this the neoFigure 2. Plan for Visual Photometer Lens and Diaphragm Type dymium liquid filter (3, p. 139) A . Squares of opal glase B . Achromatic lens, focal length 47 m m . , diameter 37 mm. or a Corning Glass filter No. C. Mirror surfaces 5920, one half standard thickD . Glass disk E . 1-mm. circle scratched on silver, so as to show black ness, which is useful in reproG. 4.5 x 0.375 inch metal bars (2). Lamp centered to lens and diaphragm M . Diaphragm ducing the color of certain bioN. Needle or drill rod lever to control diaphragm. Lamp can be moved and locked with Y and Z to regulogical stains (eosin, fuchsin, late distance from lens etc.) is also helpful. The most saturated colors of the first and second order are not alxays accurately reproduced by a monopack film, but the record so obtained should still be useful. The less saturated colors of pleochroism are usually reproduced satisfactorily. In this case, the determination of exposure is somewhat different; photometry of the image plane is a less positive criterion but it is still a satisfactory method. The illuminance is read with the analyzer and polarizer parallel. The exposure time is usually about four times the value calculated for the parallel position. This exposure factor has given surprisingly satisfactory results, although it depends upon the anisotropy of the material. The correct exposure can always be determined by the use of the Super Speed direct positive paper. There is usually little light to spare with the KO. 5B flash bulbs with polarized light; in fact, the use of more than one I flash bulb may be necessary. Too little light is transmitted by all but the clearest thin petrographic sections to allow photomicrogk 5 raphy by polarized light, even with t w o or three No. 5B flash I I 3000 5000 7000A bulbs. Probably it could be done by using large flash bulbs, WAVE LENGTH suitable lamp optics, and wide ordinary household-type tungsten COURTESY n. a. MACPHERSON (8) lamps as the alternate control light. Figure 3. Spectral Distribution of Light from Carbon1 Some photomicrographs of interference figures have also been Arc made. If a standard petrographic microscope is not available, such pictures can be made with a biological stand and crossed that can be switched into series with the lamp except during Pola-Screens. A picture of the aperture plane with the intermeasurement of its illumination. ference figure can be obtained for this photographic method by Advantage can also be taken of the fact that Kodak Super substituting the telescope accompanying phase-contrast equipSpeed direct positive paper, which is fast and easy to proces, ment for the usual ocular, but the quality of the image is rather makes an excellent black-and-white test material. I t s speed poor, oning to the simple optics of such a telescope. Better nearly equals that of Ektachrome film, when developed 1 minui e. quality in the image of the figure will be obtained by magnifying The simplest procedure is that in which the slide on the sta.:e the Rainsden disk above the ocular with a high-power magnifier consists of a simple colored specimen to be viewed by transmitted of good quality, using a device such as that described by Jelley light showing some clear background in the field and without ( 4 ) . In any case, if only a small crystal is present in the field, and excessive brightness contrast. Most stained cross sections of all light not coming through it is excluded by an iris diaphragm biological materials fall in this class. In this case, the illumina(or a piece of paper bearing a central hole) lying in a field plane, tion from the N o . 5B flash bulbs is usually adequate; in fact, a illumination will undoubtedly not be sufficient for flash technique. density of 0.9 was needed in the beam for a magnification of 50X lLLUMINATION FOR PHOTOMICROGRAPHY on the film, with a 16-mm. apochromatic objective. With a camera taking No. 620 film, the magnification is usually reduced Light Sources. For color photomicrography in general, lamps may be used whose light quality is correct or can to about one third of the visual magnification with the same 3/16"
IN
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ANALYTICAL CHEMISTRY
easily be corrected for the color film used, or the illumination of a lamp already in use far microscopy may be converted to the correct quality.
For the fist case, 3200' K. tungsten lamps can be used with all Type B color films, such as Kodachrome and Ektachrome Type B films, and with a Kodrtk calor compensating filter CC 4 with Kodachrome film, Type A, but a suitable lamp house and lamp Dondenser are required. The carbon arc light source is unusually reproducible from lamp to lamp, if the lamp is burned a t the same amperage and voltage. The distance between the carbons controls the voltage. This arc bas proved satisfactory, providing the line voltage is reasonably stable. A spectral-distribution curve of the cmhon arc illumination is shown in Figure 3. Ohviously, the cyanogen line adds much excess ultraviolet, but aside from this i t has excellent blaclc-hod7 distribution. The color temperature of the direct ourrent arc with an 8-mm. carbon at 4.5 amperes is ahout 3645" K. An ultraviolet filter consisting of a 2% solution of sodium nitrite in a water cell 10 mm. thick is advised, together with the Kodak color compensating filter CC13 for Type A film, or CC13 and CC15 far Type B. The second case is usually p r e f e r a b l e t h a t is, the use of ribbon filament and other tungsten lamps already used far microscopy. However, the quality of illuminatinu from the ribbon filament lamp varies from lamp to lamp, so that eaoh lamp must be calibrated. Color Temperature Meter. A laboratory-type photoelectric photometer can be used to make a color temperature meter, such a6 is shown in Figure 4, whereby the lamp can be set a t the correct color temperature comparatively quickly and accurately. The performance of the instrument depends upon the assumption that the spectral quality of the illumination from the lamp is closely expressed by some black-body curve--e.g., as formulated by the Wien-Plitnok equation. Therefore, changing the quality of the light by changing the electric current or by using a good photometric filter merely alters the curve of the system that is applicable, so that the curve o m be determined by determination of the ratio of any two ordinates, preferably a t widely separated wave lengths. Such a dichroic mechanism is inadequate for measuring or setting general illuminstion quality, where the nonstandard illumination is not of black-body quality.
Figure 4.
Color T e m p e r a t u r e Meter Assembly
current: a linear relation is then obtained through this uoint if the logarithm of the color temperature is plotted agai'nst the logarithm of the mtio of the voltage and amperage for the 500watt 3200" K. Mszda. lamps. The graph line, as determined from a number of bulbs, follows the equation: Log CT
=
1.042 log
(3+
2.025
If only alternating current is available, care must be taken t o alter the current and hence the temperature of the lamp with a rheostat and not an autotransformer, as the latter complicates the relations. In this case, the best relittion to use is the plot of the log of the color temperature against the log of the voltage directly across the lamp. Here the equation is: log CT = 0.4025 lag V 2.684. When several lamps rated for 115 volts were used, the curve passed through lag CT value of 3.501 for 110 volts, through 3.445 a t 80 volts, aud through 3.395 a t 60 volts.
+
Spectral Defects in photomicrography. In photomicrography, an extended opticill system, not chromatically correoted, is usually interposed between the lamp and the subject. This optical system usually changes the quality of the illumination that reaches the specimen from that which left the lamp; the quality e m no longer be expressed by a black-body curve, and cannot be correoted by varying the lamp current to change the temperature of the lamp. To regain the quality of illumination satisfactory for the film, it is muoh simpler to compensate for the error. Moreover, because there are just three spectral oompanents of the color films to be used, it is only necessary to satisfy these t h e e , with compensating filters, that the original balance has heeu regained. However, the spectral quality will now he different. Photometry of Image Plane. I n photomicrography, the image to be photographed is formed in a plane in spsee that m y readily be accessible for inspection and analysis. Then the sensitive film surface is put directly into this plane. Obviously, if we knew the characteristics of this illuminated plane, qualitatively and quantitatively, we would always obtain good photographs and would not need to know anything concerning the formation or causes of the illumination defects. The determination of illumination characteristics is, of course, photometry. I n photometry with a type of photocell used in photography, such as far the determination of exposure, we deal with three photoreceptors, (1)the photocell, (2) the eye, aud (3) the film, all having different spectral sensitivities. The spectral sensitivity of the eye is involved even in measuring with a photoelectric meter because all standard units of illumination are in visual terms. Therefore, in determining a required illumination value for the film, with mother photoreoeptor than the eye, such terms as foobeandle can validly be used only for a specific illuminat,ion
V O L U M E 21, NO. 4, A P R I L 1 9 4 9 quality or color temperature. The visual component involved with the usc of standard light units must be removed again, as is usually done during calibration, by measuring the so-called “effective foot-candles.” A photocell that had a spectral sensitivity identical FTith that of the film to be used would measure correctly for any quality of illuminatiori, but standard illumination terms could not validly be used. The spectral sensitivity of many barrier-layer cells is relatively close to that of Type B color films, especially those cells that are particularly blue-sensitive. The author has been able to use barrier-layer cells with a variety of color-compensating filters without changing the exposure constants; this is not possible with either visual photometers or with those using emission-type photocells. QUAiXTITATIVE A N L Y SIS OF IMAGE PLANE ILLUMINATIOS
Criteria. When a negative film is exposed, the rendition of “shadow detail” should govern the selection of the exposure that is given ( 5 ) . On the other hand, in exposing a reversal film, the illuminance of the brightest significant area of the image in the camera is a sufficient criterion (6) of the correct exposure. hloreover, a photographic pretest with a negative material is a n unreliable criterion that may not bear a constant, relation to a n exposure on a reversal material. I t is fortunate that the brightest area can be used as the criterion for the correct exposure of a reversal film, because it not only is the criterion that requires the least sensitive photometer, but in the common case of transmitted illumination, a large area of the highlight (clear field) can often be obtained by merely sacking the specimen temporarily out of the field. This allows t,he use of barrier-layer cell photometers, which require an appreciable area for measurement. For ordinary reflected light illumination, a highlight measure is obtained by substituting a white paper for the specimen; for specular (vertical) illumination, a first-surface mirror or unetched specimen can be substituted. That the average illuminance, obtained by photometering the whole image field containing the specimen, is not a good criterion can be illustrated as follows: With transparent illumination, assume that a field contains only one blood cell stained to exhibit a rather large brightness range. There viould be an optimum exposure for a color film that would not be changed if there 11-ere two cells in the field, or three, four, etc., until the whole field was covered except for the interstices betx-een the cells. In the meantime, the reading on a photometer covering the field would steadily decrease. If a specimen, such as a thin tissue or petrographic section, covered the whole field and if its brightness range were well within the latitude of the filni, the exposure still would not need to be changed from that determined by photometry of the clear field. The brightest area of the specimen image may be too small t,o be measured directly except by the method described later. If the specimen is a relatively thick section, a measurement, or an estimate, should be made of the illuminance of the brightest area, unless a photographic pretest is made. A measurement is preferable, if t,he size of the area allows it, and settles at once the questions discussed below. If the brightness range of the image is small, measuring the illuminance xith the specimen in place’ will probably yield a good picture, especially if the exposure is decreased over that from the clear-field criterion. The amount will depend upon the brightness level desired in the picture-Le., a specimen varying only in dark tones might be exposed only about one fourth as much as by the highlight criterion. Unfortunately, if the brightness range is not small, the readings of a photometer having a relatively large sensitive area are seriously affected by the relative areas of dark and light portions. Surprisingly good estimates of the illuminance of the highlight areas can be made in fractions of the illuminance of the clear field as measured by the photocell, using one edge of the specimen for the purpose, even though that is not included in the field to be photographed. A photographic density step wedge is an aid to such a judgment, if laid on the clear field but near the edge of the specimen image as a comparison. Such a step wedge is furnished with the Kodak Densiguide. The fraction of the clearfield illuminance involved is the reciprocal of the antilogarithm of the density used for matching-Le., if a density of 0.6 seems to reduce the brightness to the same level, the high-light brightness of the image is one fourth that of the clear field.
471
The illuminance of the brightest area may be measured with a photometer, of either visual or photoelectric type, that is capable of measuring a small spot, without the need of permanent calibration of the instrument, if a calibrated barrier-layer cell photometer is available. The latter should be used to read a clear field at a higher intensity level but one of about the same spectral quality (to bring it within the sensitivity reading of suchaphotometer) and then both high- and low-intensity fields read with the small-spot photometer. Thus, the illuminance of the spot on the specimen is measured as a fractional value of that of the higher intensity and hence is in terms of the spectral sensitivity of the barrier-layer cell only. With this method, the lamp of a visual photometer need be constant for only a short period of time. For pictures by polarized light, a factor of four greater than the clear-field criterion with parallel polarizers often gives good results, but the factor is fundamentally a function of the properties of the specimen. Photographic Pretest Method. .4 photographic determination of the exposure, in which the familiar stepped exposure series (3, page 94) is made, is fundamentally an excellent method, although in routine work it is somewhat slower than that with a photometer. The latter method is dependent upon the photographic calibration inherent in such a test. For Kodachrome and Ektachrome films, Type B, the most convenient and satisfactory black-and-white reversal material to use is Kodak Super Speed direct positive paper, as its processing is remarkably fast. I t has a speed of about 4 2 less than these two Type B films. It is, however, highly green- but not red-sensitive, and hence the exposure should not be judged from the red areas. For this purpose, its first development time should be 1 minute. This same material is probably most convenient for making pretests for Type A Kodachrome by cutting it into strips and placing the strip in the back of the camera. For this purpose, however, a black-and-white reversal 35-mm. film, Kodak direct positive panchromatic film, can be used that allows a better comparison to be made (6). Exposure Calculation, Value of E,. Some definite exposure time, E,, represents the correct exposure for the color film. This value is a constant within the intensity range for which the wellknovn Bunsen reciprocity law,
E,
=
exposure = intensity X time
is valid, and it is used for the calculation of exposure time, or with flash lanips for calculation of the proper intensity. For Kodachrome and Ekkchrome professional films, Type B, the value of E, is about 0.20 foot-candle-second for a photomicrographic transparency containing some clear background and at an exposure level of about 1 second (0.2 foot-candle). This value should produce some density in the background areas, so that no detail viill be burned out,. For Kodachronie film, Type A, the value of E, is about 0.15 foot,-candle-second for such a photomicrograph. This is the clear-field criterion previously discussed. These values represent basic minimum exposures, but they are also the values most frequently used. Use of Photoelectric Photometers. The common hand or amateur type of photoelectric photometer can sometimes be used, especially at a point closer to the microscope than the image plane, but special calibration is then required. The details of this method have been described (6). These hand photometers are especially valuable as primary calibration instruments for a more sensitive but less stable spot photometer, including simple visual types. Inasmuch as the sensitivity of the advantageous barrier-layer cell type is especially dependent upon that of the galvanometer, and photomicrography is done in a laboratory, it is worth while to utilize a much more sensitive galvanometer, even a wall type. Semiportable photometers using such photocells and giving readings down to about 0.05 foot-candle are available. Figure 5 shows a null-point photometer, utilizing a barrierlayer cell and having an amplification factor of about 10,000to 1,
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ANALYTICAL CHEMISTRY
I
' a.
1
-;-I Figure 6 . Circuit for Null-Point Photometer
Figure 5. Null-Point Photoelectric Photometer, Utilizing Barrier-Layer Photocell. w i t h Enolosine Panels Removed '
R. 2-volt atorage battery C. Inferohsngeablo barrier-layer cell G . Hubioon Type L galvanometer 0.0005 . microamp. per rnrn. nA. Weston rnilliamrfer. 0.1511.5/15 milliamp. SI.Microswitch, mxu gal"., single-pols
double-thror S . Double-pole 5-pch o n rotary switch, range selscta I=
..... .' (In: ... ...... which was constructed utilizing a CUWIII r~gu~ uc\ , e LBUUIILmended by the Weston Instrument Company. It uses a divided circuit plus a battery. It is not a "bucking circuit"; the photocell behaves as if there were no resistance and thus gives a very linear response. The principal cost is that of two meters, one a sensitive Ruhicon galvanometer. The mechanical design could be made appreciably simpler if semiportahility (by means of a typewriter table) were not important and if a wall gdvanometer could be used. It would also he cheaper to use a simple microammeter with outside shunts instead of the threerauge meter (mA, Figure 6). The instrument can be used down to ahout 0.001 foot-candle. Above 2.5 foot-candles, the battery circuit is cut out and the photocurrent is read directly. Photometers employing amplification of a vacuum-tube photocell are by far the mast sensitive type available, as they allow a small area t o be measured. This sensitivity is sometimes a great advantage, but is offsetby the different spectral sensitivities of all available tubes from that of the photographic film. It is most convenient t o use them with a harrier-layer cell type as the primary standard, so that, in effect, the spectral sensitivity of thc latter is used.
The quality of illumination in the image plane may be more or less degraded from that of the lamp and it, is advantageous to compensate rather than t o correct it. Only one or two primary color filters are needed for comneusatiou 1\loreover, it is a sim,.. plifying factor, in determining a correct filter, 60 ao so ,~y seiec~mg one filter in a concentration series of filters. such &s the Kodak CC compensating filter series. Liquid color filters can he used 8s such a series; they are especially suit,able in photomicrography and they can he discarded each time. A number of such filters for this purpose have been described ( 8 ) ; of these, concentrated stock solutions of a magenta and a blue liquid filter are being furnished by the Kodak Research Laboratories because they are both among the most useful and the most difficult t o prepare. This group of filters has the useful property of affecting only one color component of the film at a time.
..
Sa. Double-pole dsouble-throw switch, high.-10" .,s Single-polo ingle-thmw - e t c h , bsI t t
rotary
roesle
ticular optical arrangement in use. usually limited for m y one individual.
In the application of such a compensation system, several questions a t once arise: How shall we determine whether the illumination is correctly balanced for the film since the eye is no judge, and haw shall we determine the nature aur1 amount of comrequired? pelisation Obviously the color filnn, with the blue, gre en, and red 'spec^P :+" tra.1 "-"":+:..:+:-" ucLIIIu.vIuI~Lcomponent emulsions, . Its . Is own best xiterion. Photographic Deter1nination of Compensatron mequemenrs. Whenagoodgraytone is obtained by exposure of the calor film to a white or clear hackI:round, as by a series ,f exposures, the fmt ,auestian is answered. This must he done by z preliminary ealihrstion test of the parThe need for such tests is
_..
'
In determining the quantity of compensation needed-i.e., selecting the particular color-compensating filter required-it is best to eliminate all effects that might be due to the particular batch of photographic film and processing procedure employed. This can be done by utilizing the same hatch of film for the pretest that will he used for the photomicrography and by making all the test exposures on one sheet of film. For this purpose, a set of m a s h should he cut from the black paper that comes with the film, each mask hearing a slot that will expose one vertical strip of the color film at a time. A stepped exposure wedge (8) can then be made to the clear field on each strip area independently of the others, so that each strip represents a different illumination quality. The appearance of the resulting stepped wedge is illustrated by Figure 7, except that, if a blue filter were being tested, for instance, the color of each strip might vary across the sheet from yellow, representing an original off-balance, to deep blue, and some intermediate strip would he either a good gray or a t least close to the best yellowblue balance. * Photoelectric Determination of Illumination Quality. The photographic stepped-wedge method of testing the illumination quality for photomicrography involves a delay, owing t o the time required for photographic processing, before the results are known. With a photoelectric method, the results are available at once. Such & photoelectric method should fulfill the following requirements: (1) There must he three photosensitive components having the same spectral sensitivities, respectively, as the color film. I n this case, they would always return to the same balance or ratio whenever the film did so. (2) This system must he verv sensitive. as onlv a fraction of the illumination ipectrum is used for each reading.
Such a device has been made, utiliaing a commercial Photovolt Model 512 photoelectric photometer containing a No. 929 cell which is unusually sensitive to green aud red wave lengths. (The Photovolt Corporation upon special request c m furnish cells with such a n unusudly long wavelength sensitivity with this photometer.) It is necessary to hold the photometer in a
V O L U M E 21, NO. 4, A P R I L 1 9 4 9
.~ .~~~~~ . ~~~~~~~~~o~ ~~
473
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This assembly has been fastened i n t o a hoard whiih slips into the back of the camera alternative t o the ground glass, so that adjustment of the bellows length is simple. The theoretical sp,ectral transmission of the three filters that would make the action of t,he nhotoeell match that of the three ComDonents in Kodachrome ~ r o ~ e s s i o nfilm. d TYDC B. and in Kodactkome film. Type A, has .....~ . .
.. ~., ,.~ The largest error is in the red filter, because& long wake-length cutoff is needed but is not available m a suitable colored compound. For Kodschrome film, Type A, the same green and red commercial filters that were used for Type B m e the best available approximations t o the theoretical ones, but in the case of the blue filter another one must he chosen. In this case, the commercially available filter most closely resembling the theoretical blue one is a composite of Xodak Wratten filters 32A and 43, and Pittsburgh Plate Glass filter 2043, apprommately 2 mm. thick. The glass filter is used to absorb the infrared transmitted by the gelatin filters. ~
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variables to he plotted, but these a& reduced t o two by taking the ratio of the other two to the blue readings, However, because a direct photographic effect is shown by a negative, and we are dealing with reversal photographic processes, the inverse of the ratios are more desirable to u s e i . e . , the blue-green and blue-red ratios. The values of these ratios must be determined fairly accurately (10-inch slide rule) to satisfy the eye that the color balmce of the film is correct. A fourth variable is the cancentration variation of a color filter series such as would be proposed for compensation. A graph can he made to show the effect of the variation in the
Figure 7.
Stepped-Wedge Test for Filter Concentration
0.6
0.5 0.6
0.7 0.8 0.9 1.0 1.1
1.2 1.3 1.4 1.51.6 1.7
'/2.Blue/Red Figure 8.
Trichroic Ratio Graph
concentration of a calor filter upon the rrttio of the galvanometer reading-Le., blue-green and blue-red-by plotting such a ratio against the concentration of the color filter, which might he one of the successive gelatin film filters in the CC series. I n this case, the photoelectric ratio is the dependent variable, and the filter concentration is the independent variable. If semilogarithm paper is used, the effect of the filter concentration may sometimes be represented by a straight line. An alternative graphical method is preferable, although i t is harder to locate the points for plotting. This method consists in plotting the two dependent variables-Le., the two rrttios--on log-log paper, for which Codex paper No. 32,230 is especially suitable. Its scale and subdivisions are approximately correct for the sensitivity requirements of this as a graphical method, but i t is necessary to divide the blue-red ratios in half in order t o plot them on the abscissascaleof this paper. This procedure does not affect the graphical method otherwise. By this method, a straight line is always obtained with variation of concentration or thickness of a color filter series, such as either the CC or the liquid color-compensating filters. The variation in concentration of the filters-i.e.. the fourth variable-is now represented as distance along the filter lines. Trichroic Ratio Method and Graphs. Because the ratios of the galvanometer reading to the color filters are frequently mentioned a new term is needed for them. Therefore, they have been called "trichroic ratios"-i.e., the blue-green and blue-red ratios. Every illumination quality in the image plane of the camera is represented by a trichroic ratio point on the log-log graph. Moreover, there is some quality of illumination that is desirable for reproduction by the color a m . This is usually chosen empirically by photography and becomes a standard reference point on the trichroic ratio graph. As the film responds somewhat differently with large differences of illumination intensity hut photocells do not, the reference standard will really he a line on the trichroic ratio graph (Figure 8). However, in every application, only one
4'14
ANALYTICAL CHEMISTRY
point on this line is of interest. The effect of the failure of the reciprocity law on the color balance is represented by a straight line on the trichroic ratio graph, for the relatively few cases tested. Figure 8 shows the vasiation of the standard balance point with a particular hatch of Xodachrome film, Type B, as the illumination in use changed from 10 foot-candles to 0.001 footcandle. Again, thespecificilluminationlevelisrepresented by variations of distance dong the line, The function of the graph can he described as follows: Assume some illumination quality in the image plane of the camera. that is not correct for photography with the color film t o he used. This will
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pensating filters, differing only in concentration, the resulting illumination qualities will he repreFigure 9. Calibration G r a p h for Triohroie Ratio Graph by some o
