ANALYTICAL CHEMISTRY
432 All this experience should lead to the microscopist’s developing into more than a specialist in microscopy. He should become able, and should be allowed, to carry his problems beyond the boundaries of his laboratory, working in the best cooperation with other members of the staff. This may mean the establishment of new standards or test methods, the modification of production practice, and the pursuit of more or less pure research, in addition to all sorta of high grade trouble-shooting. One of his chief obligations should be to convince his associates that microscopy is not’ a last resort,, to be tried when all other approaches seem fruitless, or the sample is too small, but rather a primary exploratory method to help shape the course of investigations and to keep in touch with their advance. Whether microscopy is officially part of the analytical, control, research, physics, or other division of the organization is less important than that it should be permitted to go beyond the boundaries of these, wherever its usefulness may lead. Unofficially, the microscopist serves best when he has a roving commission. EQUIPMENT
One of the problems as microscopy takes root is that of equipment. Any good microscopist should be ingenious enough to “make do” with limited apparatus and facilities for a while, and to improvise and adapt apparatus beyond its conventional uses. But there comes a time when more equipment is needed, and here
it is his obligation to justify the expenditure, not necessarily on B strict cost-accounting basis, but in terms of possible future uses. The apparatus of microscopy is inherently versatile, but it hardly pays t o try t o do everything with one or two instruments, when time is lost in rearranging special setups, or in riaiting their release for temporary problems. In times 1%hen plant instrumentation is increased so readily, there should not be reluctance t o invest in equipment to save manpower in the microscopy laboratory. Similarly, the microscopist will find that although his earlier experience has properly been in large part manipulative, and concerned with studies of methods and materials, this knowledge can best bear fruit if he is relieved of much of the noR- routine procedures. He should not have to spend his time doing ordinary preparations, measurements, or photography, if assistants under his supervision and following the methods he has worked out can relieve him for more fundamental or collaborative work. A really valuable microscopist should spend a relatively small portion of his time looking in a microscope. After all, the most important apparatus in microscopy is borne on the shoulders of the microscopist, and it functions day and night, whether above the instrument or elsewhere. This apparatus cannot be purchased; it must be created, and both the administration and the microscopist are responsible for its fullest attainment of usefulness. RECEIVED September 15, 1848.
@ chemical microecopg egmpoe2um
MICROSCOPE OPTICS L. V. FOSTER Bausch & Lomb Optical Co., Rochester, N. Y . Work in the ultraviolet region of the spectrum is being advanced by the use of achromatic microscope objectives. There are all-refracting systems which use synthetic or natural crystals having high transmission in the ultraviolet, allreflecting systems having no optical material through which the ultraviolet light must pass, and the combination of both. A great deal of work remains to be done to improve the lamps and to control the radiations used. Phase contrast will probably be added to ultraviolet systems. The use of electronic image tubes is extending use of the microscope into the near ultraviolet and the infrared.
D
URIKG the past fen. years considerable emphasis has been put on the development of the microscope for use in the ultraviolet and infrared portions of the spectrum. About 1904, Koehler (8) of the Carl Zeiss Works developed a series of quartz monochromatic objectives for use in ultraviolet light. These objectives were designed and used primarily to increase the resolving power of the microscope. The present purpose in working in the ultraviolet region of the spectrum is not only to increase resolving power but to bring out differential absorption characteristics in material. This paper describes recent developments in this field and points out the difficulties that still exist in connection with the work. In many respects work in the ultraviolet region of the spectrum is difficult. Becaus: the eye is not sensitive to wave lengths shorter than 4000 A., a conversion from the image formed by these short radiations to the longer visible rays is either through photography or by fluorescen$e, Glass does not transmit radiations much shorter than 3500 A.; if microscopy in the short ultraviolet (3500 to 2000 A,) is required, all optical parts from the source to the final image must be made from material such as
quartz, fluorite, or lithium fluoride, which transmit these rays. The amount of chromatic aberration inherent in lens systems increases as wave length decreases and, as a consequence, images lack contrast and good definition unless strictly monochromatic light is employed. This mean3 that the light source should emit only a narrow band of ultraviolet. I t is probably for this reason that Koehler designed monochromats and :sed spark sources (9) such H-,S cadmium for illuminating at 2730 A . and magnesium at 2800 A. There are some advantages to be gained in monochromatic illumination, and others in the use of broad band illumination. Both methods have their own problems. OBJECTIVES USED A T 36501.
In 1931 the writer and Trivelli (13) showed how 2bjectives could be corrected for the mercury green 5461 and 3650 A. The ordinary glasses used in microscope optics have sufficiently high transmission a t 3650 d. to permit making photographs in this part of the ultraviolet spectrum. These lenses were corrected for chromatic aberration to bring the two wave lengths,
433
V O L U M E 21, N O . 4, A P R I L 1 9 4 9
objective over the eyepiece of a microscope and used to focus the ultraviolet image formed by regular microscope objectives. The tube can be removed from the beam divider and placed a t the camera ground glass and used like a focusing magnifier prior to taking pictures. The Bausch & Lomb apochromatic objectives are so well corrected for chromatic and spherical aberration that t,hey givc excellent images in the near ultraviolet. Although they were not achromat,ized so thrtt when focused in 5461 A. they were in focus for 3650 b.,the use of the 1P25 image tube far focusing makes possible their use in this portion of the ultraviolet spectrum. FUSED QUARTZ-LITAIUM FLUORIDE OBJECTIVE
Johnson (6) developed in 1939 a 25-mm. 0.20 N.A. mieroscope objective using lithium fluoride and fused quartz. He
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IMAGE PLANE
Figure 1. Petrographic Microscope Vertical 5 X 7 camerre and eufomatio carbon arc lemp. At front bosrd end of camera i s mounted s b e a m divider with 1P25 infrared image fuh. for making ohserrsfiona through micmsoopewhsn illuminating with either infrared or ulbsriolef light
5461 and 3650 b.,into focus a t the same time. The illumination problem was solved by using the stmosphe$c pressure mercury arc, which has strong lines a t 5461 and 3650 A. Other radiations can be filtered out by glass 01.gelatiu filters. A Wratten No. 62 or No. 77A filter will out out a" the other visible and ultraviolet radiations and trausmit 5461 A., so that an accurate focus can be made with the eye. This filter can then be replaced by a Wratten No. 18A filter, which will cut out the visual and short ultraviolet and transmit only the 3650 group of lines for photography. The maximum theoretical limit of resolution with the 3650 1.line and an objective of 1.30 n u m e r i d aperture is 0.14-w as compared with 0.21 I" using green light of wave length 5461 A. Objectives of 16-, 4-, and 1.8-mm. food length were designed for this purpose
Another means for focusing the invisible ultraviolet image became available when it was learned that the 1P25 (14) infrsi red image tube used in World War I1 in the Sniperscope (11) was sensitive to the long ultraviolet radiations. A tube of this kind has been mounted with a beam divider (Figure 1) and a telescope
GLASS
FUSED
QUARTZ
FLUORITE
Figure 3. Polaroid-Grey Refleeting Objective Mede hy Miriam S. Flower, Polsmid Cmp.
53 x
2.8 mm. 0.72 N.A.
claims that this objective was sufficiently well corrected for chromatic and-spherical aberration to use i t over the range from 2550 to 4000 A. Johnson was able to focus in visible light and photograph without chmge of focus in the ultraviolet. OBJECTIVES ACHROMATIZED FOR 2650 AND 2750
Figure 2.
Fused ( Objeeti
2.5mm. 0.85P
i.
The writer and Thiel (6)described before the Optical Society of America in 1947 an achromatic microscope objective of 2.5mm. focal length, 0.85 N.A., consisting of fused quartz and fluorite elements and corrected for spherical and chromatic aberration over the region of 2650 to 2750 A. The construction of this lens is shown in Figure 2. The longitudinal
[email protected] aberration in the image space between 2650 and 2750 A. is 1.5 mm., and the Rayleigb limit for this aberration is 1.95 mm. The Koehler quartz monochromats which were usable only for single wave lengths in the ultraviolet bad chromatic aberrations for this wavelength range of about 15 mm., making i t necessary to use them in very narrow wavelength bands. The present achromatio objectives are well corrected for spherical aberration and can be used over a broad range inathe ultraviolet, so long as the band width does not exceed 100 A.
ANALYTICAL CHEMISTRY
434
seuaration between the two mirrors. However, this chanee ~
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Mirbr’systems always h& an occluded central aperture, hut Burch does not indicate how large this is for his objectives. POLAROID-GREY OBJECTIVES
During the past year D. Grey of the Polaroid Corporation designed a two-mirror objective of 10-mm. focal length and 0.40 N.A., using only spherical surfaces. The occluded aperture is 0.16 N.A. This objective is used without an eyepiece to give a magnification of about 75X, when
iective in “col&, transferred ~framthe uitriviiiit. were demonktrated by Land (18) a t the opening of tho Sloa&Kettering Institute for Cancer Research in New York in April 1948. Since this time, a second objective of 2.8-mm. focal length and 0.72 N.A. has been desiened and made. This ohiective has two snherical reflecting surfaxes and a quartz-fluoritehaublet and a flkorite refracting component to reduce the residual spherical aberration of the spherical surfaces and correct the chromatic aberration of the quartz cover slip used to protect the object. Figure 3 shows the outical construction of this objective. The reflwtina sur~
Figure 4.
Tissue Culture Metaphase Cell
Photographed at 2650 %.with objeotjss shown in Figure 3. neation 2000x
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0.21 N.A. and is used withrt4X negative type eyepieck to give~a total magnification of 212x. Quarta eyepieces of the Huygens type can be used to give higher magnifications. Megoi-
SPHERICAL REFLECTING OBJECTIVES
In 1942 Bmmberg (9) described a new method of microscopy in ultraviolet in which he used a two-mirror reflecting objective of 6-mm. focal length and 0.5 N.A. He described both a photographic instrument and a scheme for making visual observations. I n photography he makes single photographic negatives in each ai three different wave lengths in the ultraviolet aud then ahserves these three different negatives through three filters (red, green, and blue) by mems of an instrument called a chromoscope. Direct visual observations can he made by putting a special fluorescent screen in the ocular of the microscope. This screen is said to be prepared by mixing three different compounds which fluorescein three different colors. deoendine I w. o n the wave length of the incident ultraviolet radiation.‘
FOCUSING T H E ULTRAVIOLET IMAGE
These various developments in ultraviolet microscope systems carry Koehler’s early work to a more practical use. The energy existing in the single emission lines of the sparks was low and focusing had to be done by trial and error. Lavin (10)of the Rockefeller Institute for Medical Research in New ?ark used a mercury resonance lamp having a strong line a t 2537 A. with both the mouochromatic and achromatic refracting objectives.
objective and the ocular. An uppe; disk, having the same number of groups of three color filters, is located external to the
bad.
I n ‘the food plane of t h i ocular is a white screen uon-
rapid succession and, owingto the persistence of vision, win fuse them into a single “full color” image,
Monoch
Figure 5.
A . Quartz condenser to fooua mund aperture, u, thmugn water cell
prisms on quartz substage Eondenaer, SC B. Quartz condenser containing dution or nickel sulfsts and cobalt sulfate to reduce stray light
ASPHERIC REFLECTING OBJECTIVES
F. Liquid filter
I n 1947 Burch (4) of the University of Bristol described two two-mirror reflecting objectives which he had made. One of the objectives had a focal length of 7.5 mm., 0.58 N.A., and the other 3.0 mm., 0.65 N.A. He added a hemisphere to the latter objective and, by placing the object at its center, increased the numerical aperture to 0.98-that is, the numerical aperture hecame equal to the product of the index of refrraction of the hemisuhere by the numerical aperture of the mirror ob-
&agnification of 47X. The result &e a good central image hut left off-axis coma, which was then correoted by changing the
He irolnwd thiq linr w i t h a BlckstrZm ( I ) tilrrr, which is made from a solution of 1C pitins of nickel sulinir Ihrrahgdratc and 1.1 Z I R of ~ c h n l i sulfrue I.~~nr:tl.v~lmrr in 70 nil. a i distilled water. After the s&.s are di‘ssolGed, the solution is made to 100 ml. This solution must be placed in a cell 30 mm. thick, in ~~~
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direct focusing on Goss’6hbjects. Fine aetail is appareGt, h u t outlines can be seen sufficiently well by the dark-adapted eye to get a good focus
.
V O L U M E 2 1 , NO. 4, A P R I L 1 9 4 9
43s
The Bausch & Lomh Optical Co. is making a low-power finder eyepiece by mounting a fluorescent screen in the focal plane of a Ramsden eyepiece. This screen is zinc silicate dispersed in a binder and deposited on a glass disk. The eyepiece e m be used for bringing into focus and studying the gross objects in the field prior to photography. Reflecting objectives, such as Burch, Bromberg, and the 10mm. Grey design, have absolutely no chromatic aherrstion if the object is mounted in air. When a cover slip is used over the object, its optical path length differs from one wave length to another and chromatic aberration becomes evident. These objectives can he used visuslly and in either the ultraviolet or infrared without any change in focus. The 2.8-mm. Grey reflecting objective is not so well oorrected, but visual ohservations can be made, and with a very slight change in focus, photographs in the ultraviolet can he made. [This lens is corrected within less than the (ultraviolet) depth of focus, or so-called Rayleigh quarter-wave criterion. Any objective, reflective or refractive, will probably require refocus for extremely critical work in passing from visible to ultraviolet light, because the visual depth of focus is greater than the ultraviolet depth of focus for equal numerical apertures. This refocus is perhaps best done by a relatively coarse motion of the eyepiece relative to the objective.] Figure $shows a picture made using a band width of 80 A. peaked st 2650 A.
pletely and describes several filters which may have rather broad application. The writer has used a monochromator ( I & ) , such as that shown in Figure 5, and with it can completely fill tb aperture of the short-focus substage condenser with a 100 A. band and get satisfactory field illumination.
ILLUMINATION
Figure 7. Buccal Epithelial Cell
The problem of control of illumination hss never been well solved. The cadmium and magnesium spark was used by Koehler. Spark sources are not as intense as one would like, and they do not have an ahundance of strong lines. The ideal source-would he a metal disk radiating a continuum from 2000 to 4000 A. The mercury arcs are fair; the General Electric high pressure 8 6 provides a very good source in the region 2900 to 4000 A. The medium pressure mercury arcs, such as the H-3 and 8 4 , provide many spectral lines with a moderate continuous background. The lines available with these lamps are 2378. 2483,2537,2652,2804,2894,2967,3022,3130,3341, and 3650 A. The hydrogen discharge tube provides a continuum from 2000 to 4000 A. but its brilliance is not very great. I n addition to the source, there must also he a means for selecting the wave length or the band desired. The monoohromator is prohahly the best device to use, especially with a source having a strong background of illumination. Filters, such as the Backstrbm filter, can be used with sources having strong lines. Kashs. (7) has gone into this rather com-
Photographod with ~onrentionaloptics at 3650 8.
i n d an object&e lens to image a &it or diaphragm through the prisms on the microscope condenser. The prisms are provided with three leveling screws permitting adjustment for alimment and providing stahility. CONDENSERS
Both refracting and reflecting substage condensers have been used for this work. The refracting condenser must be made of material that will transmit the ultraviolet light. Crystal quarts or fused quartz is suitable. If the reflecting objectives are used with the refracting condenser, the object is apt to he flooded with light, which would lead to low contrast. It has proved most satisfactory to use a second reflecting objective as a condenser; the aperture of the condenser then matches that of the object,ive. In a11 cases of photomicrography, whether .the refracting or reflecting objectives are in use, the aperture of the substage condenser should he reduc:ed t,o ahout two thirds its full opening in order to get optimum re:suits. PHASE CONTRAST
One of the I,&mm. ail immersion objectives corrected for 3650 and 5461 was fitted with a phase-accelerating annulus for use in 3650 A. A quartz refracting condenser was equipped with the necessary stop to correspond with the aperture of the phase annulus and the two were used together a t 3650 A. S t was so convenient to use the 1P25 infrared image tube to adjust the illumination and locate and focus the object that all the work was done with the meroury arc and a Wmtton 18A filter in the ultreviolet.
4.
A photomicrograph of the mouth buccal epithelial cells in ultraviolet made with this phase contrast equipment is shown in Figure 6. Figure 7 shows a similar picture. made without phase contrast. INFRARED
Figure 6. Buccal Epithelial Cell Photo$ra,,hhed
36.50 A. in phase
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At the long wave-length end of the speotmu, I~UUIU~LUU ~r comes i ~ less. ~ Rowever, ~ t ~many ~ materials which are opaque in the
436
A N A L Y T I C A L CHEMISTRY
visual portion become transparent w h e n radiatedwith infrared (9). Microscope optics are transparent to near infrared as far as 2 fi. The sensitivity of the eye falls off "t about 7000 A. Ordinary infraredsensitive photographic mateFigure 8. Molyhdenite rials have their Interference figure shorn with 4-mm. 0.85 Deak sensitivitv N.A. obj,j,tire, is uniaxial &tabout 8000 The infrared image tube (1P25,14) used in World W&: I1 in the Sniperscope (11) has a peak sensitivity rtt about 8200A.
A".
An infrared image is formed on a photocathode, which gives off electrons proportional to the infrared radiation it receives. These electrons are accelerated in an increasing potential field
and are brought to focus on a DhosDhor screen a t the other end ~
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over the evepiece of a convent:okl ~&croscme. The source of light used &this work is a carbon arc lamp fitked with a Corning No. 2540 filter to absorb the visual light and transmit the infrared portion of the spectrum. An example of this use of infrared (Fimre 8) shows the uniaxial interference fieure of rnolvh~"~ denite, mineral opaque in visual light.
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LITERATURE CITED
(11 Biickatrbm, H. L. T.. Arkiv K m i M i n e ~ a lGeol.. . 13A (193'31 (2) Bailley, Ren~,Bull.acad.roy.Belg., l2,791-822(1938). ' (3) Brumberg. E.M..Bull. Aead. Sci. (U.S.S.R.), 6 , 3 2 4 0 (1942). (41 ( L n n . d m . 1947.41-0..~ " SOC. . . Burch. C. R.., Proe. Phvs. ( 5 ) Foster; L. V.. clnd Thiel, E. M., J . Optical SOC.Am., 38, 689-92 ~
~~~
i l O~ d P ," ~ " , . ~
Johnson. B. K., Pmc. Phgs. Soc. (London). 1939, 1034-9. Kasha, M., J . Optical SOC.Am., 38, 929-34 (1948). Koehler. August,Z. wiss. Mikroskop., 21, 129-65 (1904). Zbid.. DD. 273-304.
. L. ~, V., J . Optical SOC.Am., 21, ~
Trivelli, A. P. H., and
Foster.
,?A ,lo?,, .I_~_"__,_ (14) Zworykin, V. K., andMorton, G.A.,Zbid.,26,181 (1'336).
15, 1948. R ~ a ; ~ v eNovember o
Rpplication of Fusion Methods in Chemical Microscopy W. C . MCCRONE, Arrnour Research Foundation, Chieogo, 111. oal and research applications of old but little used technique (fusion Is) are emphasized. Many applications are possible in the fields of and phase rule research, as well as qualitative and quantitative analysis.
F.
memaas represent a group of techniques which the uucroscoplst . oan use in investigations of organic compounds, or more generally fusible compounds. This tool can be used for characterization and identification of fusible compounds, determination of phase diagrams, punty determinations, qualitative analysis, q w t i t a t i v e analysis of binary and even polycomponent systems, study of mechanism of crystal growth, and study of other changes in the solid state such as rcerydtrtlliarttion, grain growth, ttnd boundary migration. The techniques themselves include all observations made during heating of a few milligram of the material on a microscope slide, during solidification of the melt, during cooling of the preparation, as well as observations on the erystalliaed material a t room temperature. In addition, data obtained from observation of a mixed fusion with a reference substance may also be included. The hasis for the application of fusion methods was laid by Lehmann ( 4 )in 1891, when he pointed out that crystallization of a n organic compound from the melt is very characteristic. He listed a qumber of properties that can he determined on crystals from the melt, and pointed out the value of fusion methods in studying phase diagrams. Identification of fusible compounds by fusion methods permits a very rapid analysis with small quantities of material and requires relatively little specialized training or equipment. Complete op1 UD~ULY
tical crystallographic description of a given compound may require from a few hours to several days, whereas complete fusion analysis of a new compound seldom requires more than an hodr. Furthermore, a n unknown compound in a limited category-cg., organic acids or vitamins-can usually be identified in less than 5
Figure 1. Sublimate of Hexamethylenetetramine on Microsoope Slide. 50X
