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Ultraviolet studies of tetrazolium reduction in living cells
Experimental
131
Cell Research, Suppl. 7, 131-144 (1959)
ULTRAVIOLET
STUDIES OF TETRAZOLIUM IN LIVING CELLS G. Z. WILLIAMS Tke Rockefeller
Institute,
REDUCTION
and A. C. PEACOCK New York, N.Y.,
U.S.A.
AN
understanding of the. intiniate metabolic processes in. living cells is based chiefly on detailed studies of chemical reactions of cell extracts and separated particulates. These materials are most often disorganized mixtures of components of several types of cells. Tissue slices are composed of several different cell populations. Suspensions of ascites turnor cells approach the ideal situation. However, study of the orientation and localization of reactions within individual living cells has been limited by technical difficulties. Successful observation and measurement of chemical changes in living cells requires seleqtion of sensitive and specific detection methods, the avoidance of toxic chemicals and physical injury to the cells, and the recognition .an,d correction of nlethodological errors. The ultraviolet region of the spectrum has been applied by many investigators [l, 2, 51 to detect specific materials in cells. The methods available require rather long exposures to ultraviolet, rapidly damaging to fresh cells. Amplification and electronic devices which increase the sensitivity of photodetectors make it posstile now to examine living cells by low intensity ultraviolet microscopy with less injury, and to utilize absorbing chemical indicators for observation of intracellular reactions. One group of such indicators are the tetrazolium salts which are relatively non-toxic in low concentrations (0.0125 M). This substance enters the intact cell, is reduced to insoluble formazan which precipitates immediately in situ and is detectable in very small amounts by ultraviolet absorption. By proper selection of substrates and tetrazoliums and utilization of specific ultraviolet wave bands, one may readily measure and record intracellular rates and locations of specific reduction reactions.
Television Ultraviolet Microscopy Photographic methods require relatively long exposures to ultraviolet. More sensitive methods of detection by photoconductive principles employed in the television approach [3, 4, lo] were devised to decrease exposure times of cells. The ultraviolet television microscope possesses the. following advantages [6]: (a) The facility for searching and focusing with green light to avoid Experimental
Cell Research, Suppl. 7
132
G. Z. Williams and A. C. Peacock
Fig. l.-Television ultraviolet microscope. The adapter body tube directs the UV image into either the film camera above or the TV camera immediately over and left of the reflecting tjrpe microscope. Synchronizing control panel is at the left and the line-selecting oscilloscope at the right. During the operation, film motion picture camera and still camera are mounted in front of the TV screen and oscilloscope.
cell injury. After focusing, the photographic record of the television image may be made with a very short pulse of ultraviolet. (b) Utilization of the ultratube which permits the use of ultraviolet pulses violet-sensitive “vidicon” of the order of l/l00 sec. The contrast of the absorption image may be electronically enhanced to intensify faintly visible structures. (c) Approximate values of reduction rates may be determined from records of deflections of a single TV line traversing the cell image as depicted on a line-selecting oscilloscope. There are certain disadvantages to the television method. Resolution is limited even in the ultraviolet, by the separation of scanning lines. Spatial resolution with the present instrument is only about 0.2 EC.There are difficulties in maintaining reproducibility of measurements with the line-selecting device because of electronic circuit characteristics. These can be improved but require considerable and expensive modifications of the standard circuits, Thermal electronic noise is particularly annoying and difficult to reduce without sacrificing resolution. Contrast must be carefully controlled, and as in all optical methods, scattered light interferes with the accuracy of measurements. Experimental
Cell Research, Suppl. 7
133
Tefrazolium in living cells
Fig. 2.2Block
diagram
of the television
microscope
system.
Instrumentation The system consists of an ultraviolet light source, a monochromator, a microscope equipped with reflecting optics and an adaptor body-tube for directing the microscopic image into the television camera [S, 71, and is illustrated in Fig. 1. A medium pressure mercury arc and a quartz-prism monochromator are most useful. The Gray V and VII reflecting condensers and objectives are quite satisfactory and do not require refocusing with change of wave length. The television camera is equipped with a special ultravioletsensitive “vidicon” tube (RCA developmental “vidicon” C 73439). For the best image formation, the highest quality broadcast-type closed circuit television camera, amplifier and monitor are necessary (Fig. 2). Connected to a superior monitor, the line-selector oscilloscope displays a single line of the television picture raster on its cathode ray tube. This horizontal line may be moved vertically at will to traverse the cell image and transect any desired particle or structure. Deflections in this tracing are related to the density of the structures and sequential photographic records provide information for making measurements from which reaction rates may be calculated (Fig. 3). A parfocal 35 mm camera is mounted over the microscope with a moveable mirror so that accurate focusing is accomplished by television for direct ultraviolet photographs requiring 4 to 1 sec. exposures. When protection of living cells is necessary, photographs may be made of the television screen, with much shorter exposure times of & to r&s sec. To record the progress of reactions in living cells for several hours and avoid injury, it was necessary to interrupt the light and limit exposures Experimental
Cell Research, Suppl. 7
Fig. &-Stainless
steel and quartz perfusion chamber slide. The fluid depth between coverslip
and base is 10 ~1 and the surrounding
moat at the margin
permits
exchange
of medium
without
disturbing cells in the field of view.
vidual electronic components and their functions in the control system have already been described in detail [B]. The block diagram (Fig. 2) depicts the operation of the integrated system. A pulse circuit for intensifying the ultraviolet light source was included to provide short pulses of higher intensity during the open time of the light shutter.
Preparation of Specimens Liver cells (mouse and rat) were prepared from perfused liver, minced with a sharp blade and gently pressed through stainless steel mesh. Successively smaller mesh sizes were used and finally the liver brei was passed through fine weave nylon and sedimented in Earle’s salt solution without glucose or serum. One sedimentation in isotonic sucrose facilitates rapid separation of the small cell clumps and individual cells from the bare nuclei and mitochondrial suspension. Tissue cultures were grown on quartz coverslips and inverted over flat ground depressions of 10 or 30 y depth in quartz slides. The margin of the coverslip was sealed with clear, liquid rubber cement. Ascites tumor cells were enumerated with an electronic counter [9], and suspended in Earle’s medium. Four microliters were measured into the slide depression with a micrometer pipette to provide reproducible samples and uniform light path. Perfusion microchambers were made from stainless steel and quartz. The bottom of the round central chamber consists of a quartz disc surrounded by a narrow moat, which communicates externally through very small gauge polyethylene tubes. The round quartz coverslip rests on a shoulder of the rim to which it is sealed. The depth is 10 microns. The indicators or substrates may be introduced through the perfusion tubes. (Fig. 6.) In a typical experiment the tissue culture coverslip is inverted over the chamber containing the culture medium and the desired substrate. The slip is sealed in place with clear rubber cement or Diatex and immediately placed in the warm box on the microscope stage. (Fig. 7.) A representative group of cells is searched out and focused in green or blue light by observing the image on the television monitor. The density pattern of cell structures at the selected UV band is recorded from the oscilloscope line Experimental
Cell Research, Suppl. 7
Tetrazolium in living cells CLOCK OR w-4 I
I
I
TIMNO OF CONTROL CCWONENTS A I I Y llllll
I
135 I A B c D E
a
Fig. 4.- Diagram of electrical pulses of timing control components of functions of the shutters, lamp and TV blanking signals.
to indicate
critical
alignment
Fig. 5.-Oscilloscopic display of electrical signal (video) which produces the image for observation or time-lapse record (upper portion). The monitor screen unblanking pulse (negative square wave below) is adjusted to align exactly with the picture at the maximum signal amplitude and to turn on and off exactly at the beginning and end of two frames.
device blanks out the monitor picture except for the two frames during the open time of the camera shutter. Special circuits utilize the vertical blanking pulses from the synchronous generator to control the “vidicon” blanking, ultraviolet shutter opening, the interim sensitizing lamp, the kinescope blanking interval and the motion picture camera shutter (Figs. 4 and 5). The indiExperimental
Cell Research, Suppl. 7
Fig. K-Stainless steel and quartz perfusion chamber slide. The fluid depth between coverslip and base is 10 p and the surrounding moat at the margin permits exchange of medium without disturbing cells in the field of view.
vidual electronic components and their functions in the control system have already been described in detail [S]. The block diagram (Fig. 2) depicts the operation of the integrated system. A pulse circuit for intensifying the ultraviolet light source was included to provide short pulses of higher intensity during the open time of the light shutter. Preparation
of Specimens
Liver cells (mouse and rat) were prepared from perfused liver, minced with a sharp blade and gently pressed through stainless steel mesh. Successively smaller mesh sizes were used and finally the liver brei was passed through fine weave nylon and sedimented in Earle’s salt solution without glucose or serum. One sedimentation in isotonic sucrose facilitates rapid separation of the small cell clumps and individual cells from the bare nuclei and mitochondrial suspension. Tissue cultures were grown on quartz coverslips and inverted over flat ground depressions of IO or 30 p depth in quartz slides. The margin of the coverslip was sealed with clear, liquid rubber cement. Ascites tumor cells were enumerated with an electronic counter [9], and suspended in Earle’s medium. Four microliters were measured into the slide depression with a micrometer pipette to provide reproducible samples and uniform light path. Perfusion microchambers were made from stainless steel and quartz. The bottom of the round central chamber consists of a quartz disc surrounded by a narrow moat, which communicates externaily through very small gauge polyethylene tubes. The round quartz coverslip rests on a shoulder of the rim to.which it is sealed. The depth is 10 microns. The indicators or substrates may be introduced through the perfusion tubes. (Fig. 6.) In a typical experiment the tissue culture coverslip is inverted over the chamber containing the culture medium and the desired substrate. The slip is sealed in place with clear rubber cement or Diatex and immediately placed in the warm box on the microscope stage. (Fig. 7.) A representative group of cells is searched out and focused in green or blue light by observing the image on the television monitor. The density pattern of cell structures at the selected UV band is recorded from the oscilloscope line Experimental
Cell Research, Suppl. 7
Tetrazolium in living cells
Fig. 7.-Warm box on microscope stage. The tetrazolium-containing medium is contained in the syringe reservoir at the upper left. When the waste tube at the right is lowered as shown, gra vity causes fluid to flow through the perfusion chamber.
Fig. S.-Three superimposed oscilloscopic tracings of one liver cell at 5, 10 and 30 minutes alfter addition of tetrazolium. The successively lower deflections representing the liver cell indic:ate progressive increase of formazan from measurements of which a reduction rate curve mayr be calculated and constructed. tracing (control measurements). Tetrazolium is introduced into the perfusion cham ber and the progressive changes in density due to the reduction of the tetrazolium are recorded by time-lapse photography. Intracellular reduction rates are calcula ted from successive records of deflections of the line tracing. (Fig. 8.) Experimental
Cell Research, Suppl. 7
G. Z. Williams and A. C. Peacock
Fig. 9.-Intense
photochemical
reduction
Reduction
of triphenyl
Patterns
tetrazolium
by UV light at 265 my.
of Tetrazoliums
A number of tetrazolium derivatives were synthesiz6d for us by Dr. Stanley Pierce, of the University of Richmond. These compounds were grouped according to their ultraviolet absorption characteristics and reduction reactions with mouse liver homogenates and washed mitochondria. They reacted with suspensions of cells, homogenates or mitochondria, more or less rapidly, depending upon the number of nitro groups, but without apparent relation to other radicals. Thus, methyl-, methoxy-, isopropyl- and amyl-groups did not influence reduction properties. They may be divided further into classes which react best in the presence of succinate substrate, those which react most rapidly and intensely with malate or lactate and a third group which react equally well with alt three substrates. Some differences were observed in the size and distribution of intracellular formazan particles. Many tetrazoliums are reduced to formazan by the photochemical action of ultraviolet. The parent compound, triphenyl tetrazolium in saline or phosphate buffer solutions in a beam of ultraviolet was reduced to large crystals Experimental
Cell Research, Suppl. 7
Tetrazolium in living cells
139
of formazan most rapidly at 265 mp, less rapidly at 300 and 312, slowly at 360 and very slowly at 427 and 545 rnp (Fig. 9). When the compound was dissolved in a culture medium containing 5 per cent horse serum, reduction was generally retarded but still prompt at 265 mp. On the other hand, many tetrazolium derivatives, such as the 2,3-p-dinitro, 5-p:methoxy, triand the 2-p-nitro, 3-o-nitro, 5-p-methoxy, triphenyl(#38) phenyl- (#26), TABLE Tetrazolium #4
(non-NO*) #IO (non-NO,) A(17
(non-NO,, amyl) #7
(-NO,)
#21 (-NW ff25
(di-NO,) #40
(di-NO,)
I. Ultraviolet Medium” B B+S B B+S B B+S B B+S B B+S B B+S B
photochemical Character
Large crystals Large grains Fine grains Fine grains Very small, amorphous Fine, amorphous -
reduction
of tetrazoliums.
Rate
265 m,u
303 rnp
420 rnp
Rapid Rapid Slower Slow Very slow
Intense Intense -
Marked Marked Marked Slight Very slight
Slight slight Slight -
Very slow -
Slight +--Very
Slight -
-
-
Very minute, amorphous Very minute, amorphous Very minute, amorphous
-
slight---
Very slow
+--Very
slight---
Very slow
+-Very
slight----+
B+S
a B = Buffez; S = Serum.
derivatives reduced very slowly or not at all in a continuous beam even at the most active wavelength of 265 (Table I). In a tissue culture medium containing serum, compounds with the nitro groups represented by #26 and #38 reduced so slowly that there was no measurable formazan at 16 minutes of continuous irradiation. .Only slight reduction was discernible at 20 or 30 minutes; these compounds, therefore, are suitable for studies ,of intracellular reduction patterns by ultraviolet microscopy. The structural pattern of the reduction of tetrazolium to formazan within living tissue culture and ascites tumor cells was recorded for several tetrazolium derivatives (Table II). Particle size and distribution were examined in Experimental
Cell Research, Suppt. ,I
G. Z. Williams and A. C. Peacock
140
human heart, human intestine, Chang human liver, HeLa MAI, MA embryo tibroblasts and Ehrlich and Krebs 2 ascites cells. Examples of patterns of formazan production in these cells are illustrated in Figs. 12-15. In freshly separated mouse liver cells reduction in the presence of succinate is rapid and intense. The formazan particles appear first as minute grains throughout the cytoplasm in the region of the mitochondria. These grains TABLE
II. Reducfion of fefrazolium.
No. 38 tetrazolium (0.00125 &f) and the substrate in the concentration indicated were added to the ascites and culture cells and incubated at 38°C. “Intense” indicates that reduction started immediately, was marked within 10 minutes and formazan deposit was uniform and extensive throughout the cytoplasm. “Marked’‘-formazan decosit was initiated within 10 minutes, but less formazan was deposited even at 30 minutes. “Slight” indicates visible formazan, usually only in very small scattered grains or limited to formazan which accumulated in the fat droplets. Substrate
Cell type Mouse liver Krebs 2 ascites Hela MA1 KB MA fibroblasts Human intestine Human heart Chang liver
0 Moderate 0 0 0 0 a 0
Succinate (0.003 M)
Malate (0.005 M)
Lactate (0.095 M)
Intense Moderate Moderate Marked Slight Moderate Marked Slight
Slight Marked Slight 0 Moderate Moderate 0 Slight
Slight Marked Moderate 0 Moderate Moderate 0 Moderate
enlarge as the reduction progresses and the cytoplasm absorbs with increasing intensity at 300 my. Later much of the formazan transfers to the fat droplets which become violet or very black (Figs. 10 and 11). Cells,in which reduction progresses more slowly, or in which the reducing reagents are at much lower concentration, reveal varied patterns of widely scattered particles of formazan. In HeLa and ascites cells the particles frequently deposit in a peri-nuclear zone of variable dimensions. The elongated and stellate cells of fibroblasts and muscle cultures develop polar, juxtanuclear deposits of coarse grains (Fig. 12). In the cells with least reduction (Chang liver) no distinct particles in the cytoplasm occur, but the fat droplets gradually darken and finally absorb intensely, indicating progressive solution of the formazan in the fat as reduction occurs (Fig. 13). Experimeatal
Cell Research, Suppl. 7
Tetrazoliup
in liuing cells
141
Fig. lo.--Liver cell exposed to di-nitro, methoxy tetrazolium (#38), (0.00125 M), and sodium succinate (0.0034 M) for ten minutes. The minute deposits of formazan are revealed as black spots in the region of the mitochondria. Fig. Il.-Liver cell in tetrazolium are larger and more dense.
(#38),
and succinate
for 20 minutes.
The formazan
deposits
Fig. 12.-Formazan deposits in an embryonic fibroblast (tissue culture). Tetrazolium #38 (0.00125 M) and lactate (0.005 M). The granules are large, bipolar and peri-nuclear in distribution. Fig. 13.-HeLa
cell culture.
Formazan
in fat droplets.
Tetrazolium
#38 and lactate.
Experimental
Cell Research, Suppl. 7
G. 2. Williams and A, C. Peacock
142
Fig. 14.-KB cell tissue culture. The formazan is more dense, in large granules, and fills a large portion of the cytoplasm. Tetrazolium and lactate. Fig. 15.-HeLa uted patterns.
cell pre-mitotic
phase. Distribution
of formazan
particles
in small, widely-distrib-
The significance of the variations in reduction patterns which tend to be characteristic for cell types and tetrazolium derivatives is uncertain and is under further study. Determination
of Reduction
Rate of Tetruzolium
in Living
Cells
By time-lapse cinemicrophotography and photographs of the oscilloscope tracing at successive intervals, the progress of formazan deposition was followed. Reduction rates were calculated from the increasing deflections of the oscilloscope tracing of selected particles, areas of cytoplasm or fat droplets in the cells (Fig. 15). The response of the television system to UV light is very nearly linear (gamma 0.96); therefore, the amplitude of the deflection expressed as volts is a measure of the amount of light detected by the “vidicon” tube. Full deflection from black to white, V,,, represents 100 per cent transmittance. The level of the deflection peak from the black level, V,, is proportional to the amount of light transmitted by the object; thus, the density of the object is equal to log V,/Ve (Fig. 3). These measurements are relative and the light scatter error is assumed to influence both V, and V, equally. For liver cells in which the formazan deposit is dense and uniformly distributed in the cytoplasm, the conditions may be assumed to approach those in a solution of increasing density. The path length of absorption is fixed by the chamber depth of 10 p which is filled by the cell. The path length for Experimental
Cell Research, Suppl. 7
Tetrazolium in living cells
143
fat droplets in the cytoplasm may be assumed to be equal to the diameter of the spherical droplet. Thus, the rate of reduction of tetrazolium in single cells is calculated from consecutive observations of the density of the cytoplasm or fat droplet. .6
Ol
MINUTES
Fig. 16.-Rate of reduction of tetrazolium, (#38), by liver cells in presence of succinate. The upper curve represents reduction by a l-ml suspension in test tubes incubated in a water bath. The lower curve was derived from measurements of the successive deflections of the image line on the oscilloscope.
In each experiment designed to measure reduction rates in liver cells, alicylots of identical cell suspensions and tetrazolium and substrate reagents were incubated in test tubes. At similar time intervals the reaction was stopped in one tube, the cells were separated by centrifugation and the formazan was extracted and its density measured in a UV spectrophotometer. The reduction rate curve constructed from these data for cell suspensions is remarkably similar to that of the individual cell (Fig. 16). The continuing exploitation of this method will survey the patterns and rates of reduction of different tetrazolium-substrate systems in a large variety of cells. At the moment, tetrazolium derivatives are most useful and possess the following desirable properties for cytochemical studies: (a) solubility in aqueous media, (b) insolubility of the formazan, (c) relative low UV absorption of the tetrazolium, (d) intense UV absorption by particulate formazan, and (e) low cytotoxicity. Although differential reduction of tetrazolium compounds by certain substrate-enzyme systems within living cells appears likely from these preliminary results and those of many others using frozen sections and fixed tissues, the Experimental
Cell Research, Suppl. 7
144
G. Z. Williams and A. C. Peacock
exact role of tetrazolium is uncertain and the metabolic processes it indicates are obscure. Chemical studies are being continued to obtain further evidence for the nature of tetrazolium reactions.
SUMMARY
1. Television ultraviolet microscopy with time-lapse photography permits much shorter periods of exposure to ultraviolet light and facilitates studies of living cells and their intracellular reactions. Single exposures may be as The instrushort as da second compared to 6 second for direct photography. mentation is briefly reviewed. 2. The tetrazolium compounds which are reduced in association with certain substrate-enzyme reactions in cells produce ultraviolet absorbing, insoluble formazans and are useful as indicators for such reactions. 3. The individual tetrazolium reduction patterns of several derivatives in a number of different cell types were determined. 4. By oscilloscopic display of a single television scan-line traversmg the cell image, the voltage deflections may be measured and the density of the object calculated. Thus, intracellular reduction rates of the tetrazolium compounds were determined and found to agree with chemical studies in vitro. REFERENCES 1. CASPERSSON, T., Nord. Med. 7, 337 (1934). 2. .I. Roy. Microscop. Sot. 60, 8 (1940). 3. FLORY, L. E., Cold Spring Harbor Symposia Quant. Biol. 16, 505 (1951). 4. PARPART, A. K., Trans. Am. Microscop. Sot. 71, 311 (1952). 5. WALKER, P. M. B. and DAVIES, H. G., Discussions Faraday Sot. 9, 461 (1950). 6. WILLIAMS, G. Z., J. Histochem. Cyfochem. 5, 246 (1957). 7. IRE Trans. on Med. Electronics. In prefis. 8. WILLIAMS, 9. WILLIAMS, 10. ZWORYKIN,
Experimental
G. Z., NEUHAUSER, R. G., VUREK, G. G. and JOHNSTON, G. Z., PEACOCK, A. C. and MENGOLI, H. F., In press. V. K. and FLORY, L. E., Elec. Eng. 71, 40 (1952).
Cell Research, Suppl. 7
G. I., In press.
131
Cell Research, Suppl. 7, 131-144 (1959)
ULTRAVIOLET
STUDIES OF TETRAZOLIUM IN LIVING CELLS G. Z. WILLIAMS Tke Rockefeller
Institute,
REDUCTION
and A. C. PEACOCK New York, N.Y.,
U.S.A.
AN
understanding of the. intiniate metabolic processes in. living cells is based chiefly on detailed studies of chemical reactions of cell extracts and separated particulates. These materials are most often disorganized mixtures of components of several types of cells. Tissue slices are composed of several different cell populations. Suspensions of ascites turnor cells approach the ideal situation. However, study of the orientation and localization of reactions within individual living cells has been limited by technical difficulties. Successful observation and measurement of chemical changes in living cells requires seleqtion of sensitive and specific detection methods, the avoidance of toxic chemicals and physical injury to the cells, and the recognition .an,d correction of nlethodological errors. The ultraviolet region of the spectrum has been applied by many investigators [l, 2, 51 to detect specific materials in cells. The methods available require rather long exposures to ultraviolet, rapidly damaging to fresh cells. Amplification and electronic devices which increase the sensitivity of photodetectors make it posstile now to examine living cells by low intensity ultraviolet microscopy with less injury, and to utilize absorbing chemical indicators for observation of intracellular reactions. One group of such indicators are the tetrazolium salts which are relatively non-toxic in low concentrations (0.0125 M). This substance enters the intact cell, is reduced to insoluble formazan which precipitates immediately in situ and is detectable in very small amounts by ultraviolet absorption. By proper selection of substrates and tetrazoliums and utilization of specific ultraviolet wave bands, one may readily measure and record intracellular rates and locations of specific reduction reactions.
Television Ultraviolet Microscopy Photographic methods require relatively long exposures to ultraviolet. More sensitive methods of detection by photoconductive principles employed in the television approach [3, 4, lo] were devised to decrease exposure times of cells. The ultraviolet television microscope possesses the. following advantages [6]: (a) The facility for searching and focusing with green light to avoid Experimental
Cell Research, Suppl. 7
132
G. Z. Williams and A. C. Peacock
Fig. l.-Television ultraviolet microscope. The adapter body tube directs the UV image into either the film camera above or the TV camera immediately over and left of the reflecting tjrpe microscope. Synchronizing control panel is at the left and the line-selecting oscilloscope at the right. During the operation, film motion picture camera and still camera are mounted in front of the TV screen and oscilloscope.
cell injury. After focusing, the photographic record of the television image may be made with a very short pulse of ultraviolet. (b) Utilization of the ultratube which permits the use of ultraviolet pulses violet-sensitive “vidicon” of the order of l/l00 sec. The contrast of the absorption image may be electronically enhanced to intensify faintly visible structures. (c) Approximate values of reduction rates may be determined from records of deflections of a single TV line traversing the cell image as depicted on a line-selecting oscilloscope. There are certain disadvantages to the television method. Resolution is limited even in the ultraviolet, by the separation of scanning lines. Spatial resolution with the present instrument is only about 0.2 EC.There are difficulties in maintaining reproducibility of measurements with the line-selecting device because of electronic circuit characteristics. These can be improved but require considerable and expensive modifications of the standard circuits, Thermal electronic noise is particularly annoying and difficult to reduce without sacrificing resolution. Contrast must be carefully controlled, and as in all optical methods, scattered light interferes with the accuracy of measurements. Experimental
Cell Research, Suppl. 7
133
Tefrazolium in living cells
Fig. 2.2Block
diagram
of the television
microscope
system.
Instrumentation The system consists of an ultraviolet light source, a monochromator, a microscope equipped with reflecting optics and an adaptor body-tube for directing the microscopic image into the television camera [S, 71, and is illustrated in Fig. 1. A medium pressure mercury arc and a quartz-prism monochromator are most useful. The Gray V and VII reflecting condensers and objectives are quite satisfactory and do not require refocusing with change of wave length. The television camera is equipped with a special ultravioletsensitive “vidicon” tube (RCA developmental “vidicon” C 73439). For the best image formation, the highest quality broadcast-type closed circuit television camera, amplifier and monitor are necessary (Fig. 2). Connected to a superior monitor, the line-selector oscilloscope displays a single line of the television picture raster on its cathode ray tube. This horizontal line may be moved vertically at will to traverse the cell image and transect any desired particle or structure. Deflections in this tracing are related to the density of the structures and sequential photographic records provide information for making measurements from which reaction rates may be calculated (Fig. 3). A parfocal 35 mm camera is mounted over the microscope with a moveable mirror so that accurate focusing is accomplished by television for direct ultraviolet photographs requiring 4 to 1 sec. exposures. When protection of living cells is necessary, photographs may be made of the television screen, with much shorter exposure times of & to r&s sec. To record the progress of reactions in living cells for several hours and avoid injury, it was necessary to interrupt the light and limit exposures Experimental
Cell Research, Suppl. 7
Fig. &-Stainless
steel and quartz perfusion chamber slide. The fluid depth between coverslip
and base is 10 ~1 and the surrounding
moat at the margin
permits
exchange
of medium
without
disturbing cells in the field of view.
vidual electronic components and their functions in the control system have already been described in detail [B]. The block diagram (Fig. 2) depicts the operation of the integrated system. A pulse circuit for intensifying the ultraviolet light source was included to provide short pulses of higher intensity during the open time of the light shutter.
Preparation of Specimens Liver cells (mouse and rat) were prepared from perfused liver, minced with a sharp blade and gently pressed through stainless steel mesh. Successively smaller mesh sizes were used and finally the liver brei was passed through fine weave nylon and sedimented in Earle’s salt solution without glucose or serum. One sedimentation in isotonic sucrose facilitates rapid separation of the small cell clumps and individual cells from the bare nuclei and mitochondrial suspension. Tissue cultures were grown on quartz coverslips and inverted over flat ground depressions of 10 or 30 y depth in quartz slides. The margin of the coverslip was sealed with clear, liquid rubber cement. Ascites tumor cells were enumerated with an electronic counter [9], and suspended in Earle’s medium. Four microliters were measured into the slide depression with a micrometer pipette to provide reproducible samples and uniform light path. Perfusion microchambers were made from stainless steel and quartz. The bottom of the round central chamber consists of a quartz disc surrounded by a narrow moat, which communicates externally through very small gauge polyethylene tubes. The round quartz coverslip rests on a shoulder of the rim to which it is sealed. The depth is 10 microns. The indicators or substrates may be introduced through the perfusion tubes. (Fig. 6.) In a typical experiment the tissue culture coverslip is inverted over the chamber containing the culture medium and the desired substrate. The slip is sealed in place with clear rubber cement or Diatex and immediately placed in the warm box on the microscope stage. (Fig. 7.) A representative group of cells is searched out and focused in green or blue light by observing the image on the television monitor. The density pattern of cell structures at the selected UV band is recorded from the oscilloscope line Experimental
Cell Research, Suppl. 7
Tetrazolium in living cells CLOCK OR w-4 I
I
I
TIMNO OF CONTROL CCWONENTS A I I Y llllll
I
135 I A B c D E
a
Fig. 4.- Diagram of electrical pulses of timing control components of functions of the shutters, lamp and TV blanking signals.
to indicate
critical
alignment
Fig. 5.-Oscilloscopic display of electrical signal (video) which produces the image for observation or time-lapse record (upper portion). The monitor screen unblanking pulse (negative square wave below) is adjusted to align exactly with the picture at the maximum signal amplitude and to turn on and off exactly at the beginning and end of two frames.
device blanks out the monitor picture except for the two frames during the open time of the camera shutter. Special circuits utilize the vertical blanking pulses from the synchronous generator to control the “vidicon” blanking, ultraviolet shutter opening, the interim sensitizing lamp, the kinescope blanking interval and the motion picture camera shutter (Figs. 4 and 5). The indiExperimental
Cell Research, Suppl. 7
Fig. K-Stainless steel and quartz perfusion chamber slide. The fluid depth between coverslip and base is 10 p and the surrounding moat at the margin permits exchange of medium without disturbing cells in the field of view.
vidual electronic components and their functions in the control system have already been described in detail [S]. The block diagram (Fig. 2) depicts the operation of the integrated system. A pulse circuit for intensifying the ultraviolet light source was included to provide short pulses of higher intensity during the open time of the light shutter. Preparation
of Specimens
Liver cells (mouse and rat) were prepared from perfused liver, minced with a sharp blade and gently pressed through stainless steel mesh. Successively smaller mesh sizes were used and finally the liver brei was passed through fine weave nylon and sedimented in Earle’s salt solution without glucose or serum. One sedimentation in isotonic sucrose facilitates rapid separation of the small cell clumps and individual cells from the bare nuclei and mitochondrial suspension. Tissue cultures were grown on quartz coverslips and inverted over flat ground depressions of IO or 30 p depth in quartz slides. The margin of the coverslip was sealed with clear, liquid rubber cement. Ascites tumor cells were enumerated with an electronic counter [9], and suspended in Earle’s medium. Four microliters were measured into the slide depression with a micrometer pipette to provide reproducible samples and uniform light path. Perfusion microchambers were made from stainless steel and quartz. The bottom of the round central chamber consists of a quartz disc surrounded by a narrow moat, which communicates externaily through very small gauge polyethylene tubes. The round quartz coverslip rests on a shoulder of the rim to.which it is sealed. The depth is 10 microns. The indicators or substrates may be introduced through the perfusion tubes. (Fig. 6.) In a typical experiment the tissue culture coverslip is inverted over the chamber containing the culture medium and the desired substrate. The slip is sealed in place with clear rubber cement or Diatex and immediately placed in the warm box on the microscope stage. (Fig. 7.) A representative group of cells is searched out and focused in green or blue light by observing the image on the television monitor. The density pattern of cell structures at the selected UV band is recorded from the oscilloscope line Experimental
Cell Research, Suppl. 7
Tetrazolium in living cells
Fig. 7.-Warm box on microscope stage. The tetrazolium-containing medium is contained in the syringe reservoir at the upper left. When the waste tube at the right is lowered as shown, gra vity causes fluid to flow through the perfusion chamber.
Fig. S.-Three superimposed oscilloscopic tracings of one liver cell at 5, 10 and 30 minutes alfter addition of tetrazolium. The successively lower deflections representing the liver cell indic:ate progressive increase of formazan from measurements of which a reduction rate curve mayr be calculated and constructed. tracing (control measurements). Tetrazolium is introduced into the perfusion cham ber and the progressive changes in density due to the reduction of the tetrazolium are recorded by time-lapse photography. Intracellular reduction rates are calcula ted from successive records of deflections of the line tracing. (Fig. 8.) Experimental
Cell Research, Suppl. 7
G. Z. Williams and A. C. Peacock
Fig. 9.-Intense
photochemical
reduction
Reduction
of triphenyl
Patterns
tetrazolium
by UV light at 265 my.
of Tetrazoliums
A number of tetrazolium derivatives were synthesiz6d for us by Dr. Stanley Pierce, of the University of Richmond. These compounds were grouped according to their ultraviolet absorption characteristics and reduction reactions with mouse liver homogenates and washed mitochondria. They reacted with suspensions of cells, homogenates or mitochondria, more or less rapidly, depending upon the number of nitro groups, but without apparent relation to other radicals. Thus, methyl-, methoxy-, isopropyl- and amyl-groups did not influence reduction properties. They may be divided further into classes which react best in the presence of succinate substrate, those which react most rapidly and intensely with malate or lactate and a third group which react equally well with alt three substrates. Some differences were observed in the size and distribution of intracellular formazan particles. Many tetrazoliums are reduced to formazan by the photochemical action of ultraviolet. The parent compound, triphenyl tetrazolium in saline or phosphate buffer solutions in a beam of ultraviolet was reduced to large crystals Experimental
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Tetrazolium in living cells
139
of formazan most rapidly at 265 mp, less rapidly at 300 and 312, slowly at 360 and very slowly at 427 and 545 rnp (Fig. 9). When the compound was dissolved in a culture medium containing 5 per cent horse serum, reduction was generally retarded but still prompt at 265 mp. On the other hand, many tetrazolium derivatives, such as the 2,3-p-dinitro, 5-p:methoxy, triand the 2-p-nitro, 3-o-nitro, 5-p-methoxy, triphenyl(#38) phenyl- (#26), TABLE Tetrazolium #4
(non-NO*) #IO (non-NO,) A(17
(non-NO,, amyl) #7
(-NO,)
#21 (-NW ff25
(di-NO,) #40
(di-NO,)
I. Ultraviolet Medium” B B+S B B+S B B+S B B+S B B+S B B+S B
photochemical Character
Large crystals Large grains Fine grains Fine grains Very small, amorphous Fine, amorphous -
reduction
of tetrazoliums.
Rate
265 m,u
303 rnp
420 rnp
Rapid Rapid Slower Slow Very slow
Intense Intense -
Marked Marked Marked Slight Very slight
Slight slight Slight -
Very slow -
Slight +--Very
Slight -
-
-
Very minute, amorphous Very minute, amorphous Very minute, amorphous
-
slight---
Very slow
+--Very
slight---
Very slow
+-Very
slight----+
B+S
a B = Buffez; S = Serum.
derivatives reduced very slowly or not at all in a continuous beam even at the most active wavelength of 265 (Table I). In a tissue culture medium containing serum, compounds with the nitro groups represented by #26 and #38 reduced so slowly that there was no measurable formazan at 16 minutes of continuous irradiation. .Only slight reduction was discernible at 20 or 30 minutes; these compounds, therefore, are suitable for studies ,of intracellular reduction patterns by ultraviolet microscopy. The structural pattern of the reduction of tetrazolium to formazan within living tissue culture and ascites tumor cells was recorded for several tetrazolium derivatives (Table II). Particle size and distribution were examined in Experimental
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G. Z. Williams and A. C. Peacock
140
human heart, human intestine, Chang human liver, HeLa MAI, MA embryo tibroblasts and Ehrlich and Krebs 2 ascites cells. Examples of patterns of formazan production in these cells are illustrated in Figs. 12-15. In freshly separated mouse liver cells reduction in the presence of succinate is rapid and intense. The formazan particles appear first as minute grains throughout the cytoplasm in the region of the mitochondria. These grains TABLE
II. Reducfion of fefrazolium.
No. 38 tetrazolium (0.00125 &f) and the substrate in the concentration indicated were added to the ascites and culture cells and incubated at 38°C. “Intense” indicates that reduction started immediately, was marked within 10 minutes and formazan deposit was uniform and extensive throughout the cytoplasm. “Marked’‘-formazan decosit was initiated within 10 minutes, but less formazan was deposited even at 30 minutes. “Slight” indicates visible formazan, usually only in very small scattered grains or limited to formazan which accumulated in the fat droplets. Substrate
Cell type Mouse liver Krebs 2 ascites Hela MA1 KB MA fibroblasts Human intestine Human heart Chang liver
0 Moderate 0 0 0 0 a 0
Succinate (0.003 M)
Malate (0.005 M)
Lactate (0.095 M)
Intense Moderate Moderate Marked Slight Moderate Marked Slight
Slight Marked Slight 0 Moderate Moderate 0 Slight
Slight Marked Moderate 0 Moderate Moderate 0 Moderate
enlarge as the reduction progresses and the cytoplasm absorbs with increasing intensity at 300 my. Later much of the formazan transfers to the fat droplets which become violet or very black (Figs. 10 and 11). Cells,in which reduction progresses more slowly, or in which the reducing reagents are at much lower concentration, reveal varied patterns of widely scattered particles of formazan. In HeLa and ascites cells the particles frequently deposit in a peri-nuclear zone of variable dimensions. The elongated and stellate cells of fibroblasts and muscle cultures develop polar, juxtanuclear deposits of coarse grains (Fig. 12). In the cells with least reduction (Chang liver) no distinct particles in the cytoplasm occur, but the fat droplets gradually darken and finally absorb intensely, indicating progressive solution of the formazan in the fat as reduction occurs (Fig. 13). Experimeatal
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Tetrazoliup
in liuing cells
141
Fig. lo.--Liver cell exposed to di-nitro, methoxy tetrazolium (#38), (0.00125 M), and sodium succinate (0.0034 M) for ten minutes. The minute deposits of formazan are revealed as black spots in the region of the mitochondria. Fig. Il.-Liver cell in tetrazolium are larger and more dense.
(#38),
and succinate
for 20 minutes.
The formazan
deposits
Fig. 12.-Formazan deposits in an embryonic fibroblast (tissue culture). Tetrazolium #38 (0.00125 M) and lactate (0.005 M). The granules are large, bipolar and peri-nuclear in distribution. Fig. 13.-HeLa
cell culture.
Formazan
in fat droplets.
Tetrazolium
#38 and lactate.
Experimental
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G. 2. Williams and A, C. Peacock
142
Fig. 14.-KB cell tissue culture. The formazan is more dense, in large granules, and fills a large portion of the cytoplasm. Tetrazolium and lactate. Fig. 15.-HeLa uted patterns.
cell pre-mitotic
phase. Distribution
of formazan
particles
in small, widely-distrib-
The significance of the variations in reduction patterns which tend to be characteristic for cell types and tetrazolium derivatives is uncertain and is under further study. Determination
of Reduction
Rate of Tetruzolium
in Living
Cells
By time-lapse cinemicrophotography and photographs of the oscilloscope tracing at successive intervals, the progress of formazan deposition was followed. Reduction rates were calculated from the increasing deflections of the oscilloscope tracing of selected particles, areas of cytoplasm or fat droplets in the cells (Fig. 15). The response of the television system to UV light is very nearly linear (gamma 0.96); therefore, the amplitude of the deflection expressed as volts is a measure of the amount of light detected by the “vidicon” tube. Full deflection from black to white, V,,, represents 100 per cent transmittance. The level of the deflection peak from the black level, V,, is proportional to the amount of light transmitted by the object; thus, the density of the object is equal to log V,/Ve (Fig. 3). These measurements are relative and the light scatter error is assumed to influence both V, and V, equally. For liver cells in which the formazan deposit is dense and uniformly distributed in the cytoplasm, the conditions may be assumed to approach those in a solution of increasing density. The path length of absorption is fixed by the chamber depth of 10 p which is filled by the cell. The path length for Experimental
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Tetrazolium in living cells
143
fat droplets in the cytoplasm may be assumed to be equal to the diameter of the spherical droplet. Thus, the rate of reduction of tetrazolium in single cells is calculated from consecutive observations of the density of the cytoplasm or fat droplet. .6
Ol
MINUTES
Fig. 16.-Rate of reduction of tetrazolium, (#38), by liver cells in presence of succinate. The upper curve represents reduction by a l-ml suspension in test tubes incubated in a water bath. The lower curve was derived from measurements of the successive deflections of the image line on the oscilloscope.
In each experiment designed to measure reduction rates in liver cells, alicylots of identical cell suspensions and tetrazolium and substrate reagents were incubated in test tubes. At similar time intervals the reaction was stopped in one tube, the cells were separated by centrifugation and the formazan was extracted and its density measured in a UV spectrophotometer. The reduction rate curve constructed from these data for cell suspensions is remarkably similar to that of the individual cell (Fig. 16). The continuing exploitation of this method will survey the patterns and rates of reduction of different tetrazolium-substrate systems in a large variety of cells. At the moment, tetrazolium derivatives are most useful and possess the following desirable properties for cytochemical studies: (a) solubility in aqueous media, (b) insolubility of the formazan, (c) relative low UV absorption of the tetrazolium, (d) intense UV absorption by particulate formazan, and (e) low cytotoxicity. Although differential reduction of tetrazolium compounds by certain substrate-enzyme systems within living cells appears likely from these preliminary results and those of many others using frozen sections and fixed tissues, the Experimental
Cell Research, Suppl. 7
144
G. Z. Williams and A. C. Peacock
exact role of tetrazolium is uncertain and the metabolic processes it indicates are obscure. Chemical studies are being continued to obtain further evidence for the nature of tetrazolium reactions.
SUMMARY
1. Television ultraviolet microscopy with time-lapse photography permits much shorter periods of exposure to ultraviolet light and facilitates studies of living cells and their intracellular reactions. Single exposures may be as The instrushort as da second compared to 6 second for direct photography. mentation is briefly reviewed. 2. The tetrazolium compounds which are reduced in association with certain substrate-enzyme reactions in cells produce ultraviolet absorbing, insoluble formazans and are useful as indicators for such reactions. 3. The individual tetrazolium reduction patterns of several derivatives in a number of different cell types were determined. 4. By oscilloscopic display of a single television scan-line traversmg the cell image, the voltage deflections may be measured and the density of the object calculated. Thus, intracellular reduction rates of the tetrazolium compounds were determined and found to agree with chemical studies in vitro. REFERENCES 1. CASPERSSON, T., Nord. Med. 7, 337 (1934). 2. .I. Roy. Microscop. Sot. 60, 8 (1940). 3. FLORY, L. E., Cold Spring Harbor Symposia Quant. Biol. 16, 505 (1951). 4. PARPART, A. K., Trans. Am. Microscop. Sot. 71, 311 (1952). 5. WALKER, P. M. B. and DAVIES, H. G., Discussions Faraday Sot. 9, 461 (1950). 6. WILLIAMS, G. Z., J. Histochem. Cyfochem. 5, 246 (1957). 7. IRE Trans. on Med. Electronics. In prefis. 8. WILLIAMS, 9. WILLIAMS, 10. ZWORYKIN,
Experimental
G. Z., NEUHAUSER, R. G., VUREK, G. G. and JOHNSTON, G. Z., PEACOCK, A. C. and MENGOLI, H. F., In press. V. K. and FLORY, L. E., Elec. Eng. 71, 40 (1952).
Cell Research, Suppl. 7
G. I., In press.