- Email: [email protected]
A multiple field time-lapse cinemicrography apparatus
0 1919 by Academic Press Inc. Experimental
Cell Research 47, 357-362 (19G7)
A MULTIPLE
FIELD
357
TIME-LAPSE
CINEMICROGRAPHY
APPARATUS J. ENGELBERG,
J. BROOKS,
W. MURRELL
and P. N. RAO
Department of Physiology and Biophysics, University of Kentucky, Lexington, Ky 40506, U.S. A.
Received December 13, 1966
T -1
IME apse cinemicrography, a technique which has led to results of singular beauty in biology, has one tedious aspect to it-only the cells in a given microscopic field are photographed in a given experiment. When cell samples of greater size are required, the experiment must be repeated over and over again. This difficulty can be obviated by having several time-lapse units in simultaneous operation in a given laboratory. The cost of duplicating the equipment and the space required to house it, however, make this approach uneconomical if not impossible for many laboratories. In 1959, in a conversation with Dr P. I. Marcus in Denver, Colorado, these considerations led to the idea of an apparatus in which several different fields would be repeatedly photographed in succession. The problem associated with maintaining several fields in focus without human intervention or complicated instrumentation, led us to abandon the idea at the time. Several years later it occurred to one of us, however, that the construction of a relatively simple form of such an apparatus was indeed possible for use in experiments where only low levels of magnification were involved. The description of this apparatus follows: The depth of focus of a number of microscope objectives is given in Table I. It is to be noted that a tolerance of 0.001” (25 ,u) is readily obtainable in the construction of a piece of apparatus in a machine shop. Higher tolerances can, of course, be obtained with greater care and expense. This suggests, a priori, that it should be possible, by relatively simple means, to construct a multiple field time-lapse apparatus in which a 2.5 x objective is used for observation on mammalian cells (diameter N 15 p). For statistical studies (determination of doubling times, duration of mitosis, survival of irradiated or otherwise damaged cells) the resolution afforded by such an objective is ample. A schematic diagram of the multiple field time-lapse apparatus devised for this purpose is shown in Fig. 1. Tissue culture cells were grown in a plastic Cooper dish.l The dish (1Z)2 was clamped onto stage (22) which could be 1 Falcon Plastics, 1.0s Angeles, Calif., U.S.A. 2 Italic figures refer to numbers in Fig. 1. Experimental
Cell Research 47
J. Engelberg,
358
J. Brooks, W. Murrell
and P. N. Rao
rotated about its axis. The optical axis of the microscope (20) was displaced from the axis of rotation of the stage. Thus, as the stage rotated new fields were brought into view by the microscope. The stage rotated in intermittent increments of about 36 angular degrees so that ten separate fields were brought beneath the microscope objective for each revolution of the stage. These ten TABLE (After
I. Approximate R. Barer, Lecture
depth of focus of microscope objectives.
Notes on the Use of the Microscope, Thomas, Springfield, 1956.) Numerical aperture
Magnification 2.5 10 40 100 a Estimate
0.08 0.2 -0.3 0.65 - 0.85 1.2 -1.3
p. 21.2nd ed.,
Depth of focus (micra) 45a c. 10 1-2 0.5
by extrapolation.
fields were photographed over and over again as the stage revolved in the course of an experiment. The rotation of the stage and the precise, reproducible orientation of each field were obtained in the following manner. A lever (2) was attached to the output shaft of a synchronous motor (1).l Each time the shaft made one revolution, the lever made one revolution. The motor assembly was set next to a brass drive plate (3). Ten brass pins (4) jutted out from the face of this plate in a symmetrical array. The pins were concentric with the axis of the plate and equidistant from one another. (The precise location of each pin on the plate is of no consequence to the precision of operation of the apparatus.) Each time the lever passed by the drive plate it engaged one of the pins and rotated the plate by approximately one-tenth of one revolution. The drive plate was geared (7, 8) to the revolving microscope stage; each time the drive plate rotated by a given amount the stage rotated by the same amount. For each revolution of the lever there existed a period during which the drive plate and stage moved. This was followed by a period (during which the lever returned to engage the next pin) when the stage was at rest. The stage and the attached culture chamber were in motion about onetenth of the time and at rest about nine-tenths of the time. During the rest period, after a suitable delay, the field was photographed. The camera was triggered via a microswitch (6) which was operated by the action of each of 1 Bodine Motor Experimental
Co., Type KYC-22RC,
Cell Research 47
6 R.P.M.
Multiple
field time-lapse apparatus
359
ten projecting Allen head screws (5) located on the face of the drive plate opposite to that on which the brass pins were mounted. The microswitch started a timing mechanism which operated the camera mechanism. By removing one or more of the Allen head screws it was possible to reduce the number of fields photographed for a given revolution of the microscope stage. MULTIFIELD
TIME-LAPSE
APPARATUS
Fig. l.-Multiple field time-lapse cinemicrography apparatus. I, Synchronous motor; 2, rotating lever; 3, drive plate; 4, pins with which rotating lever engages; 5, Allen head screws which trigger microswitch; 6; microswitch; 7, bevel gears; 8, gears; 9, ball bearing; 10, microscope objective; II, Cooper dish; 12, plano-convex condenser, F.L. 50 mm, N.A. 0.3, maximum aperture 35 mm, distance: 100 mm from object plane, 50 mm from mirror; 13, optical stop; 14, mirror; 15, green filter; 16, heat filter; 17, optical stop; 18, 19, Pyrex plano-convex condenser, 45 mm effective focal length, distance: 150 mm from mirror, 50 mm from lamp filament; 20, lamp (15 watt) filament, length of filament 10 mm; 21, revolving stage. (This semischematic diagram is drawn only roughly to scale.)
The stage was enclosed in a water jacket by means of which precise temperature control of the culture vessel was effected. The enclosure was gassed with a humidified CO,-air mixture. The optical design for the Kohler illuminating system is also shown in Fig. 1. The axis of the illuminating system was offset from the optical axis of the microscope to provide a dark-field effect by means of which the optical contrast was greatly enhanced. The cells were photographed by means of a Vinten electric motion picture camera1 which was connected to a timing system of our own design. It was found possible to maintain all ten fields in focus by the following simple expedient. The optical axis of the microscope was brought as closely 1 Vickers
Instruments,
Inc. Malden,
Mass., U.S.A. Experimental
Cell Research 47
360
J. Engelberg,
J. Brooks, W. Murrell
and P. N. Rao
as possible to the center of rotation of the stage, yet far enough away from the center to prevent adjacent fields from overlapping. Thus, the area of the Cooper dish which was photographed was a tiny one near its center, and the cells in all of the ten fields were in relative proximity of one another. This greatly minimized difficulties leading to lack of register or focus due to (a) the irregular nature of the bottom of the dish, (6) inaccuracies in the stage mechanism and (c) inaccuracies in the drive mechanism. The microscope adjustment was carried out as follows. The microscope tube (which was originally derived from an old microscope) was mounted on a carriage. The latter could be moved laterally by means of a hand-operated, finely threaded screw. The carriage permitted the microscope tube to be moved in a radial direction over the culture chamber. The tube was moved until the optical axis of the microscope coincided with the axis of rotation of the stage; at this location the objects on the surface of the culture dish did not move out of the field of view when the stage was rotated. The tube was then moved away from the axis of rotation just far enough for the adjacent fields derived from the multiple held mechanism not to overlap. By this means we were surprised to find, that though the system was designed to operate at low magnifications (a 2.5 x objective, N.A. 0.08 \vas used in our studies), the system gave indications of being capable of operating satisfactorily at magnifications obtained even with a 40 x objective. In this connection it is to be noted that the higher the magnification of the objective, the smaller the field and the closer to the center of rotation of the stage one can operate. The experiences described above seem to demonstrate that it is technically feasible to carry out multiple field cinemicrography studies with relatively simple and inexpensive instrumentation. For this technique, however, to achieve relevance with regard to routine laboratory investigations it is essential that one have a fairly efficient procedure for dealing with the resulting film. Only every tenth frame on the film corresponds to a given field. Thus, one cannot view the results of the experiment by running the film through a conventional motion picture projector. A number of approaches to this problem are under investigation. The most elegant approach would consist of reprinting the film in such a way that every tenth frame of the original appears in succession on the print. Thus, one ten-field film would be converted into ten one-field films which could then be analyzed by conventional means. Film processing laboratories1 which specialize in the manufacture of animated films have automatic equipment for converting ten-field films into one1 Cineffects, Experimental
Inc., New York, Cell Research 47
N.Y.
Multiple
361
field time-lapse apparatm
Fig. 2.-Three successive blocks of ten successive fields as photographed in the multiple field time-lapse cinemicrography apparatus using a 2.5 x (N.A. 0.08) Zeiss objective, a 10 x eyepiece and Vickers cinemicrography optics. HeLa cells are shown. Each field is about 0.5 mm across.
field films. The cost is, unfortunately, rather high-on the order of one dollar per foot of film. Alternatively, one can use a time and motion study projector-l to directly analyze the ten-field film. The frame counter on the projector can be used, by manual operation of the projector, to view only the frames corresponding to a given field. The procedure is quite tedious and tiresome and the operator must memorize previous fields to detect changes in the cells which he seeks. We hope to facilitate this process by the following means. A reversible motor will be geared to the projector drive. By means of a cam system the film will be advanced by exactly ten frames at a time. The projection of the nine intermediary frames mill be suppressed to reduce confusion. \I’e are hopeful that a system of this kind will provide a relatively economical solution to the problem of analyzing the ten-field films.2 A by-product of the operation of the multiple field apparatus is worthy of mention. The periodic motion of the rotating stage causes the liquid medium in the cell chamber to be gently stirred every few seconds. From the point of view of providing nutrient and gas exchange for the cells this is desirable. Conceivably, if this stirring were violent enough, it would cause the loosely attached mitotic cells to become dislodged from the surface of the cell cham1 Bell and Howell, Time and Motion Study Projector, 16 mm, Model 173. 2 Since the submission of this article such a projector system has been built operation shortly. Experimental
and will be in
Cell Research 47
362
J. Engelberg,
J. Brooks, W. Murrell
and P. N. Rao
ber, a condition hardly to be desired. In the Cooper dish the close proximity of the surfaces of the dish and dish cover leads to a rather thin film of medium above the cells and tends to damp the fluid flow. This damping can be increased, if necessary, by cutting down the rim of the bottom dish and bringing the two dish surfaces even more closely together. In this connection it is to be noted that near the center of the dish the fluid motion is a minimum. While we bring up here the possible advantages and disadvantages of the stirring motion of the dish, we have not in preliminary studies detected any undesirable effects of stirring. In Fig. 2 photographs obtained with the apparatus are shown to convey to the reader an idea of the focus and reproducibility of individual fields. It is to be noted that the microscope was focused only on one field and that no adjustments of focus were made while other fields were photographed. In one experiment HeLa cells were photographed through the 2.5 x objective over a 24 hr period. The resulting ten-field film was converted into ten one-field films. When these films were run through a conventional motion picture projector the fates of individual cells could be easily followed. Compared to a normal one-field film the image was slightly jumpy, reflecting no doubt minor inaccuracies in the degree of register of each frame. SUMMARY
An apparatus has been designed and constructed by means of which, using a single microscope and camera, ten separate microscopic fields were photographed in the course of a time-lapse study. The heart of the apparatus is a stage which, by means of a mechanism, was rotated about its axis in increments of about 36 angular degrees. The axis of the microscope was positioned eccentrically with the axis of the stage so that ten separate fields were presented to the microscope in succession. These fields were photographed over and over again in the course of an experiment. The successful operation of this simple apparatus illustrates that it is possible to achieve a many-fold increase in data derived from time-lapse-cinemicrography experiments in which a single camera and microscope are used. We gratefully acknowledge support of this investigation by PHS grant NIH-CA06835 from the National Cancer Institute and Research Grant E-303 from the American Cancer Society.
Experimental
Cell Research 47
Cell Research 47, 357-362 (19G7)
A MULTIPLE
FIELD
357
TIME-LAPSE
CINEMICROGRAPHY
APPARATUS J. ENGELBERG,
J. BROOKS,
W. MURRELL
and P. N. RAO
Department of Physiology and Biophysics, University of Kentucky, Lexington, Ky 40506, U.S. A.
Received December 13, 1966
T -1
IME apse cinemicrography, a technique which has led to results of singular beauty in biology, has one tedious aspect to it-only the cells in a given microscopic field are photographed in a given experiment. When cell samples of greater size are required, the experiment must be repeated over and over again. This difficulty can be obviated by having several time-lapse units in simultaneous operation in a given laboratory. The cost of duplicating the equipment and the space required to house it, however, make this approach uneconomical if not impossible for many laboratories. In 1959, in a conversation with Dr P. I. Marcus in Denver, Colorado, these considerations led to the idea of an apparatus in which several different fields would be repeatedly photographed in succession. The problem associated with maintaining several fields in focus without human intervention or complicated instrumentation, led us to abandon the idea at the time. Several years later it occurred to one of us, however, that the construction of a relatively simple form of such an apparatus was indeed possible for use in experiments where only low levels of magnification were involved. The description of this apparatus follows: The depth of focus of a number of microscope objectives is given in Table I. It is to be noted that a tolerance of 0.001” (25 ,u) is readily obtainable in the construction of a piece of apparatus in a machine shop. Higher tolerances can, of course, be obtained with greater care and expense. This suggests, a priori, that it should be possible, by relatively simple means, to construct a multiple field time-lapse apparatus in which a 2.5 x objective is used for observation on mammalian cells (diameter N 15 p). For statistical studies (determination of doubling times, duration of mitosis, survival of irradiated or otherwise damaged cells) the resolution afforded by such an objective is ample. A schematic diagram of the multiple field time-lapse apparatus devised for this purpose is shown in Fig. 1. Tissue culture cells were grown in a plastic Cooper dish.l The dish (1Z)2 was clamped onto stage (22) which could be 1 Falcon Plastics, 1.0s Angeles, Calif., U.S.A. 2 Italic figures refer to numbers in Fig. 1. Experimental
Cell Research 47
J. Engelberg,
358
J. Brooks, W. Murrell
and P. N. Rao
rotated about its axis. The optical axis of the microscope (20) was displaced from the axis of rotation of the stage. Thus, as the stage rotated new fields were brought into view by the microscope. The stage rotated in intermittent increments of about 36 angular degrees so that ten separate fields were brought beneath the microscope objective for each revolution of the stage. These ten TABLE (After
I. Approximate R. Barer, Lecture
depth of focus of microscope objectives.
Notes on the Use of the Microscope, Thomas, Springfield, 1956.) Numerical aperture
Magnification 2.5 10 40 100 a Estimate
0.08 0.2 -0.3 0.65 - 0.85 1.2 -1.3
p. 21.2nd ed.,
Depth of focus (micra) 45a c. 10 1-2 0.5
by extrapolation.
fields were photographed over and over again as the stage revolved in the course of an experiment. The rotation of the stage and the precise, reproducible orientation of each field were obtained in the following manner. A lever (2) was attached to the output shaft of a synchronous motor (1).l Each time the shaft made one revolution, the lever made one revolution. The motor assembly was set next to a brass drive plate (3). Ten brass pins (4) jutted out from the face of this plate in a symmetrical array. The pins were concentric with the axis of the plate and equidistant from one another. (The precise location of each pin on the plate is of no consequence to the precision of operation of the apparatus.) Each time the lever passed by the drive plate it engaged one of the pins and rotated the plate by approximately one-tenth of one revolution. The drive plate was geared (7, 8) to the revolving microscope stage; each time the drive plate rotated by a given amount the stage rotated by the same amount. For each revolution of the lever there existed a period during which the drive plate and stage moved. This was followed by a period (during which the lever returned to engage the next pin) when the stage was at rest. The stage and the attached culture chamber were in motion about onetenth of the time and at rest about nine-tenths of the time. During the rest period, after a suitable delay, the field was photographed. The camera was triggered via a microswitch (6) which was operated by the action of each of 1 Bodine Motor Experimental
Co., Type KYC-22RC,
Cell Research 47
6 R.P.M.
Multiple
field time-lapse apparatus
359
ten projecting Allen head screws (5) located on the face of the drive plate opposite to that on which the brass pins were mounted. The microswitch started a timing mechanism which operated the camera mechanism. By removing one or more of the Allen head screws it was possible to reduce the number of fields photographed for a given revolution of the microscope stage. MULTIFIELD
TIME-LAPSE
APPARATUS
Fig. l.-Multiple field time-lapse cinemicrography apparatus. I, Synchronous motor; 2, rotating lever; 3, drive plate; 4, pins with which rotating lever engages; 5, Allen head screws which trigger microswitch; 6; microswitch; 7, bevel gears; 8, gears; 9, ball bearing; 10, microscope objective; II, Cooper dish; 12, plano-convex condenser, F.L. 50 mm, N.A. 0.3, maximum aperture 35 mm, distance: 100 mm from object plane, 50 mm from mirror; 13, optical stop; 14, mirror; 15, green filter; 16, heat filter; 17, optical stop; 18, 19, Pyrex plano-convex condenser, 45 mm effective focal length, distance: 150 mm from mirror, 50 mm from lamp filament; 20, lamp (15 watt) filament, length of filament 10 mm; 21, revolving stage. (This semischematic diagram is drawn only roughly to scale.)
The stage was enclosed in a water jacket by means of which precise temperature control of the culture vessel was effected. The enclosure was gassed with a humidified CO,-air mixture. The optical design for the Kohler illuminating system is also shown in Fig. 1. The axis of the illuminating system was offset from the optical axis of the microscope to provide a dark-field effect by means of which the optical contrast was greatly enhanced. The cells were photographed by means of a Vinten electric motion picture camera1 which was connected to a timing system of our own design. It was found possible to maintain all ten fields in focus by the following simple expedient. The optical axis of the microscope was brought as closely 1 Vickers
Instruments,
Inc. Malden,
Mass., U.S.A. Experimental
Cell Research 47
360
J. Engelberg,
J. Brooks, W. Murrell
and P. N. Rao
as possible to the center of rotation of the stage, yet far enough away from the center to prevent adjacent fields from overlapping. Thus, the area of the Cooper dish which was photographed was a tiny one near its center, and the cells in all of the ten fields were in relative proximity of one another. This greatly minimized difficulties leading to lack of register or focus due to (a) the irregular nature of the bottom of the dish, (6) inaccuracies in the stage mechanism and (c) inaccuracies in the drive mechanism. The microscope adjustment was carried out as follows. The microscope tube (which was originally derived from an old microscope) was mounted on a carriage. The latter could be moved laterally by means of a hand-operated, finely threaded screw. The carriage permitted the microscope tube to be moved in a radial direction over the culture chamber. The tube was moved until the optical axis of the microscope coincided with the axis of rotation of the stage; at this location the objects on the surface of the culture dish did not move out of the field of view when the stage was rotated. The tube was then moved away from the axis of rotation just far enough for the adjacent fields derived from the multiple held mechanism not to overlap. By this means we were surprised to find, that though the system was designed to operate at low magnifications (a 2.5 x objective, N.A. 0.08 \vas used in our studies), the system gave indications of being capable of operating satisfactorily at magnifications obtained even with a 40 x objective. In this connection it is to be noted that the higher the magnification of the objective, the smaller the field and the closer to the center of rotation of the stage one can operate. The experiences described above seem to demonstrate that it is technically feasible to carry out multiple field cinemicrography studies with relatively simple and inexpensive instrumentation. For this technique, however, to achieve relevance with regard to routine laboratory investigations it is essential that one have a fairly efficient procedure for dealing with the resulting film. Only every tenth frame on the film corresponds to a given field. Thus, one cannot view the results of the experiment by running the film through a conventional motion picture projector. A number of approaches to this problem are under investigation. The most elegant approach would consist of reprinting the film in such a way that every tenth frame of the original appears in succession on the print. Thus, one ten-field film would be converted into ten one-field films which could then be analyzed by conventional means. Film processing laboratories1 which specialize in the manufacture of animated films have automatic equipment for converting ten-field films into one1 Cineffects, Experimental
Inc., New York, Cell Research 47
N.Y.
Multiple
361
field time-lapse apparatm
Fig. 2.-Three successive blocks of ten successive fields as photographed in the multiple field time-lapse cinemicrography apparatus using a 2.5 x (N.A. 0.08) Zeiss objective, a 10 x eyepiece and Vickers cinemicrography optics. HeLa cells are shown. Each field is about 0.5 mm across.
field films. The cost is, unfortunately, rather high-on the order of one dollar per foot of film. Alternatively, one can use a time and motion study projector-l to directly analyze the ten-field film. The frame counter on the projector can be used, by manual operation of the projector, to view only the frames corresponding to a given field. The procedure is quite tedious and tiresome and the operator must memorize previous fields to detect changes in the cells which he seeks. We hope to facilitate this process by the following means. A reversible motor will be geared to the projector drive. By means of a cam system the film will be advanced by exactly ten frames at a time. The projection of the nine intermediary frames mill be suppressed to reduce confusion. \I’e are hopeful that a system of this kind will provide a relatively economical solution to the problem of analyzing the ten-field films.2 A by-product of the operation of the multiple field apparatus is worthy of mention. The periodic motion of the rotating stage causes the liquid medium in the cell chamber to be gently stirred every few seconds. From the point of view of providing nutrient and gas exchange for the cells this is desirable. Conceivably, if this stirring were violent enough, it would cause the loosely attached mitotic cells to become dislodged from the surface of the cell cham1 Bell and Howell, Time and Motion Study Projector, 16 mm, Model 173. 2 Since the submission of this article such a projector system has been built operation shortly. Experimental
and will be in
Cell Research 47
362
J. Engelberg,
J. Brooks, W. Murrell
and P. N. Rao
ber, a condition hardly to be desired. In the Cooper dish the close proximity of the surfaces of the dish and dish cover leads to a rather thin film of medium above the cells and tends to damp the fluid flow. This damping can be increased, if necessary, by cutting down the rim of the bottom dish and bringing the two dish surfaces even more closely together. In this connection it is to be noted that near the center of the dish the fluid motion is a minimum. While we bring up here the possible advantages and disadvantages of the stirring motion of the dish, we have not in preliminary studies detected any undesirable effects of stirring. In Fig. 2 photographs obtained with the apparatus are shown to convey to the reader an idea of the focus and reproducibility of individual fields. It is to be noted that the microscope was focused only on one field and that no adjustments of focus were made while other fields were photographed. In one experiment HeLa cells were photographed through the 2.5 x objective over a 24 hr period. The resulting ten-field film was converted into ten one-field films. When these films were run through a conventional motion picture projector the fates of individual cells could be easily followed. Compared to a normal one-field film the image was slightly jumpy, reflecting no doubt minor inaccuracies in the degree of register of each frame. SUMMARY
An apparatus has been designed and constructed by means of which, using a single microscope and camera, ten separate microscopic fields were photographed in the course of a time-lapse study. The heart of the apparatus is a stage which, by means of a mechanism, was rotated about its axis in increments of about 36 angular degrees. The axis of the microscope was positioned eccentrically with the axis of the stage so that ten separate fields were presented to the microscope in succession. These fields were photographed over and over again in the course of an experiment. The successful operation of this simple apparatus illustrates that it is possible to achieve a many-fold increase in data derived from time-lapse-cinemicrography experiments in which a single camera and microscope are used. We gratefully acknowledge support of this investigation by PHS grant NIH-CA06835 from the National Cancer Institute and Research Grant E-303 from the American Cancer Society.
Experimental
Cell Research 47