Cryomicroscopy of isolated plant protoplasts

CRYOBIOLOGY

21, 209-233 (1984)

Cryomicroscopy

of Isolated

Plant Protoplasts’

PETER L. STEPONKUS,*,2 MICHAEL F. DOWGERT,* JAMES R. FERGUSON ,t.3 AND RONALD L. LEVINt,4 *Department

of Agronomy und +Sihley School qf Meckmicul Cornell

University,

Ithaca,

and Aer-ospclc~e En,ginecv%zg, Nrb<, York 148.53

.. the direct observation of the freezing cell is the best means of obtaining information on the causes of death by freezing.”

Microscopic observations of biological specimens at subzero temperatures have been reported for over a century. Over this period, sophistication of design and precision of temperature control have continuously advanced with the development of new technologies. Early workers relied on ambient cooling of the specimen and microscope (and scientists!) by conducting their studies outdoors. To Molisch (23), the development of a cold chamber in which to place the specimen and microscope was a significant advance. Molisch (23) and Chandler (6) discuss the efforts of numerous plant cryomicroscopists in that era-Goeppert (13, 14), Sachs (29), Nageli (27), Prillieux (28), Kunisch (17), Miiller-Thurgau (24-26). Keceived November 16, 1982; accepted April 18, 1983. t This material is, in part, based on work supported by the National Science Foundation under Grant PCM8021688 and the U.S. Department of Energy under Contract DE-AC0 2-81R10917 to Peter L. Steponkus. Department of Agronomy Series Paper No. 1447. Presented in part at the 19th Annual Meeting of the Society for Cryobiology, Houston, Tex., June 277July 1, 1982. ? To whom correspondence should be addressed: 609 Bradfield Hall, Department of Agronomy, Cornell University, Ithaca, NY 14853. 3 Present address: Washington State University, Pullman, Wash. 99163. 4 Present address: Biomedical Engineering and Instrumentation Branch, National Institutes of Health, Bethesda, Md. 20205.

Somewhat later, similar approaches were used by Wiegand (40) and Akerman (1). Although crude by the standards of today, the approach was quite informative-but not without controversy. For instance, Goeppert (13, 14) observed ice formation in both the intercellular spaces and within the cells, whereas Sachs (29) observed that it almost always occurred in the intercellular spaces. This difference was reconciled by MullerThurgau (26) who observed that rapid cooling resulted in ice formation within the cell and in the intercellular spaces, whereas slow cooling only resulted in the latter. Schander and Schaffnit (30) were the first to employ a cold stage mounted on the microscope. Subsequently many stage designs, which varied in the means of cooling, were developed (see (8)). In several studies, control of temperature could be affected, but precise control of cooling and warming rates was lacking. This, however, was not considered essential because most of the reports were concerned only with the location of ice within the tissues (21, 33, 38). In 1970, Diller and Cravalho (9) detailed the construction of a cryomicroscope that employed an analog control system for a cold stage cooled by vaporizing liquid nitrogen to precisely control the temperature between 77 and 310°K at time rates of temperature change between zero and several thousand degrees per minute. The current

209 001 l-2240/84 $3.00 Copyright All

rights

‘C 1984 by Acadrmx of reproduction

in any

Prr,,. form

Inc. reserved.

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STEPONKUS

renaissance in cryomicroscopy is, in large part, due to this advance. Since then, several variants of the system have evolved, and limitations of an analog control system have been overcome with digital control systems (8, 31). Surely, Molisch would have been ecstatic. Similarly, over the past century the recording of the events during a freeze-thaw cycle has evolved from a written description and hand-drawn renderings or woodcuts (23) to still photography (5) to motion picture films (20) to video recording (4, 22), to computer-enhanced image analysis (16, 32). For a dynamic process such as cellular behavior during a freeze-thaw cycle, photographs fail to convey an accurate portrayal of the situation. High speed cinematography overcomes this deficiency and has a high degree of resolution. Film processing, however, is both expensive and time consuming. Video recordings overcome these drawbacks-but at the expense of picture resolution. There are, however, a wide range of video cameras that can be used for recording images in a manner not easily accomplished with photographic emulsions. For example, ultra-low light cameras are very useful for studies using fluorescent probes which would require extremely long exposure times for photographic emulsions. Most important, the video signal can be converted directly to digital information for computer based image analysis. In the last decade cryomicroscopy has emerged as a very powerful technique for quantifying and understanding cellular behavior during a freeze-thaw cycle. Quantifying volumetric responses as a function of the freeze-thaw protocol with respect to the incidence of intracellular ice formation has been a major concern. In recent studies, volumetric behavior of yeast cells (39), erythrocytes (lo), HeLa cells (22), mouse ova (I@, and human leukocytes (31) has been reported. Paradoxically, although it has been nearly 100 years since Mtiller-Thurgau

ET AL.

(26) observed the cooling rate dependency for intracellular ice formation in higher plants and nearly 50 years since Levitt and Scarth (19) proposed a mechanistic interpretation, quantitative studies of the volumetric behavior of higher plant cell types have only recently been reported (12). The power of the cryomicroscope, however, goes beyond its use for quantifying volumetric behavior in that it allows for the characterization of cellular lesions and the consequences of a freeze-thaw cycle on membrane structure and function (35-37). Presently, cryomicroscopy of isolated protoplasts has assumed central prominance in our laboratory. Because of the extensive use that is made of the cryomicroscope, automation of video image analysis has been of keen interest. THE

CRYOMICROSCOPE

The general design of our cryomicroscope parallels that of the MIT instrument (9) and the cold stage is similar to that reported by McGrath et al. (22). There are, however, fundamental differences in the temperature programmer and controller. The cryomicroscope system consists of (1) a cold stage, (2) a programmable voltage generator, (3) a temperature sensor, and (4) a light microscope as schematically depicted in Fig. 1. The Cold Stage

Cooling and warming of the protoplast suspension are effected by a “conduction heat transfer” stage consisting of a copper cold sink interfaced with a transparent electrical resistance heater (Fig. 2). Vaporized liquid nitrogen flows to the cold stage directly from the liquid withdrawal port of a 160-liter Dewar and serves as the refrigerant. The gaseous Nz passes through a passive heat exchanger of copper tubing and is then discharged into a “dry box” enclosing the stage, objectives, and condensor to minimize condensation on these components. In practice, the dry box con-

211

CRYOMICROSCOPY OF PLANT PROTOPLASTS

VIDEO MON I TOA

VIDEO

CASSETTE RECORDER

VI DE0 IMAGE PROCESSOR

-

d PROGRAMMABLE VOLTAGE GENERATOR

VIDEO MONITOR -

THERMOCOUPLE CONDITIONER

PSEUDO-DERIVATIVE FEEDBACK CONTROLLER

STRIP CHART RECORDER MICROSCOPE HEAT EXCHANGEI~

FIG. 1. Schematic diagram of the cryomicroscope system.

sists of a plastic shroud fastened around the objective nosepiece and extended to the microscope base. The center of the copper substage is milled out to a thickness of 0.305 mm (0.012 in.) in a circular shape and a OS-cm viewing port is bored through the center. This design allows for the use of close working distances condensers and yet still provides for adequate heat transfer. A glass coverslip (140 km thick), the underside of which is coated with tin oxide (Coming Glass Works, Corning, N.Y.), is positioned over the viewing port and serves as an electrical resistance heater. The copper substage is first encased in Mylar tape to electrically insulate it from the resistance heater. The coverslip is then attached to the Mylar surface with a silver-based, electrical conducting paint (Dynaloy 340, Hanover, N.J.) at two opposite edges through which the coverslip is electrically connected to the controller.

The specimen temperature is measured with a 5-Frn (0.0002 in.)-thick copper-constantan foil thermocouple (RDF Corp., Hudson, N.H.) positioned so that the measuring junction is positioned in the center of the viewing region on the upper surface of the coverslip heater. The leads of the thermocouple (200 pm wide) extending up to the viewing part are secured to the stage with clear nail polish (Maybelline, Hard & Fast). Once the heater has been attached, electrical leads connected, and the foil thermocouple positioned and secured, the entire stage-except for the viewing port-is masked with Mylar tape. This precaution precludes spurious short circuits induced by seepage of electrolytes and provides ample protection for the fragile thermocouple leads. Rapid warming of the copper substage at the completion of a programmed freezethaw cycle is effected by circulating hot air

212

STEPONKUS ET AL. A

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through the copper substage. Compressed air is passed through a drier and molecular sieve (Lab Clear gas filter, VWR, Rothester, N.Y.) and then through a heat exchanger of copper tubing wrapped with electrical heater tape which is “T”ed into the refrigerant lines entering the substage.

In this manner, warming and cleaning of the stage in preparation for the next specimen requires less than 5 min. The Programmable

Voltage Generator

A specialized digital voltage generator provides an analog, 0- to 5-V signal which

CRYOMICROSCOPY

OF PLANT

duplicates the desired freeze-thaw protocol. The processing unit determines whether an ascending or descending voltage will be imposed, when to stop at a selected voltage, and for what length of time the voltage should remain constant. The generator is capable of providing a reference signal for linear cooling and warming over the range of + 56 to - 200°C with 4096 stepping divisions (0,0625”C/division) in either direction. Cooling and warming rates between 0.012 and 8000”Cimin may be selected from fixed selector switches or varied continuously over this range. Cooling and warming to four preset temperatures with isothermal hold periods can be programmed with the provision for manual intervention at any time. The Temperature Controller The thermocouple signal is transmitted to a thermocouple conditioner which provides for cold junction compensation, linearization, and amplification of the signal (Model 4150-l 187, Action Instruments Co., San Diego, Calif.). The thermocouple wire leading to this device is shielded to prevent stray signals and noise from obscuring the millivolt level signal. Upon leaving the device, the signal is linearized between 0 and 5 V and corresponds to a temperature range of -200 to +56”C. The “conditioned” thermocouple signal and the reference signal from the voltage generator are used as inputs to an analog integral plus pseudo-derivative feedback (PDF) controller which regulates the rate and amount of energy dissipated in the form of heat by the tin oxide coating of the lower coverslip. The PDF controller was determined to provide superior controlled response to changes in the reference signal when compared with more conventional control algorithms, such as proportional control. The comparator compares the voltage levels between the reference and feedback inputs to provide an output voltage whose

PROTOPLASTS

213

magnitude depends upon the difference between the input voltage levels. This voltage is called the error which must be acted upon to make the two input voltages equal. The error voltage is then integrated before being passed through another summing or comparing operational amplifier. This summing junction has an output which depends on the magnitudes of the integrated error signal and pseudo-derivative of the feedback signal. This provides the input to a power transistor which regulates the voltage applied to the resistance heater upon the cryomicroscope stage. However, diodes ensure that no power is dissipated by the heater when the desired temperature is cooler than the measured specimen temperature. The PDF controller is capable of maintaining a constant conditioned signal from the thermocouple within 2.0 mV which corresponds to approximately O.OS”C-which is comparable to the stepping divisions of the voltage generator. Accuracy of the complete system at the thermocouple junction is of the order of ?O.l”C using calibrated thermocouples. The Light Microscope The cold stage is routinely fitted on a Nikon Biophot (Ehrenreich Photo-Optical Ind., Garden City, N.J.) equipped with brightfield, phase contrast, or differential interference contrast optics. For cryomicroscopy, differential interference contrast (DIG) optics are far superior to phase contrast optics for resolution of the specimen in the ice matrix. With phase contrast there is considerable error in determining cell boundaries due to the characteristic halos which are accentuated in the ice matrix. Both episcopic and diascopic fluorescence illumination are available. For isolated protoplasts (20 to 30 km in diam), a 20 or 40 x objective and 5 x photo eyepiece are routinely used. Transmission through the HFM photomicrographic attachment increases the final magnification by 1.25 x .

214

STEPONKUS

ISOLATED

Temperature Control

PROTOPLASTS

Isolated protoplasts have proven to be an excellent system in which to study cellular and subcellular aspects of freezing injury and cold acclimation in higher plants (3437, 41, 42). Protoplasts behave as ideal osmometers over a wide range of osmolalities determined from 0.3 to 3.0 Osm and 0.4 to 6.0 Osm for nonacclimated and acclimated protoplasts, respectively (12). Over this range of osmolalities the protoplasts remain spherical facilitating quantitative microscopic studies of volumetric behavior during a cooling/warming cycle. Further, water efflux is not impeded by cell walls or tissue organization. A significant advantage is that survival of isolated protoplasts frozen in vitro in a suspending medium parallels the hardiness (nonacclimated vs acclimated) of the tissue from which the protoplasts were isolated. Most important, since damage to the plasma membrane is of primary concern, functional characteristics of the plasma membrane in response to a freezethaw cycle can be studied directly and observed in isolated protoplasts at the most elementary level of cell organization with the plasma membrane still intact. USE

AND

THE

PERFORMANCE

ET AL.

OF

CRYOMICROSCOPE

Quantitative analyses of cellular responses at subzero temperatures-especially determination of volumetric behavior-require precise control of temperature and cooling/warming rates. For example, in a protoplast suspension of 0.50 Osm, the fractional osmotic volume of the protoplast may decrease from 0.93 at - l.o”C to 0.47 at -2.O”C. Thermal precision is largely dependent on the instrumentation, but is easily diminished by inappropriate working procedures. Image quality is significantly influenced by the choice of optics and user techniques. For both thermal precision and image quality, cryomicroscopy transcends the realm of the technologist and enters the realm of the artisan.

The temperature control achieved for cooling and warming rates of 10 and lOo”C/ min is presented in Fig. 3. With this particular stage design, maximum cooling and warming rates are limited to hundreds of degrees per minute. Three characteristics of such temperature profiles are important and should be noted: (1) the linearity of the cooling and warming rates without loss of temperature control at either the freezing or melting point of the suspending medium-when control is most difficult due to the latent heat of fusion; (2) “crisp” thermal transitions upon entering or leaving isothermal periods; and (3) minimal oscillations in temperature during isothermal periods. Additionally, no surges or pulses in the ice front could be observed microscopically during either cooling/warming or an isothermal period. During isothermal periods, the digitized signal of the specimen temperature varied 2 O.OOYC. Thermal Gradients Thermal characteristics are largely dependent on the design of the system. There are, however, several factors in the use of any system that must also be considered. Most notably, thermal gradients on the cold stage must be ascertained and selection of cells for observation should be within an acceptable temperature range-not just within the field of view. Considering the entire area of the specimen (5 mm in diam), the thermal gradients were circular with a significant influence (colder) of the thermocouple. Consequently a method was employed to determine isotherms in the viewing area without perturbation of the temperature profile by the presence of a probe. An estimate of isotherms was made by first balancing the controller so that the ice-water interface of distilled water at 0°C was located at a specified location in the field-the proximal end of the thermocouple junction. The temperature was then lowered in 0.25”C decrements

CRYOMICROSCOPY OF PLANT PROTOPLASTS

TIME

215

(mid

FIG. 3. Chart recording of specimen temperature for programmed cooling rates of 10 and IOOWmin to - 40°C.

from 0 to - l.o”C and the location of the ice-water interface was photographed with the interface considered the 0°C isotherm (Fig. 4). The isotherms are skewed in the region of the Cu portion of the thermocouple junction. This is because the foil thermocouple is fastened to the surface of the copper cold sink and protrudes into the viewing area and because of the relatively higher thermal conductivity of Cu vs constantan. Using the 20x objective, a 1°C temperature gradient occurred in the entire field of view with a gradient of 0.25”C/40 pm near the thermocouple. As cellular volumetric relations are extremely temperature dependent just below the freezing point, only cells within 40 km of the established 0°C isotherm were used for observation. With such a constraint, the cellular temperature was within 0.25”C of the inferred temperature.

Ice Nucleation Excessive supercooling of the suspending solution and the need for “seeding” of the solution were obviated by using a coverslip whose diameter was slightly smaller than the diameter of the viewing hole (ca. 5 mm). A l.O- to 2.0+1 sample was then loaded (specimen thickness ca. 50 to 100 km), and the coverslip positioned so that it covered the area of the lower heater coverslip which was not in contact with the copper cold sink. During sample loading and cooling of the substage to the desired temperature, the specimen temperature was maintained at +5”C. During cooling of the substage ( - 50 to - 75°C for these studies), some water vapor condensed on the Mylar covering of the copper substage. This condensate froze in the form of minute crystals which extended to the perimeter of the pro-

216

STEPONKUS ET AL.

toplast suspension. Due to the thermal characteristics of the stage, the specimen within the perimeter of the upper coverslip remained unfrozen until the programmed cooling of the specimen was initiated. Thereupon, nucleation of the protoplast suspension occurred and precluded supercooling of the protoplast suspension and the need for seeding by other means. Osmotic Gradients Precise control of temperature is required for an accurate inference of the osmotic potential of the extracellular solution during a freeze-thaw cycle. Even with precise temperature control, however, osmotic gradients may arise due to other conditions. With our protocol, osmotic gradients may be introduced by either the manner of nucleation or by desiccation due to the extremely small sample volume used. When nucleation of the suspending medium occurs along the sample periphery and advances inward, an osmotic gradient will be formed. The influence of this gradient on the volumetric behavior of the protoplasts under consideration will depend on the distance the protoplast is from the ice interface and the time of exposure. The influence of this was determined by loading the sample while the substage temperature was approximately 5°C and immediately measuring the cell diameter. The substage was then cooled to -50°C while the specimen was maintained at + 5°C. This normally requires approximately 3 to 5 min. Following this routine, volumetric contraction of the protoplast in response to this gradient was not measurable. The extent of desiccation of the small sample volume was determined by com-

FIG. 4. Thermal gradients on the cold stage as evidenced by displacement of the ice-water interface of distilled water at (a) 0°C. (b) -0.25”C, (c) -O.WC, (d) -0.7X, (e) - l.OO”C. Ten divisions of the graduated reticle = 20 km.

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paring fractional protoplast volumes before and after a nonlethal freeze-thaw cycle to - 5°C using protoplasts from acclimated tissue. Various cooling rates were used with a subzero isothermal period of 3 min. Following warming, the protoplasts were held at + 5°C for 5 min. The post-thaw fractional protoplast volume was 0.983 * 0.045 (n = 28), indicating that desiccation of the specimen was minimal. Thus, the need for “sealing” the sample coverslip was not necessary and allowed for the manner of nucleation described above. Specimen Image and Size Determination The quality of the specimen image at subzero temperatures previously published varies tremendously, with either the cell totally obscured by ice or exceptional clarity, and bears little relationship to the sophistication of the cold stage. Figure 5 shows a rye protoplast during cooling and warming under conditions where intracellular ice formation was precluded (cooling to -5°C at lO”C/min). The protoplast image remains sharply defined upon freezing of the suspending medium with the edge easily resolved. During dehydration, the shape of the protoplast can be approximated as a sphere. In Fig. 6, protoplasts subjected to a cooling protocol that results in intracellular ice formation (cooling to - 15°C at 16”Umin) are shown. Intracellular ice formation is characterized by the typical “flashing” phenomenon which is the result of nucleation of gas bubbles (34). With DIC optics, the gas bubbles are readily apparent. Several factors contributed to the quality of the image obtained. The use of differential interference contrast optics adds significantly to the optical resolution and is superior to phase-contrast optics. With phase contrast there is considerable degradation of the image due to the characteristic halos due to light diffraction (Fig. 7). In a frozen matrix this is especially apparent and detracts considerably from the

PLANT

217

PROTOPLASTS

image quality. More importantly, however, the halos around the cell preclude an accurate identification of the true cell boundary which could have serious repercussions in automated analysis of phase contrast images (e.g., (32)). The freeze-thaw protocols used with isolated protoplasts encompass cooling rates sufficiently slow (1 to 20”Umin) and the manner of nucleation of the suspending solution is such that ice enters the viewing area as a single ice front rather than many small dendrites. At faster cooling rates, or after excessive supercooling, dendritic ice formation causes much visual distortion. Most important, however, is the specimen thickness. Even using DIC optics and slow freeze-thaw protocols, the frozen image deteriorated significantly as sample thickness was increased. Routinely, samples of 1 to 2 (~-1 were used so that the formation of multiple ice planes was precluded. Care was taken, however, to ensure that the specimen volume was large enough to preclude wedging of the protoplasts between the coverslips. This was ascertained by the free movement of the protoplasts prior to freezing and after thawing of the suspending medium. The calculated thickness of the applied solution is approximately 50 to 100 pm, assuming a l- to 2-1*1sample volume and a 5-mm-diam coverslip. THE

VIDEO

SYSTEM

A schematic of the video components that are available for interfacing with the cryomicroscope is shown in Fig. 8. The components are used in various configurations depending on the experimental objective. The minimum configuration consists of a video camera, cassette recorder, and monitor. In all configurations, the video signal from the camera is combined with a time and temperature signal generated by a video character generator (Model V240TW, Vicon Industries Inc., Farmingdale, N.Y.). The compensated, linearized, and amplified signal from the stage thermocouple is digi-

218

STEPONKUS ET AL.

CRYOMICROSCOPY

OF PLANT PROTOPLASTS

tized (Model 54, Digitin, Van Nuys, Calif.) and transmitted to the character generator. The timer within the video character generator is started simultaneously with the temperature controller at the start of a freeze-thaw cycle. As a result, temperature (O.Ol”C) and time (0.1 set) are recorded directly on each video frame (e.g., Figs. 9 and 10) to facilitate analysis of time- and temperature-dependent phenomena. Video Cameras

Currently, five video cameras are available for use with the cryomicroscope. Initially, a relatively inexpensive monochrome CCTV camera (Model TE-44, General Electric, Syracuse, N.Y.), 3/4-in. video cassette recorder (Model CR 606OU, JVC, Maspeth, N.Y.), and monochrome monitor (Model VlO, Electrohome Ltd., Ketchener, Ontario) were used. With a monochrome image, however, a significant amount of image contrast due to inherent color differences is not available. This is a distinct disadvantage in cryomicroscopy and is especially obvious when using DIC optics. Therefore, a color camera (Model WV 3700, Panasonic Co., Secaucus, N.J.) with an NTSC (National Television Standards Commission) composite video output is routinely used for cryomicroscopy in the minimum configuration. This particular camera has a remote control unit which allows for a relatively compact and light camera readily interfaced with the microscope. Minimum illumination required is only 15 fc and good color rendition is obtained at the relatively low light levels encountered in DIC microscopy. The camera is interfaced with a 3/4-in. video cassette recorder (VCR) (Model NV-9400, Panasonic Co.) and displayed on an NTSC color monitor (Model RV 3701, World Video, Inc.,

219

Boyertown, Pa.) which employs the Sony Trinitron System. When controlled with the start switch on the video camera, recording can be interrupted without the usual video distortion because of an automatic editing function and capstan servo system. Horizontal resolution of the video camera is 250 lines. Although higher resolution cameras are available, the horizontal resolution of 3/4-in. video cassette recorders and NTSC color monitors is typically 250 lines. Therefore higher resolution cameras offer little advantage if video analysis is limited to visual observations of recorded video information. For video image processing, the horizontal resolution of the video camera should correspond to the resolution of the video digitizer, which is 512 x 5 12 pixels (picture elements) in our system. Therefore a camera with a minimum horizontal resolution of 512 lines would allow for the maximum resolution of image processor. For this purpose a Hamamatsu C-1000-1 Chalnicon (Waltham, Mass.) camera with 700 lines of resolution is routinely used. The camera has a relatively low light sensitivity (400 nA/lx) and is well suited for DIC optics. For ultralow light fluorescence microscopy, a silicon intensified tube (SIT) (Hamamatsu C-IOOO12) which has an extremely low light sensitivity (20,000 nA/lx) with only slightly less horizontal resolution (500 lines) is used. Both camera heads are controlled by a single camera control unit (CCU). For computerenhanced cryomicroscopy, the video output from the CCU is routed through the temperature/time character generator and then routed in parallel to a VCR/monitor for direct recording and to the video image processor for the initial video digitization. Although the quality and contrast of the monochrome image as viewed directly on a

FIG. 5. Dehydration of a nonacclimated protoplast during cooling to - 5°C at a rate of 10”Cimin in 0.53 Osm CaC& + NaCl at (a) 5.O”C, (b) - l.O”C, (c) -5°C; during an isohold period at -5°C for (d) 1.0 min, (e) 2.0 min, (f) 5.0 min; during warming at (g) 0”. (h) +5.O”C; and during an isohold period at +5”C for(i) 5 min, (j) 10 min. Scale = 10 km.

220

STEPONKUS

ET AL.

CRYOMICROSCOPY

OF PLANT

high resolution monitor is superb and lends itself to real time video image processing, color contrast-so useful in cryomicroscopy-would be a significant improvement. Three approaches for color separation and subsequent video image processing are possible. A relatively inexpensive NTSC color composite camera can be used with a RGB (red, green, blue) decoder to present three separate primary color images to the processor or a high resolution monochrome camera can be used with a computerized filter wheel assembly for generation of the primary color images. Alternatively, a high resolution three-tube camera with prismatic color separation with separate red, green, and blue (RGB) outputs can be used. Although the latter is the most expensive alternative, it appears the most suitable for cryomicroscopy. A high resolution (500 lines) three-tube (RGB) (Chalnicon) prism camera (Model DK 5000, Hitachi Ltd., Woodbury, N.Y.) provides a superb image. Video Image Processor

A video image array processor (Model 6424V0, DeAnza Systems, San Jose, Calif.) allows for digital processing of the video image. Originally designed for analysis of LANDSAT satellite images, the system is equally well suited for the analysis of microscopic images. The system is provided with I/O drivers and diagnostic and utility programs that operate under DEC RSX-1 1M operating systems. Additionally, software is available for image processor support, filters and transforms, graphics, and file access to disk and tape storage which expedites the implementation of image processing routines. Within the array processor, each incoming video frame is digitized into a 512 x 512 array of pixels with &bit intensity values in video frame time (li30th set). The

PROTOPLASTS

221

results can be viewed directly or stored in any one of three 5 12 x 512 x &bit RAM refresh memories for future manipulation or storage. Each of the refresh memories has its own roam, zoom, and intensity transformation table. A digital video processor based on two &bit arithmetic/logic units allows for a variety of data processing operations on up to 1 Mbyte of image data in one video frame time including calculation of sum or difference of two images and constants or functional modification of one or two images through intensity transformation tables. Additional hardware features include a 4-bit graphics overlay channel with scroll, zoom, and intensity transformation table; a hardware cursor generator to provide two cursors in eight different modes; joystick and interface; alphanumeric overlay generator; a dual rate (525 and 559) line output; and external sync control. The system allows for real time arithmatic operations such as multiply, add, subtract, compare, and reciprocal divides. Intensity transformation and look-up tables provide for contrast enhancement, intensity thresholding, slicing, and pseudo-coloring in real time. Operations such as digital filtering, convolutions, correlation, edge detection, and edge enhancement can be done in multiple frame times in the image processor alone. Only more complex operations require manipulations in the microprocessor memory. The image processor is controlled by a dedicated LSI 1l/23 microprocessor with 192-kbyte memory and Q-verter interface. A floppy 27-Mbyte fixed disk (Model VC2FFD, SMS, Mountainview, Calif.) and a magnetic tape transport and imbedded formatter (Digi-Data, Jessup, Md.) are interaced with the microprocessor. The system operating software is stored on the fixed disk which can also receive digitized

FIG. 6. Intracellular ice formation in a nonacclimated protoplast cooled at a rate of 16”Cimin in 0.53 Osm CaC12 + NaCl at (a) +5”C, (b) - l”C, (c) - lo”C, (d) - ll”C, (e) - 12”C, (fJ - 13”C, (g) - 14”C, (h) -15°C. Scale = 10 Fm.

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STEPONKUS ET AL.

FIG. 7. Intracellular ice formation in nonacclimated protoplasts suspended in 0.53 Osm CaC12 + NaClz at - 15°C as observed with (a) differential interference contrast optics and (b) phase contrast optics. Scale = 20 km.

images from the refresh memories. For permanent storage, the video images are off loaded onto magnetic tape, or in some instances, onto floppy disks. The system is accessed through either a VT100 terminal or DEKwriter (Digital Equipment, Maynard, Mass.). Additionally, a plotter (Model 7470A, Hewlett Packard, San Diego, Calif.) is available for graphics hard copy. Output from the video image processor is observed on a high resolution RGB monitor (Model C-3910, Mitsubishi Electric, Compton, Calif.) or, if only a monochrome image is desired, a high resolution monochrome monitor (Model V 10, Electrohome Ltd.). While a monochrome output can be recorded directly on a standard 3/4-in. video cassette recorder, recording of a color image requires that the RGB output be converted to an NTSC composite video signal by using

an NTSC encoder (Model 801, Lenco Electronic, Jackson, MO.). IMAGE PROCESSING APPLICATIONS FOR CRYOMICROSCOPY

Cellular Mensuration Volumetric behavior during a freeze-thaw cycle. Quantitative analysis of cell volumetric behavior by cryomicroscopy is an extremely arduous task. Typically, the freeze-thaw cycle is recorded on video tape and subsequently cell dimensions are determined by “freeze-framing” the video tape at fixed time or temperature intervals and using a linear scale held to the screen of the video monitor. Data collection is slow and, in practice, exceeds the amount of time required for the original freeze-thaw cycle by a factor of 2. As a result, the number of

223

CRYOMICROSCOPY OF PLANT PROTOPLASTS

RGB MONITOR

NTSC

COLOR

rt

FIG. 8. Schematic diagram of the video image prcessing system. NTSC. National Television Standards Commission color camera; SIT, silicon intensified tube: RGB, three-tube (red, green, blue) prism camera.

observations in such studies is quite low (e.g., (16, 18, 32)) or, if many cells are analyzed at frequent intervals (12), a great investment of time is required. Recently, Diller and co-workers (7,8, 11) have reported on computerized recognition of cells and their measurement. The method is, however, quite involved and time consuming in that 35-mm negatives are used as source images for video digitization. Several nontrivial computer operations are required for edge detection, noisy edge re-

moval, shape detection, and boundary tracking and cannot be done on video images in real time. To reduce the time required, image compression by pixel averaging is done-but diminishes image detail. All of these operations are necessary in attempting to automate cryomicroscopic video observations because the image is extremely “cluttered” due to boundaries within the ice matrix that intersect the cell. Simpler techniques based on thresholding the image intensity relative to the back-

STEPONKUS ET AL.

a

FIG. 9. Semiautomated volumetric analysis of an acclimated protoplast during cooling to - 10°Cat a rate of S”C/min in 1.03 Osm CaClz + NaCl. Video image corresponds to a 83 x 83-pm field of view.

CRYOMICROSCOPY OF PLANT PROTOPLASTS

FIG. 10. Acclimated protoplast cooled and warmed in a 1.03 Osm sorbitol soution. (a-c) digitized differential interference contrast image, (e-f) corresponding images after digital filtering. Photographs were taken of the video image displayed on a high resolution monitor. Video image corresponds to a 83 x 83+m field of view.

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ground intensity are of limited value because of fluctuations in image intensity during the freeze-thaw cycle. We have elected to take a semiautomated approach which allows for real time analysis of cellular volumetric behavior during a freeze-thaw cycle. With the video image processor, a circular cursor-the radius and position of which are controlled with a joystick-is generated in the overlay plane and superimposed on the digitized video image. The circle is described by drawing equal chords, the number of which can be varied between 3 and 50. Routinely a circle described by 6 chords is used for mensuration during a freeze-thaw cycle. When using a 40 x objective and 5 x photo eyepiece, the video image displayed on the monitor corresponds to a 83 x 83pm field of view. The overlay plane in which the cursors are generated is a 512 x 512pixel matrix. Thus, the measuring divisions correspond to 0.16 km (0.32 pm for 20x objective and 0.06 pm for 100x objective). The radius of the protoplast under observation is determined prior to initiation of cooling. During the freeze-thaw cycle, the operator continuously outlines the protoplast image by adjusting the radius of the circular cursor and, if necessary, its position. From the cursor coordinates, the radius, volume, and surface area of the protoplast are calculated, assuming sphericity, and continuously displayed in the annotation margins of the video image. Time and temperature, displayed directly on the specimen image, are generated by the character generator, the output of which is also converted to BCD and routed to the microprocessor. At a specified time or temperature interval or on operator command, the calculated cellular parameters are saved in data files on the fixed disk. Following completion of the freeze-thaw cycle, data analysis, including fractional cell volumes, fractional osmotic volumes, and the extent of supercooling of the intracellular solution at the specified time or temperature intervals are

ET AL.

printed out with graphic plots available for hard copy or video display. Thus, a real time analysis of cellular volumetric behavior is possible. This procedure is demonstrated in Fig. 9. In this particular example, a protoplast isolated from cold acclimated rye leaves is shown during cooling to - 10°C at a rate of 5”Umin. A 40 x objective was used and the video image corresponds to an 83 x 83-km field of view. All values (except gray scale) displayed in the annotation margin are in micrometers. In this particular example, the initial volume of 24,303 pm3 decreased to 16,894 urn3 during cooling to - 10°C and then decreased to 11,023 pm3 during the subsequent isothermal period at - 10°C. Accurate determinations of volumetric behavior during a freeze-thaw cycle require that deviations from sphericity are minimal and that protoplast boundaries are well defined for precise positioning of the cursor. Most commonly, protoplasts remain spherical during cooling at large fractional volumes (Figs. 9a-e). When excessively dehydrated, however, deviations from sphericity are apparent (Fig. 90. With DIC optics the protoplast boundary is well defined and generally no edge enhancement is required. Image quality, however, is strongly influenced by the composition of the suspending medium and thickness of the suspension. For example, the quality of the image of protoplasts cooled in sorbitol (Figs. lOa-c) is decreased considerably when compared with the image observed for protoplasts suspended in CaC12 + NaCl (Fig. 9). In the frozen sorbitol matrix, the protoplast boundary appears quite blurred and, in some regions, difficult to discern in the presence of ice. Further, in this particular instance, the DIC polarizer was intentionally set at a less than optimum setting to maximize the glare on the left side of the protoplast (Fig. 10a). In such instances, digital filtering of the image can be used for edge enhancement (Figs. lOd-f). In this case, a filter with a kernel of

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0 -63 - 127

63 0 -63

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127 63 0

was used to enhance the edges of the protoplasts. Such a filter is directional and will emphasize regions where there is a more or less abrupt change in gray level-indicating the end of one region and the beginning of another. The effect and directionality of the filtering operation is best appreciated and easily comprehended by examination of the time and temperature characters in the video image which have a light interior (256 gray level) with a dark border (0 gray level) displayed against a midrange (90) gray level. The protoplast boundary also presents a discontinuity or edge. As a result of filtering, the protoplast edges are defined and readily discerned from the solution channels in the ice matrix (Figs. lOd-f). Further, in the region that was obscured by the glare (Fig. IOa) the protoplast edge can be discerned (Fig. 10d). Ultra-Low Light Microscopy The use of fluorescent probes to study membrane behavior during a freeze-thaw cycle or as a result of osmotic or thermal perturbations alone is often limited by the low light levels encountered. Recording the response by photomicrography requires long exposure periods of many seconds. In some instances the required exposure time can be reduced by increasing the intensity of the excitation illumination, but this will increase the rate of photobleaching of many fluorescent probes. In contrast, an ultra-low light video camera using a silicon intensilied tube (Hamamatsu C-1000-12) allows for video microscopy at extremely low light levels. In fact, the intensity of the excitation light may be decreased substantially to minimize photobleaching and allow for continuous observation over long periods of time. When combined with a video image array processor, relatively simple arithmetic manipulations can be done in real time

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so that exceptional resolution at extremely low light levels can be achieved. For example, video frame averaging is an effective way to improve the signal to noise (S/N) ratio at low light intensities. In Fig. 11 a protoplast labeled with concanavalin A conjugated with fluorescein is shown when various numbers of frames (1 to 256) are averaged. Averaging of 64 frames results in a significant improvement in the S/N ratio observed in one frame. This requires approximately 2 sec. Further averaging of 128 or 256 frames results in only small improvement because the S/N ratio increases linearly with the square of the number of frames averaged. To further improve the image, the background can be subtracted (Fig. 12~) and the contrast of the resultant image enhanced (Fig. 12d) by transforming the narrow range of gray scale (0 to 25) in the subtracted image over a wider range of the available gray scale (0 to 256). A 3-D wire-frame plot of the image intensity surface (8 x g-pixel sampling interval) quantitatively illustrates the very low S/N ratio in one video frame (Fig. 13a), the improved S/N ratio after averaging 64 frames (Fig. 13b), the lowered gray level due to background subtraction (Fig. 13c), and contrast enhancement by gray scale transformation (Fig. 13d). High Resolution Light Microscopy Recently, there have been several reports of video-enhanced polarized light and differential interference contrast microscopic images (2, 3, 15). The resultant image quality is greatly improved and the conventional Abbe limit of resolution can be exceeded by at least a factor of 2 so that resolution of 100 nm is possible (2). As a result, the method allows for the viewing of cells at a magnification of 7000 or more without the sensation of “empty magnification” (3). These improvements have been achieved with a high resolution video camera (Hamamatsu C-1000-1) in which the gain and black pedestal levels can be conveniently

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FIG. 11. Ultra-low light microscopy of a nonacclimated protoplast labeled with concanavalin A conjugated with fluorescein illustrating the improvement in signal to noise ratio as a function of the number of video frames averaged. Video images recorded with a SIT camera and digitized. Photographs were taken of the video image displayed on the monitor.

adjusted to optimize image contrast at relatively high light levels, With the addition of a video image array processor further improvements can be made. For instance, the primary limitation of the technique is the increased video noise in a single video frame. This can be reduced by averaging video frames-as demonstrated in Fig. 11. Another undesirable consequence of the video-enhancement tech-

nique is the appearance of dirt on and/or imperfections in the optical surfaces at high video gains. With the video image processor, such imperfections in the optical surfaces can be removed by first digitizing the video image of the field of view without a specimen and then subtracting this image from the specimen image. This can be done in real time. Additionally, with digitized images further increases in contrast can be

FIG. 12. (left) SIT video image of protoplast labeled with concanavalin A conjugated with fluorescein. (a) 1 frame, (b) 64 frames averaged, (c) 64 frames averaged minus background, (d) 64 frames averaged minus background and contrast enhanced. FIG. 13. (right) Three-dimensional wire-frame of the image intensity surface of the video images shown in Fig. 12.

CRYOMICROSCOPY

OF PLANT PROTOPLASTS

better optimized and various digital filtering techniques can be used for edge enhancement. In Fig. 14a a high resolution image of a protoplast illustrates the high incidence of cytoplasmic vesicles observed in nonacclimated protoplasts subjected to hypertonic conditions. (As a 100x objective and 5 x photo eyepiece were used, the image corresponds to a 33 x 33-km field of view with each pixel corresponding to 0.06 pm. On the video monitor, a magnification of 8000 is achieved). These vesicles are a consequence of the subduction of the plasma membrane during protoplast contraction (35-37, 41, 42). In this image, contrast was first improved by the appropriate gain and black pedestal settings on the video camera control units and four frames were averaged. A histogram of the gray level (0 to 256) for the entire 512 X 512 matrix is shown in Fig. 14b. A histogram of the gray level across a single line (horizontal white line in the image) is shown in Fig. 14~. In Fig. 14d, the contrast was further enhanced by digital processing. In the region of the vesicles, the darkest areas had a gray scale of 60, while the lightest were 180, with a background of 120. Therefore, the gray scale of each pixel was multiplied by 2.0 and then a value of 110 was subtracted. The “stretching” of the gray scale in the 512 x 512-gray scale histogram (Fig. 14e) and the line slice histogram (Fig. 14f) quantitatively show the greater increase in contrast achieved by this approach. In Figs. 14a and d, the boundaries of the vesicles appear somewhat blurred. To improve their sharpness, the edges were en-

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hanced by using a linear digital filter to emphasize the edges or changes in gray level (Fig. 14g). The filtered image (Fig. 14g) was then subtracted from the original image (Fig. 14a). This image was then contrast enhanced (Fig. 14j). As a consequence the contrast and edges of the cytoplasmic vesicles have been increased. In the line slice histogram (Fig. 141), the effect of edge enhancement is manifested as sharper changes in gray level across the line scan whereas the increased contrast is manifested as a greater amplitude in the change in gray levels. Although these examples of video image processing entail relatively simple operations, they significantly increase the power of cryomicroscopy. Image enhancement is but one obvious advantage. Quantitative image analysis whether it involves automated mensuration capabilities, definition of cell boundaries in an ice matrix, or high resolution light microscopy adds a new and exciting dimension to cryomicroscopy. SUMMARY

It has been nearly 100 years since MtillerThurgau (26) employed cryomicroscopy to identify the cooling rate dependency of intracellular ice formation. Since that time cryomicroscopy has advanced from the “ice age” when Molisch (23) packed his microscope in ice to the “space age” of today when computer hardware developed for space satellite imagery is used for cryomicroscopic image analysis. Although interest in cryomicroscopy has been sporadic in the intervening period, current interest is at a high level-largely as a result of the refine-

FIG. 14. High resolution microscopy of a nonacclimated protoplast suspended in a hypertonic (I .OO Osm) solution of CaClz + NaCl showing a proliferation of cytoplasmic vesicles. (a) Digitized video image, (d) contrast enhancement of digitized video image, (g) digital filtering for edge enhancement, and tj) subtraction of the filtered image from the original image followed by contrast enhancement. Video image corresponds to a 33 x 33-p,rn field of view. Gray level histograms of entire 512 x 512 image for (b) digitized video image, (e) contrast enhanced image, (h) filtered image, and (k) filter subtracted and contrast-enhanced image. Gray level intensities for a single line demarked by the horizontal white line across (c) the digitized image, (f) the contrast-enhanced image, (i) the filtered image, and (1) the filter subtracted and contrast-enhanced image.

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ET AL.

14

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FIG. l&C

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ment in the cryomicroscope design by Diller and Cravalho (9). The increased sophistication in cryostage design and precision of temperature control allow for quantitative studies of cell behavior during a freeze-thaw cycle. Not only does quantitative video image analysis facilitate this task, but it provides for increased resolution of cellular and subcellular responses during the freezethaw cycle. Most importantly, cryomicroscopy presents a researcher with a panorama of cellular behavior within which existing facts can be placed in perspective and from which future experiments can be more accurately focused. ACKNOWLEDGMENT

We wish to acknowledge the able assistance of Mr. C. C. Fogelin in development and refinement of the computer software used in these studies. The analog temperature controller used to regulate the cryomicroscope specimen temperature was designed, built, and tested by R. C. Ricketson of the Mechanical Engineering Department at Cornell University in consultation with Professor R. M. Phelan. REFERENCES

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34. Steponkus, P. L., and Dowgert, M. F. Gas bubble formation during intracellular ice formation. Cryo-Letters 2, 42-47 (1981). 35. Steponkus, P. L., Wolfe, J., and Dowgert, M. F. Stresses induced by contraction and expansion during a freeze-thaw cycle: A membrane perspective. In “The Effect of Low Temperature on Biological Membranes” (G. J. Morris and A. Clarke, Eds.), pp. 307-322. Academic Press, New York/London, 1981. 36. Steponkus, P. L., Dowgert, M. E, Evans, R. Y., and Gordon-Kamm, W. Cryobiology of isolated protoplasts. In “Plant Cold Hardiness and Freezing Stress” (P. H. Li and A. Sakai, Eds.), Vol. 2. pp. 459-474. Academic Press, New York, 1982. 37. Steponkus, P. L., Dowgert, M. F., and GordonKamm, W. J. Destabilization of the plasma membrane of isolated protoplasts during a freeze-thaw cycle: The influence of cold acclimation. Cryobiology 20, 448-465 (1983). 38. Stuckey, I. H., and Curtis, 0. F. Ice formation and the death of plant cells by freezing. P/ant Physiol. 13, 815-823 (1938).

39. Ushiyama, M., Cravalho, E. G., Diher, K. R., and Huggins, C. E. Intracellular water content of Saccharomyces cerevisiae during freezing at constant cooling rates. Cryobiology 10, 517-518 (1973). 40. Wiegand, K. M. The occurrence of ice in plant tissue. Plant World 9, 25-39 (1906). 41. Wolfe, J., and Steponkus, P. L. The stress-strain relationship of isolated protoplasts. Biochim. Biophys. Acta 643, 663-666 (1981). 42. Wolfe, J., and Steponkus, P. L. Mechanical properties of the plasma membrane of isolated plant protoplasts: Mechanism of hyperosmotic and extracellular freezing injury. P/ant Physiol. 71, 276-285 (1983).