The Cell Is My Test Tube

Micromanipulator Tables In the early 1970s, researchers showed that macromolecules retained activity when injected into living cells. Since then, studies involving the activity or fate of any number of natural and synthetic macromolecules as well as transplanted organelles have created an informative field. Injected material can be studied directly, or longer-term effects such as transformation can be monitored; with some materials it is even possible to reisolate and characterize the injected material after incubation in the cell. Conceptually, manipulating single cells and injecting material into cells is very simple--secure the cell at the injection site, then use a very tiny needle to inject a small volume of material through the cell wall or membrane. In practice, though, it is a much harder proposition. Reproducibly handling minute volumes and eliminating all sources of vibration are two of the biggest concerns. For those operating on a tight budget, vibration problems can be handled by a simple trip to the local hardware store. A couple of 2 sq. ft. paving slabs on top of a bit of 1/2 in. dense rubber foam do a decent job of damping most vibrations inherent in a typical lab. For very sensitive procedures, however, a good pneumatic vibration isolation table will be essential. Another important consideration is the site chosen to set up the apparatus. If possible, find a corner in the basement as far from any machinery as possible, preferably one that shares structural components of the building, away from doors and high-traffic corridors and out of direct drafts. Many people use phase-contrast microscopy for micromanipulation and patch-clamp work, but some applications call for fluorescence or other techniques. An inverted microscope is usually the best choice, since one needs plenty of room above the sample for all the equipment. Also, many upright microscopes focus by moving the stage, which will disrupt the carefully positioned instruments when refocused. While it is possible to fix the stage and move the microscope during focusing, most inverted microscopes already use this arrangement. As video technology becomes more affordable, many researchers are also including a video camera, easing sample monitoring and allowing frame capture for record keeping and analysis. Getting to the Point--Micropipettes The proper injecting device for a specific application must often be custom made. Pipette pullers make micropipettes from glass capillary pipettes. MicroData Instruments manufactures two different pipette pullers: the PMP-100 and the PMP-100D. The PMP-100D is a pneumatically operated, NiCr coil-heated, programmable unit that produces tips from both ends of the pulled stock. The machine will pull injection needles with tips as fine as 0.1 µm. Pull and temperature profiles that work well for a type of stock can be saved for future use. Because the PMP-100D allows precise control of taper, it can also pull voltage-clamp and intracellular electrodes that are highly tapered (more conical). The PMP-100 model is similar, but also pulls multibarrel micropipettes. Narishige International lists a full line of pipette pullers for a variety of applications. The company will also work with customers to create a custom machine for special applications. Many of its products are CE certified for greater quality assurance. For many applications, quartz and fused silica micropipettes and electrodes are preferable. Quartz micropipettes are nonfluorescent and have superior electrical properties. They can have thinner walls and penetrate tougher cell walls than borosilicate pipettes. Since the melting point of quartz is too high for use with metal filament heaters, Sutter Instruments uses a CO2 laser as the heating source on the P-2000 pipette puller. The P-2000 is a versatile, programmable instrument that is optimized for a 1 mm tube or rod; a separate (factory configured) option allows work with smaller diameter capillaries and optical fiber. Sutter also manufactures filament-based pipette pullers (the P-87 and P-97 and the basic P-30). For certain applications, beveling the tip of the injecting device can improve electrical measurements and ease injection by reducing the tip diameter and creating sharper points. Sutter's BV-10 micropipette beveler uses a conventional plate lapping (grinding) approach to bevel tips and electrodes from 0.1 to 50 µm. Narishige International offers the EG-40 and EG-400 pipette grinders for high-precision shaping. Beveling of all tips can be monitored using a microscope; electrode beveling can be further monitored using electrical impedance. An alternative or complementary approach to grinding the tip is to use a microforge such as Research Instruments' MF42 or Narishige's selection of four models. Microforges create specific shapes at the ends of glass tubes, such as square ends, lumen reductions, and bends. The microforge can also be used to flame-polish surfaces. Downsizing--Micromanipulators Micromanipulators can be used to move cells around or hold them in place and to position microelectrodes and micropipettes. Most manual micromanipulators (for example, Singer's Mk I Micromanipulator) use levers to translate and reduce fourfold the movements of a joystick. Joystick control makes the Mk I easy to use. Damped bearings reduce shake, and careful counterbalancing ensures the tool stays put when released. Mirror-image pairs are available. Micromanipulators facilitate relatively fast pick-up, carry, and put-down operations under the microscope; it's surprising just how precise you can be with only fourfold reduction of your movements. But even with 0.5 mm joystick positioning accuracy, there is still only ~100 µm precision. For applications that need more precise tool positioning, researchers will need to use translation stage-based equipment. Tritech Research, for example, produces the microMAGNIPULATOR, which has a magnetic x-y stage. A needle holder can be smoothly positioned either by hand or using turnscrews and magnetically locked down. This type of unit can be moved with great control, even while engaging the magnetic lock; fixed-base, micrometer screw-controlled translation stages are therefore best for precision work. Narishige International offers more than 65 micromanipulators, including models incorporating hydraulic oil/water, electric, multiaxis, and other features. Sutter Instruments' MM-33 (also available from Drummond Scientific) is an example of a high-precision, compact x-y-z translation stage. The micrometer screw scales read to 0.1 mm in the y and z axes and to 0.001 mm in the x axis, although actual positioning is far more precise. Controls are located on the back of the unit, which allows one to array several around an experiment. Left- and right-handed configurations are available. Research Instruments also has a range of micromanipulators, with positional accuracies to 0.5 µm; models such as the TDU500 and TFU40 are small enough to mount directly on the microscope stage. Eppendorf's Micromanipulator 5171 There are also a number of electronic micropositioners available, such as Eppendorf's Micromanipulator 5171. This joystick-controlled unit is programmable, allowing the user to set appropriate speed ranges for the movement. An automatic home function eases tool exchange. The 5171 can only be used with inverted microscopes, making it challenging to work with multiple units. Eppendorf has incorporated similar positioning technology into several of its cell transfer, cell injection, and electrophysiology systems. Just a Little Pin Prick--Microinjection Harvard Apparatus' PLI-90 picoliter injector is a basic automated microinjector. It will deliver femtoliter to microliter volumes through a single micropipette using pressurized gas. The injection can be triggered using a foot switch, and the injection time can be precisely controlled. Positive pressure systems, such as the PLI-90, allow the fluid in the syringe to be balanced with the fluid around (or in) the target cell and also facilitate high-pressure tip-cleaning pulses. The PLI-100 model has all of the features of the PLI-90 but adds two built-in vacuum systems--one allows the injection tip to be filled from the tip (important when reagents are limited), while the second holds the target cell in place with a gentle vacuum applied to a large pipette. Tritech Research has a similar system: The basic microINJECTOR controls the duration of the injection pulse using a foot switch and has an additional Pulse-length Control Module allowing pulse settings from 0.1 to 20 seconds. The unit also has a Backfill Vacuum module that allows filling from the needle tip, and the Holding Pipette Controller adds a vacuum-hold component to the system. MicroData Instrument's PM2000(B) is a vacuum-hold machine that controls up to four injection pipettes, allowing multiple injections and combined injection/perfusion experiments. The PM2000 is a single-pipette version. Narishige International produces a full line of microinjectors, both manual and automatic. The IM-3 injector uses a standard Hamilton syringe. The IM-26-2 and IM-55-2 have been recently redesigned to be more compact and maneuverable than earlier designs. For those doing repeated injections and aspirations, the IM-300 motor-driven injector can be preprogrammed with different times and pressures. Stoelting's PM-20 Piezoinjector helps minimize cell damage during intracellular injection. Piezo materials expand in response to an applied current, and the piezoinjector uses this phenomenon to produce rapid acceleration over very short (1 µm) distances. This acceleration is also precisely aligned with the pipette axis, allowing the cell to be punctured with minimal compression. Fast withdrawal, along precisely the same path, is also thought to minimize cell damage by preventing adhesion and tearing. Burleigh Instruments also uses the piezoelectric effect in its PiezoDrill. An electronic amplifier generates adjustable pulses (frequency, duration, amplitude, and bandwidth) that are piezo-translated into cutting movements at the tip. Research Instruments has followed a similar path with the Sonic Sword Zona Penetrator for oocyte injection. Using an action described as analogous to sonication, the sonic sword removes blockages and debris from the micropipette and reduces oocyte trauma during injection. Environmentally Friendly To minimize cell stress during manipulation, several companies produce equipment to control the cells' environment during microscopy. Bioptechs is a leader in this area, specializing in "live cell microscopy environmental control systems." The Bioptechs Focht Chamber System 2 (FCS2) is a closed-cell system consisting of a chamber (the environmental optical cavity) that is assembled from eight parts. A key feature of FCS2 is the slide surface; the nonsample side is coated with Indium Tin Oxide and contacts two electrodes, allowing even and nearly instantaneous heating of the sample. An objective heater is useful for experiments that involve optical coupling between the lens and the sample (immersion oil or similar). FCS2 also includes a stage adapter for the environmental chamber and controllers for the chamber and objective heater. Various chamber configurations are available, including versions with access ports for perfusion studies and open versions that allow probe access to the samples from above. Tritech Research makes the relatively inexpensive DigiTherm™ Temperature Control Kit that includes a controller and a pair of Peltier Effect thermoelectric heat pumps that can be mounted directly to heat or cool microscope stages, various samples, and vessels. Narishige International's MT-1 Biowarmer comes in two configurations (Nikon or Olympus) with a compact design that allows multiple holders for hydraulic tubing to be positioned around the specimen. Arcturus Engineering's PixCell II laser-capture microdissection system While perfusion is outside the scope of this article, several other companies, such as ALA Scientific, which also has a strong presence in microtool fabrication and electrophysiology, market temperature control and perfusion equipment for microinjection studies. Drummond Scientific's Nanoject II Microinjector Single-Cell EEG--The Patch Clamp Erwin Neher and Bert Sakmann were awarded the Nobel Prize in physiology or medicine in 1991 for their pioneering work in patch clamp techniques, which allowed researchers to study the electrophysiological properties of biological membranes. Patch clamp techniques involve attaching a microelectrode pipette to a membrane to give a highly resistive seal (on the order of 109 (omega), a "gigaseal") and measuring the small (pA) currents that result from changes to membrane-bound structures such as voltage-dependent and receptor-activated ion channels. There are many techniques that involve different membrane-electrode configurations (inside out, outside out, whole cell, perforated patch), but they all rely essentially upon the same concept--a patch pipette is sealed to the target membrane with a micromanipulator, and electrical signals are studied with a low-noise amplifier and data recorder. Dagan Corp.'s 3900A patch/whole cell clamp measures capacitance rather than resistance, as has been traditionally done in patch clamp experiments, deriving the current signal from the capacitance charge. Measuring capacitance avoids some problems, such as thermal noise, and improves the signal-to-noise and dynamic range seen with a conventional instrument. The 3900A can be expanded with a number of options such as the 3910 Bilayer expander and the 3911 Whole Cell Expander, each of which can drive a variety of different probes. Dagan also produces the inexpensive PC ONE general patch clamp and the more specialized BVC-700 high-speed current clamp. Warner Instrument Corporation also has a wide range of patch clamp amplifiers for a number of general and specialized applications. Its PC-501A whole cell/patch clamp amplifier is a resistive feedback instrument that is suitable for many single-channel experiments. The PC-505A is a more sophisticated amplifier that is ideal for high-speed, low-noise patch clamp and whole cell applications. Both the PC-501A and the PC-505A can operate in multiple modes and accommodate a number of different headstages for different applications. Warner also manufactures some amplifiers for more focused studies, such as the OC-725C for Xenopus oocyte patch clamp experiments and the EC-800 and EC-825 single and dual channel epithelial voltage clamps. Other companies have designed products for specific applications. Axon Instruments, for example, produces patch clamps that are particularly suited for neuroscience use (but can be used by any electrophysiologist as well), including the Axopatch 200B (capacitor-feedback patch clamp), the Axoclamp 2B (voltage and current clamp), the Geneclamp 500B (voltage, current, and patch clamp), and the Axopatch 1D (resistor-feedback patch clamp). The above innovations make single-cell manipulation easier and more efficient, and laser-based techniques for dissecting and capturing cells are adding even more tools to the modern cell biologist's repertoire. Before too long it seems likely that the marriage of computational prowess with robotic advances will be adapted to automate some aspects of cell dissection, perhaps making it possible to harvest greater quantities of subcellular components for further analysis, and maybe even ending the days when lengthy ultracentrifuge spins are required for cell fractionation. Further Reading L. Luo et al., "Gene expression profiles of laser-captured adjacent neuronal subtypes," Nature Medicine, 5:117-22, 1999. K.O. Greulich, D.L. Farkas (ed.), Micromanipulation by Light in Biology and Medicine: The Laser Microbeam and Optical Tweezers, Boston, Birkhauser, 1999. N. Simone et al., "Laser capture microdissection: opening the microscopic frontier to molecular analysis," Trends in Genetics, 14:272-6, 1998. M. Berns, "Laser scissors and tweezers," Scientific American, 278:62-7, 1998. H. Yin et al., "Transcription against an applied force," Science, 270:1653-7, 1995. B. Sakmann, E. Neher (ed.), Single Channel Recording, New York, Plenum Press, 1995. J. Celis, Microinjection and Organelle Transplantation Techniques: Methods and Applications, London, Academic Press, 1986. A. Ashkin, "Acceleration and trapping of particles by radiation pressure," Physics Review Letters, 24:156-9, 1970. Star Wars Meets Cell Biology: Laser-Assisted Microscopic Manipulation In the early 1970s researchers such as Arthur Ashkin began to use lasers to trap and manipulate cells and particles. But as long ago as that may seem, many would be surprised to find out that much of this technology can be traced back to the microbeam studies done in the early 1900s involving tightly focused conventional light sources. Today's laser tweezers and laser scissors are the direct descendants of Arthur Ashkin's pioneering work. The physics behind the laser tweezer is, at least on a superficial level, easy to understand. Light's dual nature as a particle-wave means it has momentum and can therefore exert force on an object. Also, small objects that do not strongly absorb at the wavelength employed tend to diffract the laser beam. Changing the direction of the momentum-carrying photons transfers some of the momentum to the target object; when the geometry is exactly right the particle will migrate toward the beam. The numbers are harder to pin down precisely, but a few tens of milliwatts of laser power might produce a force of a few piconewtons; insignificant compared to, say, the brakes on your car but quite adequate to stop a fleeing E. coli in its tracks. Skeptical? One look at RNA polymerase playing tug-of-war with a laser tweezer-held styrofoam bead on the Web site of Jeff Gelles from the Department of Biochemistry at Brandeis University may change your mind (see www.bio.brandeis.edu/~gelles/stall). The force exerted by the laser tweezer is proportional to the power of the laser beam. However, you can't simply use more power for a greater effect because some of the laser energy is absorbed by the target. If too much energy is absorbed by, for example, a bacterial cell, the cell will die or might even explode--a process appropriately called opticution. Using nonabsorbed wavelengths, for example, 1064 nm from a NdYAG laser, helps prevent this problem. However, using relatively well-absorbed wavelengths, such as 337 nm UV light from a nitrogen laser, can lead to an entirely different phenomenon. Single photons that are absorbed ultimately just heat the sample, but high-energy photons can break chemical bonds and do more extensive damage in a process often called photoablation. Photoablation can eliminate particles, excise or cut specific structures, even punch tiny short-lived holes in the cell membrane. This technique, which strongly complements laser tweezers, is often referred to as laser scissors. Cell Robotics of Albuquerque, N. Mex., has the lion's share of the laser-assisted microscopic manipulation market. Its Cell Robotics Workstation comprises four units: a microscope workstation, LaserTweezers® 980/1000 for optical trapping, LaserScissors™ 390/20 for microdissection and microsurgery, and CellSelector™ for isolating single cells and delivery of picoliter-volumes of reagents. Each of the four components is also available separately. The LaserTweezers 980/1000 uses a 1 W, continuous-wave, infrared diode laser at 980 nm. The module, a fixed-beam design, focuses a single beam into a three-dimensional trap. In a fixed-beam design, the trap is stationary relative to the microscope objective while the microscope stage is moved. The other main design--the flying beam--often allows the trap to be interactively moved with a joystick while the objective and stage remain fixed. The precise position of the trap in the z plane (the height above the microscope slide) can be controlled, and the trap can be switched on and off and adjusted in intensity using software controls. Using the LaserTweezers is as simple as positioning the structure of interest under the optical trap using the x-y microscope stage, turning on the laser, moving the stage to bring a second structure into position, and turning off the laser. LaserScissors 390/20 uses a nitrogen laser with a dye head to produce a pulsed 390 nm beam. The intensity of the pulses, the repetition rate (from one to 20 pulses per second), and pulse mode (single pulse or repetitive pulse) as well as the size and z position of the cutting spot can all be adjusted in the program. Using the LaserScissors is as simple as using the tweezers--move the structure to be photoablated under the guide mark using the x-y stage and blast away. CellSelector allows you to move material from one culture to another. It uses no flexible or expandable tubing, so volumes are accurately controlled. The unit has a hinged design and a precise z stop, so a cell or structure can be withdrawn from one sample, the unit rocked out of the way, a second sample placed under the microscope, the CellSelector moved back into position, and then the captured sample ejected. With the Cell Robotics Workstation, a cell from one culture can be isolated using the LaserTweezers optical trap, captured using CellSelector, released into a second culture, moved alongside a second cell using the optical trap, and fused to the second cell using LaserScissors. Arcturus Engineering Inc. (Mountain View, Calif.) has put lasers and microscopy to a different use. Its PixCell II™ laser-capture microdissection system uses a nondamaging infrared solid-state laser. A key feature of the system is the CapSure™ film, a thermoplastic polymer film that can be layered onto a tissue section. To use the system, the researcher aims the laser at a cell of interest and fires the laser. The light activates the polymer film, which sticks to the target cell. The film can then be lifted away from the tissue section with the cell of interest, leaving all other material behind. Single cells and collections of similar cells can be collected using single shots; an "autofire" mode allows whole tissue regions to be collected. After capture, the isolated cells can be subjected to DNA, RNA, or protein analysis, and as the entire process can be videocaptured, a recorded image of the tissue morphology and selected cells is readily available. Bob Sinclair can be contacted at bobsinclair@tech-write-edit.com.

The Cell Is My Test Tube

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