Live-Cell Microscopy Environmental Control for Mammalian Specimens
by Daniel C. Focht
One of the most exciting aspects of biology today is found in the small world of mammalian live-cell microscopy (MLCM). The ability to observe and quantify mammalian live-cell phenomena is made possible largely due to the recent advancements in technological tools for imaging. These advancements include microscopes, computers, detection systems, dyes, reagents and micro-observation techniques. We all know there is an unlimited wealth of dynamic information just waiting to be discovered relative to cell behavior, physiology, and morphology.
Typically, live-cell experiments can be classified into two categories; developmental studies to establish natural behavior, or induced change to study the effects of a controlled factor. Nearly all disciplines of cell research, from the university level to pharmaceutical companies, have embellished the benefits of this dynamic form of live-cell imaging. There is, however, one area of live-cell imaging which, until recently, has not seen technological advancements contemporary with the other imaging technologies.
This area is the environmental control of the specimen with respect to the limitations of optical microscopy. Frequently investigators equip themselves with the most advanced microscopes, computers, imaging programs, detectors, mass storage devices, and exotic peripherals they can afford, then cobble together a marginal micro-observation system. Because the correlation between in vitro and in vivo phenomena is paramount for mammalian live-cell biology, it is of the utmost importance to accurately simulate the host conditions of the isolated specimen during live-cell microscopy. Therefore, it follows that the accuracy of the data and thus the strength of the data's support should not be weakened through the data acquisition process. This article describes several new and contemporary technologies which will greatly simplify the micro-observation process for nearly all mammalian live-cell experiments.
In applications where single cell analysis is appropriate, the specimen must to be contained in an optical enclosure. The two most commonly used methods of micro-observation are the open culture dish and closed system observation.
Open Dish Observation
In applications where cells are being observed with low numeric aperture (N.A.) objectives, an open culture dish is frequently used. This is optically very similar to the observation of cells typically using phase microscopy in a plastic culture flask. The sophistication of today's experiments are far more demanding on the cell's environment, especially as it relates to microscopy. For example, during long-term developmental or induced change studies it is necessary to accurately simulate the host conditions on the microscope stage without limiting the resolution of the microscope and maintain the simultaneous flexibility to change variables. This simulation should include control of temperature, medium, pH and environmental toxicity. Medium and pH can be controlled by regulated perfusion. The toxicity of the artificial environment has to be evaluated against compatibility with the specimen. Presuming the artificial environment is non-toxic or biologically inert, one of the most difficult factors to control has been temperature. Even with all the relevant papers on the importance of accurate temperature control and its effect on data, it should be readily apparent that temperature is a critical factor in most mammalian live-cell experiments.
When using high N.A. applications most biologists have had to rely on peripheral stage warmers and the use of carefully supported coverslips to attain optical compatibility and simultaneous temperature control. Many times this meant sacrificing the accuracy of the thermal control, and/or adequate perfusion of the cells resulting in either inaccurate data, due to compromised cells or poor images. The following outline demonstrates the limitations of the traditional methods of open dish micro-observation.
- Conventional culture dishes are made of plastic which is known to have the following characteristics:
- Non-uniform optical surface which degrades the image.
- Strain in the optical surface due to the injection molding process which prohibits the use of polarization optics.
- Thickness of the bottom surface prohibits the use of high N.A. objectives.
- Inefficient heat transfer from the source to the cells resulting in long temperature stabilization cycles.
- Traditional culture dish warmers are peripheral heating devices that must radiate heat through two, poor, thermally conductive mediums (air & plastic) therefore resulting in:
- Thermal inaccuracy.
- Non-uniform temperature distribution.
- Excessive thermal transfer time during thermal recovery (on the order of minutes).
- Thermal expansion of the metal surface causing vertical displacement which is most apparent during 3D imaging or confocal applications.
One solution to the above problems is to observe cells in culture maintained in a patented Bioptechs Delta T live-cell, micro-environmental system. This new device utilizes a thin-film coating of a transparent, electrically-conductive coating applied to the bottom surface of a glass substrate which is then incorporated into a hybrid disposable culture dish structure optimized for live-cell imaging. This technique provides temperature control directly to the cells through a thermistor feedback loop which applies an electrical current through the coated underside of the glass substrate. The thermal response using this technique is as fast as 1 degree C/sec. The Bioptechs controller utilizes special safety circuitry that protects the cells and regulates the control current intelligently within seconds making it possible to compensate for temperature changes that occur in the dish due to surface evaporation or perfusion. This technique offers high resolution imaging capabilities through a uniform glass surface free of strain and is available in a variety of thicknesses, including the popular No.1.5 coverglass for high N.A. applications. The dish environment is compatible with all modes of microscopy including, but not limited to, brightfield, darkfield, phase, DIC, fluorescence, reflection interference and confocal.
The Delta T technique provides the basis for many adaptations of the basic principal. After becoming familiar with the accuracy, ease of use and superior optical images obtainable in a Delta T system, it is easy to envision and put into practice numerous derivations for specific applications other than isolated cell culture. The following are examples of some of these uses:
-
In tissue slice, natural or artificial membrane observation, the specimen can be easily suspended at an optical reference plane in the dish at the appropriate focal distance from the objective for ease or observation.
-
This approach also works well with isolated solutions for ion channel detection or cell migration studies.
-
The Delta T can also be used for brain slice observation on an inverted microscope with a brain slice adapter. With this device the brain slice can be observed in a temperature controlled optical chamber which can be continuously perfused during high resolution microscopy. This is of particular importance for induced change and toxicology studies.
-
The Delta T is also very useful for micro-observation of smooth muscle cells in conjunction with the Kent Scientific Rectilinear Force Transducer. With the Kent transducer a section of muscle tissue can be attached to the transducer and lowered into the Delta T, enabling simultaneous reading of the force value and correlating with optical observation on an inverted microscope.
As you can see, this system was designed with the live-cell microscopist in mind and readily becomes an indispensable addition to the microscope. There are a wide variety of adapters and specimen carriers available that enable Delta T users to take advantage of its superior capabilities.
Closed-System Chambers
A closed system chamber is required when there is a need for complete isolation of the specimen from the outside world or the ability to perform more advanced modes of microscopy than can be accomplished in an open dish. In this case, cells are placed in a temperature controlled, perfusable, optical cavity or plated onto a coverslip which is part of this cavity for microscopy. There are several traditional configurations of these closed system chambers commercially available. Nearly all of them utilize the same basic characteristics.
Traditional closed system chambers provide two optical surfaces separated by a perfusion ring sealed with gaskets. This "sandwich" is then clamped together by several other structures. With this type of enclosure the perfusion rate, volume, and optical suitability for various modes of microscopy are interrelated. In most cases, perfusion with this configuration results in a turbulent jet stream which tends to dislodge the cells and there is a trade-off for optical compatibility with all modes of microscopy. Furthermore, temperature control is usually achieved by the use of peripheral heaters or warmed air blown across the stage. In either case temperature control is not reliable nor controlled within an acceptable range.
When selecting a closed-system chamber the following factors need to be considered:
- Optical compatibility for the intended modes of microscopy.
- Temperature control, including uniformity.
- Volume of chamber.
- Perfusion.
- Volume exchange rate for perfusion.
- Cell surface shear.
- Imaging aperture.
- Thermal effect of optically coupled (oil) high N.A. objectives.
A solution to these problems is provided by the patented Bioptechs microaqueduct perfusion technique. Microaqueduct flow, the basis of the Bioptechs FCS2 closed-system, live-cell micro-observation chamber, is achieved by incorporating perfusion grooves into one of the optical surfaces which defines the optical cavity, thereby eliminating the perfusion ring common to most other chambers and defining the optical cavity with only one gasket separating the perfusion slide from the coverslip. The physical configuration of these grooves produce a laminar flow region in the optical cavity. The single gasket design allows the user to define the size, volume, thickness and shape of the optical cavity. In addition, microaqueduct perfusion provides large aperture flow inputs and outputs eliminating the problem of volume exchange rates. To further enhance the performance of this design, Bioptechs also includes the electrically-conductive, thermal control coating on the microaqueduct slide thus adding thermal uniformity to the chamber even during experiments with period of no flow.
This allows the specimen, adherent cells on a coverslip, to be maintained safely in a temperature controlled optical environment which is compatible with all modes of microscopy, including but not limited to low and high N.A., transmitted, brightfield, darkfield, phase, DIC, and reflected modes of fluorescence as well as confocal.
High N.A. Objectives
In high N.A. imaging it should be noted that the optical medium used to couple the objective to the coverglass will act as a heat sink. If the temperature is not also regulated, this will result in a temperature gradient across the field of as much as 5 degrees. The microscope manufacturers have not yet met the live-cell researchers' needs by providing integrated temperature control for the objective so an external objective heater must be used.
In order to accurately control the temperature of the objective, it is necessary to overcome the constant drain of heat from any thermally conductive mass such as the nosepiece or microscope frame. The thermal characteristics of all objectives and microscopes vary considerably making it necessary to provide an efficient transfer of heat to the objective with an intelligent controller. Such a system would sense the temperature of the objective at a point close to the specimen and regulate the heat applied to the objective while taking into account the thermal mass of the objective and the ambient conditions. A patented device for this purpose, which partially surrounds the upper portion of the objective central tube with a heating band and measures the thermal transfer in a gap formed between the ends of the heating bands is available from Bioptechs, Inc.. The controller regulates the heater current in such a manner that it maintains the temperature to within 0.1 degree C. Additionally, storing the objective in a 37 degree C enclosure when not in use will reduce the detrimental effects of thermal cycling between physiological and room temperatures. Bioptechs also provides enclosures of this nature.
Perfusion
Perfusion is necessary for one of the following two reasons; either experiments on live cells take place over a time span greater then cells will tolerate without fresh media or the experiment is one in which chemical change is induced through perfusion. In both cases it is necessary to have a method of perfusing cells in their micro-observation environment which will not impede the experiment or the acquisition of electrical, chemical or optical data. Due to the small
fluid volume in live-cell chambers, the diaphragm affect produced at the unsupported aperture of the coverslip and the sensitivity to shear stresses of the cells, selection of a perfusion system requires great care and in some cases a trade-off.
There are three basic methods of perfusion -- gravity flow, manual injection with a syringe or mechanical pumps. Gravity flow is very inexpensive but difficult to control at flow rates necessary for microscopy. Manual syringes are ideal for adding growth factors, inhibitors or other periodic small volume fluids. Mechanical pumps are the most reliable and are available in two popular forms; motorized syringe and peristaltic. The syringe pump is limited in volume for long term experiments and subject to flow variations on a micro-flow scale due to temporary sticking of the plunger. Before deciding which type is the most appropriate for the application consider the following:
-
Flow rate.
-
Uniformity of flow rate.
-
Range of flow rate.
-
Quality of flow.
In view of these factors, Bioptechs recommends the Micro Perfusion pump for perfusion during microscopy. The Micro Perfusion pump is a peristaltic pump small enough to be held in the palm of one hand. It uses a tachometer regulated DC motor and a multi-stage, step-down gear box to drive the roller spindle resulting in a flow profile free from sudden pulsations typical with most peristaltic pumps. It is equipped with an internal speed control. It can also be interfaced to a computer through an analog interface. The pump can be configured to provide single tube perfusion for closed chambers or dual tube for continuous self-leveling perfusion in an open chamber.
Summary
The micro-observation approaches described in this article demonstrate the commitment of Bioptechs, Inc. to providing state-of-the-art solutions for live-cell microscopy environmental control. Bioptechs techniques are rapidly becoming the standard for live-cell observation. Bioptechs live-cell observation products have wide ranging applications in cancer research, pharmaceutical studies and basic science, as well as direct medical uses in IVF and clinical pathology. Bioptechs welcomes your questions and suggestions.
About the Author
Daniel Focht, founder and president of Bioptechs, Inc. is an
opto-mechanical/electronic engineer. He began his career in optics when
affiliated with the Carl Zeiss Corporation, then furthered his experience in
live-cell microscopy when employed by Carnegie Mellon University to develop
automated microscopy systems for live-cell imaging. Dan is currently designing
and developing new and more advanced systems for live-cell research.