Live-Cell Microscopy Environmental Control for Mammalian Specimens
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
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.
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:
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.
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.
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 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.
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:
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.
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