Radiant energy – Inspection of solids or liquids by charged particles – Electron microscope type
Reexamination Certificate
2000-06-22
2002-09-03
Anderson, Bruce (Department: 2881)
Radiant energy
Inspection of solids or liquids by charged particles
Electron microscope type
C250S310000, C250S440110, C250S441110, C250S442110, C250S306000
Reexamination Certificate
active
06444982
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to the field of environmental scanning electron microscopes, and methods for using the same.
BACKGROUND OF THE INVENTION
Conventional Scanning Electron Microscopes (CSEMs) require most samples to be dried of all water, and then coated with metal or carbon. This treatment generally precludes the study of dynamic events, such as the effects of dissolution.
In contrast, Environmental Scanning Electron Microscopes (ESE microscopes) and similar variable pressure microscopes, allow samples with a high moisture content to be imaged. Within an ESE Microscope, the samples are imaged by introducing water vapor into the chamber, and ionizing the vapor cloud directly over the sample. By controlling both the chamber pressure and the sample temperature, the sample can be maintained in a water saturated state.
SUMMARY OF THE INVENTION
In order to evaluate the effect of a substance on a sample, it is desirable to view a single sample at various time intervals after being exposed to the substance in a dissolution bath. For example, the dissolution characteristics of controlled released pharmaceuticals are often critical to the pharmaceutical's usefulness. Moreover, it is often important to monitor the dissolution of controlled release pharmaceuticals for extended time periods (e.g. 8, 12, or 24 hours or more).
Since the controlled release pharmaceuticals are moist during dissolution, it is advantageous to view these pharmaceuticals using an ESE microscope or other variable pressure microscope. This approach, however, has a number of drawbacks. First, the controlled release pharmaceutical sample is subject to damage when it is transferred from the dissolution bath to the ESE microscope. Second, once a sample of the pharmaceutical is removed from the dissolution bath for viewing with the ESE microscope, it can not be returned to the dissolution bath.
To alleviate these problems, conventional ESE microscope's offer a “peltier stage” which is mounted in the ESE microscope specimen chamber and which allows moisture to be condensed onto a sample by controlling the temperature of the peltier stage. In this manner, the peltier stage can be used to provide a “dissolution bath” of water for a sample. The peltier stages, however, are inadequate for evaluating the dissolution characteristics of pharmaceuticals for a number of reasons.
For example, current peltier stages are too small to hold a pharmaceutical tablet and, since they operate by condensing moisture onto the sample from the atmosphere within the ESE microscope, they cannot provide the desired degree of “mixing” for an effective dissolution experiment. In addition, since they operate on a condensation principle, it is not possible to use these stages to conduct dissolution experiments with other dissolution media, such as simulated gastric fluid or simulated intestine fluid.
Moreover, in order to conduct a dissolution experiment with a peltier stage, the ESE microscope must first cool the stage so that enough water condenses into the sample well of the peltier stage to immerse the sample in water. Then, in order to image the sample, the stage must be heated sufficiently to evaporate the water in the well so that the sample can be imaged. This process has a number of disadvantages. First, rather than allowing the sample to be maintained at a desired temperature (for example, 98.6° F., 37° C.) throughout the experiment, the sample must be repeatedly cooled to cause condensation, and then heated to cause evaporation. As a result, it is not possible to simulate the dissolution experiment of the human body. In addition, the condensation/evaporation technique becomes increasingly impractical as the size of the sample, and therefore the amount of water to be condensed and evaporated, is increased.
It is also known to deposit a sample into a sample cup located in the ESE microscope Specimen chamber, and to introduce liquid into a sample cup by using a syringe or similar device. Such a method, however, also fails to provide the desired degree of mixing, and, moreover, is inadequate for long term automated experiments because an operator must be present to refill the sample cup with liquid. Moreover, since this technique requires removal of the water by evaporation, it suffers from the same deficiencies as the peltier stage described above.
In accordance with the present invention, a system is provided for imaging, in an ESE microscope or other variable pressure microscope, a single sample at various time intervals during dissolution of the sample in a liquid. The system includes a sample chamber having a sample well. The sample well includes an first fluid port and a second fluid port for forming a dissolution bath in the sample well. In accordance with the system according to the present invention, the sample chamber is placed into the specimen chamber of the ESE microscope and a sample is deposited into the sample well of the sample chamber. Preferably, the sample well is large enough to fully immerse a typical pharmaceutical sample which is prepared as a solid oral dosage form (e.g. tablets from <5 mg to 1000 mg). The sample is immersed in a liquid which flows through the sample well via the first and second fluid ports during a dissolution cycle. The liquid is then drained from the sample well via one of the first and second fluid ports during a draining cycle, and then, during an imaging cycle, the sample is imaged by the ESE microscope. The dissolution cycle, the draining cycle, and the imaging cycle all occur while the sample well is inside the specimen chamber of the ESE microscope. By immersing the sample in a flowing liquid, a mixing effect is achieved which promotes dissolution of the sample because it reduces or eliminates the boundary zones which would otherwise form around the sample and impede dissolution. Moreover, since the sample well is filled and drained while it remains in the specimen chamber, a single sample can be imaged at various stages of dissolution by draining the well, imaging the sample, and then refilling the well at predetermined time intervals. In addition, the sample chamber in accordance with the present invention is not limited to using water as the dissolution fluid. Other dissolution media, such as simulated gastric fluid or simulated intestine fluid, can also be used.
Preferably, the second fluid port of the sample well is elevated relative to the first fluid port. This construction provides a number of additional advantages including i) preventing overflow of the well; and ii) providing a “sipping” effect which causes the level of water in the well to rise and fall, thereby enhancing the mixing effect. In accordance with this embodiment, the sample well is filled by coupling a source of dissolution fluid to the first fluid port during the dissolution cycle, and then coupling the first fluid port to a drain line during the draining cycle to drain the fluid from the sample well. A vacuum source (such as a pump) could also be coupled to the drain hose to more quickly and effectively drain the fluid from the sample well. This can be implemented in any known manner. For example, a three port valve could be used, with one port coupled to a water faucet, one port connected to a drain hose, and the other port connected to the first fluid port of the sample well. The valve could then be actuated in any known manner to couple the water faucet to the input port during the dissolution cycle, and to couple the drain hose to the first fluid port during the draining and imaging cycles. The valve could be actuated mechanically or electrically (or in any other known manner), and the actuation could be triggered manually by the operator, or automatically via, for example, a computer or other automatic control system.
In accordance with a further aspect of the invention, a passage at least partially surrounds the sample well, and the passage is coupled to a heating and/or cooling source to provide for temperature control of a sample placed in the sample well. Prefer
Mitchell James A.
Palermo Philip J.
Anderson Bruce
Davidson Davidson & Kappel LLC
Euro-Celtique S.A.
Wells Nikita
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