Patent Application
20130002848
The invention generally relates biological imaging. More specifically, the present invention relates to a stage adaptor for imaging a biological specimens.
Over the past several decades the ability to achieve detailed images of biological specimens has substantially improved. The ability to capture real-time images of biological processes, such as morphogenesis in a cell, has contributed greatly to our understanding of how biological systems work and how we can alter or manipulate these processes.
Along with advances in imaging, advances have also been made in developing cell and tissue culture systems that more closely represent the in vivo condition and can be more easily studied to test different compounds and parameters. For example, the chick embryo chorioallantoic membrane (CAM) is an established model system to evaluate various in vivo parameters.
The CAM model has been used to study bacterial invasion (Adam R et al., Int J Med Microbiol., 2002, 292(3-4):267-75). This in vivo model has also been used to study angiogenesis and the effects of anti-angiogenesis agents (Richardson M et al., Current Drug Targets-Cardiovascular & Haematological Disorders, 2003, 3:155-185). Vascular changes, such as vascular leakage, can also be studied in this model (Pegaz B et al., J Photochem Photobiol B, 2005, 80(1):19-27). The CAM also serves as a good model for the study of some ocular diseases (Lange N et al., Invest Opthalmol Vis Sci., 2001, 42(1):38-46). In addition, transplantation of heterologous cells and tissues to the chick embryo is used to evaluate many different parameters of tumor growth and also to evaluate anti-neoplastic agents (Richardson M et al., supra). The natural immuno-deficiency of the chick embryo makes it a good host fora variety of cells and tissues. The CAM model system conveniently and inexpensively reproduces many of the characteristics of tumors in vivo, such as tumor mass formation, angiogenesis, infiltration and metastases.
CAMs are incubated either in ovo, or ex ovo as a shell-less culture. In both cases, the embryos must be maintained at temperature and humidity levels that are higher than those in the normal atmosphere of a lab. The embryos are preferably kept in an incubator at a temperature of about 37° C. and having approximately 60-70% humidity. These atmospheric conditions provide a hostile environment for other equipment, such as a microscope. As such, it is difficult to image the live embryos for extended periods of time and do intravital time-lapse video microscopy. It is not practical to bring an imaging device to the embryo in the incubator. Moreover, the risk of damaging a specimen by bringing the embryo to an imaging device limits its routine use. Thus, while the chick embryo CAM model has proven useful as a model for studying various biological activities such as vascularization, tumor metastases, etc, its use has been limited due to the need to carefully move chick embryos in and out of an incubator for observation.
Furthermore, imaging the CAM in ovo presents difficulties in obtaining a clear, high quality microscopic image of the specimen being studied. Although imaging the CAM ex ovo does provide a better opportunity to obtain such images, it is difficult to maintain the environmental conditions necessary to support the life of the embryo for any significant amount of time outside the incubator.
According to an aspect of the present invention there is provided a microscope stage adaptor for imaging a biological specimen. The adaptor comprising: a housing; a vented chamber contained within the housing; and, a removable lid for covering the vented chamber. The surface of the lid in contact with the environment comprises a depression for receiving an objective from a microscope, wherein an aperture is provided at the apex of the depression for viewing inside the vented chamber.
In one embodiment, the removable lid covers only the vented chamber. In this case, a second lid may be provided that covers at least a portion of the housing.
In another embodiment, the removable lid covers the vented chamber and at least a portion of the housing.
Each of these lids can be supported by compressible posts or retractable pins. Moreover, in each of the above cases, the removable lid can be transparent.
In a further embodiment, the depression is conical in shape.
In yet a further embodiment, the aperture is covered by a cover slip.
In an alternative embodiment, a heating element for controlling the internal of the housing is provided. In some cases, the heating element wraps around the vented chamber.
In another embodiment, the adaptor further comprises at least one probe to sense environmental conditions in the housing. The probe can comprises a sensor to detect the temperature in the housing and/or a sensor to detect the humidity in the housing. The probe can also be connected to a controller that adjusts the environmental conditions in the housing in response to signals from the probe. In this case, it may be desirable to include a channel beside the housing to hold the controller.
The stage adaptor of the present invention is preferably used for imaging the chorioallantoic membrane of a developing amniote incubated in a shell-free system, wherein the amniote is a chick embryo.
According to another aspect of the present invention, there is provided an integrated stage adaptor and imaging system comprising a stage adaptor as defined hereinabove interfaced with an imaging device.
In one embodiment, the imaging device is a photomicroscope. In another embodiment, the imaging device is a video-microscope. In either case, the imaging device is adapted to process fluorescent images. The integrated system can also be interfaced with a computer program for analysis of images.
According to a further aspect of the present invention, there is provided a stage adaptor as described essentially as above having at least two vented chambers contained within the housing. Each chamber covered by a removable lid.
According to another aspect of the invention there is provided a method for imaging a biological specimen. The method comprising the steps of: transferring the biological specimen to a stage adaptor as defined hereinabove; positioning the stage adaptor in the optical axis of a microscope lens; and imaging the biological specimen through the microscope.
In one embodiment, the imaging device is a photomicroscope. In another embodiment, the imaging device is a video-microscope. In either case, the imaging device is adapted to process fluorescent images. The integrated system can also be interfaced with a computer program for analysis of images.
The stage adaptor and related systems and methods described above are preferably used for imaging the chorioallantoic membrane of a developing amniote incubated in a shell-free system, wherein the amniote is a chick embryo.
These and other features of the invention will become more apparent from the following description in which reference is made to the appended drawings wherein:
The stage adaptor of the present invention can be used to image a number of different biological specimens. For example, the stage adaptor can be used to image living organisms, such as rodents and other laboratory animals, biopsied tissue, such a human breast tissue, and a variety cell and tissue cultures. For the purposes of the present discussion, the stage adaptor will be described with reference to its use in imaging the chorioallantoic membrane of a developing amniote incubated in a shell-free system. However, it should be readily understood that the stage adaptor could be used in a multitude of different applications.
The chorioallantoic membrane (CAM) is a vascular membrane found in the eggs of some amniotes, such as, but not limited to, chicks and quails. The present invention will be described with reference to chick embryos, since these are the most commonly studied organism for this purpose. However, the stage adaptor described herein could be used to study the CAM of other amniotes grown in a shell-less system.
The stage adaptor described herein can be used for imaging the CAM of a developing amniote incubated in a shell-less or ex ovo culture system. Auerbach et al., in Developmental Biology, 1974, 41: 391-394, the contents o which is incorporated herein, describe such an exemplary shell-less culture system. In brief, eggs are cracked on day 3 or 4 of incubation and the yolk sac placed in a Petri dish, or other suitable container. The shell-less eggs are incubated or grown in an environment with a temperature of about 37° C. with 60-70% relative humidity. After cracking, the CAM develops on the surface of the yolk sac thus permitting the whole of the organ to be available for observation of morphogenesis and growth or a response to an intervention.
The stage adaptor described herein allows for the CAM of the incubating chick embryo to imaged without having to sacrifice the chick. The stage adaptor containing the specimen, i.e. the chick embryo in a container that supports growth of the embryo, allows for real-time imaging of the CAM over a period of time or on separate occasions.
As shown in
As shown in
In operation, the space 80, defined by the wall of the chamber 24 and the housing 12, is at least partially filled with water, or some other equivalent liquid that can be used to maintain the humidity in the chamber 24. This space 80 can be overlaid by a covering structure 85 to minimize the amount of moisture lost to the surrounding environment. Preferably, the humidity of the chamber 24 is maintained at a level that is similar to that of the incubator, which the specimen is normally housed. The humidity is preferably maintained at about 55% to about 65% humidity, more preferably about 60% humidity.
In order to maintain the humidity of the chamber 24, a lid 40 is provided, which covers the space defined by the chamber 24 (see
As shown in
The temperature of the stage adaptor 10 may be maintained during operation by one of several different methods. For example, the ambient temperature of the room in which the microscope is housed can be adjusted to maintain the stage adaptor at a certain desired temperature. Moreover, the stage of the microscope may be enclosed in a temperature controlled housing. Furthermore, the ambient temperature of the housing may be maintained by including one or more heating elements within the housing. In one embodiment, a heating element 100 surrounds the specimen. Either alone or in conjunction with the heating element 100, the temperature can be maintained by a heating element 101 positioned in the water. Either heating element 100 or 101 may be associated with the base of the housing. Alternatively the heating elements 100, 101 can be associated with the lids 40, 85. The temperature is preferably maintained at between about 35° C. to about 40° C., more preferably about 37° C. to about 38° C. The temperature is preferably maintained by a thermostat-controlled, insulated heating element.
A probe 28 can be provided to sense the conditions within the vented chamber 24 or the space 80 between the wall of vented chamber 24 and the walls of the housing 12. The probe monitors the temperature and sends a signal to a heating element controller/thermostat 30 to maintain the temperature at a predetermined setting. The probe 28 may also monitor the humidity levels in the chamber 24 or the space 80.
The controller 30 may be freely attached via its wires to the stage adaptor 10. Alternatively, the stage adaptor 10 includes a receptacle for holding the controller 30 or the controller may be built into the stage adaptor housing 12. The controller 30 provides a visual display 32 of the temperature and/or humidity in the vented chamber 24.
In one embodiment, as shown in
The stage adaptor 10 can be adapted for use as part of an integrated incubation and imaging system. The integrated system includes an imaging device interfaced with computer hardware and software for displaying and analyzing images. Various types of microscopes can be used as the imaging device. For example, the controlled environment stage adaptor may be associated with an MOT fluorescent microscope, a confocal microscope, etc. or any other upright microscope. It can be used for identification or development studies, toxicity, or any in vivo test that can be assessed using a CAM model. Proprietary computer program products and algorithms may used to display and analyze the images.
An exemplary integrated stage adaptor and imaging system according to the invention is shown in
A plurality of stage adaptors may be mounted on a moveable stage whereby the photomicroscope records changes on a rotating or sequential basis from one embryo to another. This enables several agents to be monitored over a specific time period. Alternatively, multiple embryo chambers can be included in a single controlled environment stage adaptor and the photomicroscope(s) can take sequential images of the embryos.
All citations are hereby incorporated by reference.
The present invention has been described with regard to one or more embodiments. However, it will be apparent to persons skilled in the art that a number of variations and modifications can be made without departing from the scope of the invention as defined in the claims.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/CA2011/000038 | 1/14/2011 | WO | 00 | 9/14/2012 |
| Number | Date | Country | |
|---|---|---|---|
| 61295363 | Jan 2010 | US |
