Patent Description:
Especially in the field of microscopic examination of living samples like cells, it is of great interest to keep the sample as long as possible under favourable and stress-free environmental conditions. To this end, incubators are used for generating a microclimate adapted to the sample to be examined. Existing incubators can be distinguished in stage top incubators, on the one hand, and cage incubators, on the other hand.

Cage incubators comprise a climatic chamber enclosing the entire microscope such that a large volume needs to be incubated. Access to the working area for placing or manipulating the sample is impaired since the microscope itself is located within the cage incubator. Further, it is hardly possible to equip a microscope with a cage incubator in a space-saving manner. On the other hand, a stage top incubator provides a small volume to be incubated as the stage top incubator only encloses the sample itself and is placed onto the microscope stage. Even if a stage top incubator has minimum space requirements, access to the sample is impaired since the sample is surrounded by a sealed box which would have to be opened, thus, destroying the incubation atmosphere within the box. While a cage incubator has a high energy and gas consumption, a stage top incubator provides a small closed incubated room including connected supply conduits for supplying the desired incubation atmosphere.

US patent application <CIT> relates to a culture microscope that comprises an incubator chamber with a hinged lid, a tray with sample-holding holes for holding sample containers, an LED light source with corresponding optics, a CCD sensor with corresponding optics, and a gas pipe that is part of the lid, with the gas pipe being used to supply mixed air from a tank to the incubator chamber.

European patent application <CIT> relates to a tissue culture microscope apparatus, which comprises a sample chamber with a hinged cover, a specimen tray, light sources, an imaging device with corresponding optics, and an electromagnetic valve for adjusting a CO<NUM> concentration inside the culture space, with the electromagnetic valve being part of the hinged cover. The tissue culture microscope apparatus is an inverse transmitted-light microscope and the imaging optics are inside a buffer chamber which is separate from the sample chamber.

US patent application <CIT> relates to a climate chamber, which can be put on top of a microscope.

<CIT> titled "Systems for Performing Cellular Analysis and Related Devices for Conditioning Environments Adjacent Chips in Such Systems" is prior art pursuant to Article <NUM>(<NUM>) EPC and discusses systems and devices used for conditioning the environment surrounding chips in cellular analysis. In such a device, an external heater/chiller can be used to heat/chill the chips.

<CIT> titled "OPTICAL MICROSCOPE" discusses the reduction of size and production costs for an optical microscope apparatus that can block light or maintain the specimen environment. The provided apparatus includes a microscope with a stage for mounting the specimen, a transmission-illumination optical system, and a detection optical system. It also includes a housing that surrounds the microscope, consisting of a fixed housing and a movable housing. The fixed housing comprises a duct connected to an external carbon-dioxide-gas supplier.

In view of the drawbacks described above, there is a need for an improved incubation solution in microscopes, such a solution particularly providing the possibility of retrofitting an existing microscope with an incubation facility.

Embodiments of the present inventive concept provide an inverse transmitted-light microscope according to claim <NUM>.

The present inventive concept can thus provide a microscope including a relatively large sample chamber which can be incubated and, at the same time, which provides easy access to the sample itself without accepting the disadvantages involved with existing incubation solutions as described above. As a dedicated sample chamber is formed by a separated housing section, a larger incubation room can be designed compared to stage top incubators, at the same time avoiding a bulky cage incubator arrangement. Further, the door/lid/window of the housing section enables easy access into the sample chamber for placing or manipulating the sample on the microscope stage.

In an advantageous embodiment, the housing section of the microscope housing is configured such that, when the lid is closed, the sample chamber is sealed. By sealing, particularly hermetically sealing the sample chamber, any losses of incubation atmosphere due to leakage can be minimised.

In an advantageous embodiment, the housing section encloses, at least partly, the microscope stage and a working area for placing the sample on the microscope stage. The microscope stage of the microscope typically comprises a microscope stage tabletop movable in an x-y-direction including corresponding driving means. For placing the sample on the microscope stage, particularly on the microscope stage tabletop, a working area above, to the left and to the right of the sample is provided allowing access to the sample for a user, a robot and/or any manipulators. Frequently, next to the upper side of the microscope stage/microscope stage tabletop a working surface is provided serving as a shelf space for any instruments or other samples to be examined. In this context, it is advantageous if the housing section is bounded downward by the working surface including an upper side of the microscope stage, e.g. the microscope stage tabletop.

The illumination optics typically comprises an (LED) light source and a collimator lens as the main components and possibly other optical elements, like filters etc..

The housing section may be bounded lateralward and upward by said lid in form of a hinged lid or hinged door and/or by side walls of the housing section.

In an advantageous embodiment, the interface comprises at least one opening, also meant to be a duct, which is configured to receive a conduit, also meant to be a tube or pipe, which itself is connected to the external incubation environment conditioning unit. In a different embodiment, the interface comprises at least one opening, also meant to be a duct, including a conduit, also meant to be a tube or pipe, which itself is connectable to the external incubation environment conditioning unit. The at least one conduit (or tube or pipe) serves the purpose of supplying incubation atmosphere into the sample chamber, the incubation atmosphere being adapted to the sample to be examined. In the above embodiments, the at least one conduit may be connected to the conditioning unit either directly, e.g. by welding or by a flange connection, or indirectly, e.g. by using an adapter which may be mounted to the backside of the microscope for carrying the load of the conditioning unit. As discussed later, the at least one conduit may also comprise sensors or sensor leads for sensors detecting parameters of the incubation atmosphere and/or cables and lines for a communication between the microscope controlling unit and the conditioning unit controller.

In an advantageous embodiment, the at least one opening is configured to be closed in a light-tight manner when the microscope is operated without the external incubation environment conditioning unit. This measure avoids scattered light invading from the outside into the sample chamber.

It is advantageous if the interface comprises a plurality of openings for respective conduits for supplying and discharging incubation atmosphere. Such an arrangement can ensure a homogeneous distribution of incubation atmosphere within the sample chamber.

While it is possible to have one conduit for supplying and another conduit for discharging incubation atmosphere, it is also possible if at least one conduit is formed in a two-part form with a split cross-section area which defines two fluid paths separated from each other in the longitudinal direction for supplying incubation atmosphere through one of the two fluid paths and for discharging incubation atmosphere through the other one of the two fluid paths. By this arrangement optimal circulation of the incubation atmosphere can be accomplished.

It is advantageous if at least one conduit comprises fins at its end facing the sample chamber. Such fins can influence the flow directions and either induce a desired flow direction of the incubation atmosphere flowing through the sample chamber or generate a turbulent flow within the sample chamber.

In some examples, said interface comprises at least one opening including a conduit, which is connectable to the external incubation environment conditioning unit. As already mentioned above, it is advantageous if the microscope comprises at least one sensor for sensing at least one parameter of the incubation atmosphere, particularly at least one of the sensors being integrated, at least partly, into a conduit of said at least one conduit for supplying incubation atmosphere to the sample chamber. More particularly, such sensors can be located in a conduit and/or in the sample chamber itself. Sensors for detecting the humidity (H<NUM>O) and/or the contents of nitrogen (N<NUM>) and/or oxygen (O<NUM>) and/or carbon dioxide (CO<NUM>) and/or for detecting the temperature of the incubation atmosphere should be provided.

It is advantageous if the interface is located at a backside of the housing section and thus the incubation environment conditioning unit to be connected directly or indirectly to the interface (see above) is mounted to the backside of the housing section and thus to the backside of the microscope. This arrangement ensures that examination, as well as changing and manipulating the sample are not hindered by the incubation environment conditioning unit. Further, it makes retrofitting of a microscope with such an incubation environment conditioning unit easier.

The microscope is an inverse transmitted-light microscope, wherein the housing section encloses, at least partly, the microscope stage of the microscope and a transmitted-light illumination unit as the illumination optics of the microscope, and wherein a second housing section is provided which second housing section encloses, at least partly, the imaging optics of the microscope. With such an arrangement, the microscope is essentially divided in two parts, a first part being formed by the first housing section comprising e.g. the top side of the microscope stage and the illumination optics, and a second part in form of a second housing section enclosing e.g. the microscope objective and an image detector of the microscope and, possibly, the lower side of the microscope stage. Parts of the illumination optics as well as parts of the imaging optics, like a camera or a display displaying the microscopic image of the sample, can also be located outside the first and second housing section, respectively.

By providing two different separated housing sections within the microscope housing, it is possible to incubate and/or air-condition and/or temperature-control both housing sections independently from each other. While the first housing section is incubated, the second housing section can simply be air-conditioned by supplying air or another atmosphere preferably at a predetermined temperature. This is particularly advantageous in case of using immersion objectives for microscopic examination of a sample.

In a second aspect of the present inventive concept, a system for microscopic examination of the sample is provided, said system comprising a microscope according to the above first aspect of the inventive concept and an external incubation environment conditioning unit connected to the microscope via said interface of the microscope (<NUM>), the external incubation environment conditioning unit (<NUM>) being external to the microscope.

In an advantageous embodiment of the second aspect of the present inventive concept, the incubation environment conditioning unit comprises at least one of the following connections: a connection for supplying H<NUM>O, a connection for discharging H<NUM>O and/or incubation atmosphere, a connection for supplying N<NUM> and/or O<NUM>, a connection for supplying CO<NUM> into the sample chamber. In live cell imaging, the incubation environment conditioning unit should typically be able to control temperature, humidity and CO<NUM> content of the incubation atmosphere. To this end, separate connections for supplying H<NUM>O and CO<NUM>, remainder air, should be present. The resulting incubation atmosphere is supplied through the at least one conduit placed into or being part of the microscope interface into the sample chamber. Typically, the corresponding connections into the sample chamber are combined, e.g. in the form of said conduit(s). It is, however, also possible to provide air, humidity and CO<NUM> through separate connections into the sample chamber. On the other hand, it is also desirable to conduct hypoxia experiments where an oxygen deficiency in the incubation atmosphere is present. An oxygen deficiency is typically established by supplying nitrogen (N<NUM>) into the incubation atmosphere. It is also advantageous to have a connection for discharging H<NUM>O and/or incubation atmosphere in order to uphold a circulation of atmosphere and/or to refresh the atmosphere.

In an advantageous embodiment, the microscope comprises a controlling unit that is configured to control functional components of the microscope for controlling the microscopic examination of the sample. Such functional components typically are the illumination optics, the imaging optics and the microscope stage of the microscope. In this embodiment, the controlling unit is further configured to control an operation of the external incubation environment conditioning unit when the external incubation environment conditioning unit is connected to the interface. Cables and lines for communication between the microscope controlling unit and the conditioning unit controller can be guided through one or more of the above openings of the interface. This embodiment enables a very user-friendly operation of the microscope including the incubated sample chamber. Particularly, the microscope including the microscope interface and the connected incubation environment conditioning unit can be controlled through a universal software interface having a graphical user interface (GUI) which is preferably adapted to the various possible kinds of experiments. In case of e.g. an hypoxia experiment, the user can set the desired temperature, humidity and CO<NUM> content as well as the desired O<NUM> content of the incubation atmosphere through corresponding buttons in the GUI. Preferably, default values for the respective parameters are shown in the GUI, which can be changed by the user. Furthermore, parameters of functional components of the microscope can be set through the same GUI, e.g. the kind of sample, the illumination wavelength, camera parameters etc. After a sensor has detected the closing of the lid such that the sample chamber is sealed, generation of the user-defined incubation atmosphere starts by introducing air with predetermined contents of H<NUM>O and CO<NUM> into the sample chamber. Additionally, N<NUM> is introduced for displacing O<NUM> in order to reduce the O<NUM> content. A part of the incubation atmosphere may escape through leaks and/or through a connection/conduit for discharging incubation atmosphere. At the same time, the functional components of the microscope are set to the desired settings for proper microscopic examination/imaging of the sample. Once the settings and the incubation atmosphere are accomplished, examination/imaging of the sample by the microscope is carried out.

In a last aspect, the present inventive concept relates to a method for equipping or retrofitting an inverse transmitted-light microscope with an external incubation environment conditioning unit for incubating a sample chamber of the microscope. Said microscope comprises a microscope housing enclosing an illumination optics, a microscope stage and an imaging optics, further an integrated sample chamber located within the microscope housing and formed by a separated housing section within said microscope housing. The housing section encloses, at least partly, the microscope stage of the microscope and a transmitted-light illumination unit as the illumination optics of the microscope. The microscope housing comprises a second housing section that encloses, at least partly, the imaging optics of the microscope, the second housing section being separated from the housing section. The housing section comprises a lid providing direct access to the microscope stage for placing the sample in the sample chamber. Further, the housing section comprises an interface for connection of the external incubation environment conditioning unit to the sample chamber, the interface being arranged outside and apart from the lid. The interface is configured to provide a connection between the external incubation environment conditioning unit and the sample chamber such that the environmental conditions in the sample chamber can be controlled when the external incubation environment conditioning unit is connected to the interface.

Equipping the microscope with the external incubation environment conditioning unit comprises connecting the external incubation environment conditioning unit to the microscope via the interface to establish a system according to the second aspect of the present inventive concept.

It is pointed out that the features described above in relation the microscope according to the first aspect and in relation to the system according to the second aspect of the present inventive concept represent an analogous description of the corresponding features of the two aspects and of the method according to the third aspect of the present inventive concept.

It should be noted that features of the above examples as well as of the examples explained below can, within the scope of the appended set of claims, - wholly or in part - be combined to other examples not explicitly mentioned herein, nevertheless being part of the present disclosure.

<FIG> schematically shows in a perspective view an embodiment of a microscope <NUM> for microscopic examination of a sample <NUM> arranged on a microscope stage <NUM>. A microscope housing <NUM> encloses an illumination optics <NUM>, the microscope stage <NUM> and an imaging optics <NUM>. An integrated sample chamber <NUM> is located within the microscope housing <NUM> and formed by a separated housing section <NUM> within said microscope housing <NUM>. The housing section <NUM> comprises a hinged lid <NUM> which provides direct access to the microscope stage <NUM> for placing the sample <NUM> in the sample chamber <NUM> onto the microscope stage <NUM> and for exchanging samples <NUM> and/or manipulating samples <NUM> when the lid is opened. The embodiment shown in.

<FIG> is an inverse transmitted-light microscope <NUM> where the transmitted-light illumination optics <NUM> is arranged within the housing section <NUM>, while the imaging optics is located below the microscope stage <NUM> in a second housing section <NUM>. The imaging optics <NUM> typically includes a microscope objective and an image detector as the main components. The image detector usually comprises a camera which generates microscopic images which are typically displayed on a display screen <NUM> outside the microscope housing <NUM>.

The construction of the microscope housing section <NUM> allows - after closing the lid <NUM> - to form a dedicated sample chamber <NUM> which constitutes a preferably sealed space which can be incubated such that during microscopic examination/imaging of living samples <NUM> like cells, the sample can be kept under favorable and stress-free environmental conditions. To this end, the housing section <NUM> comprises an interface <NUM> for connection of an external incubation environment conditioning unit <NUM> to the sample chamber <NUM>. The interface <NUM> is configured to provide a connection between the conditioning unit <NUM> and the sample chamber <NUM>, such that environmental conditions in the sample chamber <NUM> can be controlled when the conditioning unit <NUM> is connected to the interface <NUM>.

In the embodiment shown, the interface <NUM> as a part of the housing section <NUM> comprises two openings <NUM> in the backside of the housing section <NUM>, each opening <NUM> being configured to receive a conduit <NUM> (see also <FIG> and <FIG>). Incubation atmosphere can be introduced through at least one of the conduits <NUM> into the sample chamber <NUM>. Depending on the leakage of the sample chamber <NUM>, a part of the incubation atmosphere is allowed to escape the sample chamber <NUM>. On the other hand, a part of the incubation atmosphere can be withdrawn from the sample chamber <NUM> via another one of the conduits <NUM>.

Suitable incubation atmospheres comprise air with a predefined content of H<NUM>O (relative humidity) and a predefined content of CO<NUM> (carbon dioxide). It is also desirable to conduct hypoxia experiments with a deficiency of oxygen in the atmosphere. Typically, the temperature of the incubation atmosphere can be set in a range between ambient temperature up to <NUM>, the CO<NUM>-range is set between <NUM> to <NUM>%, and the O<NUM>-range is between <NUM> to <NUM>%. The humidity must be balanced to ensure that potential condensation is avoided or at least does harm neither the microscope <NUM> nor the conditioning unit <NUM> nor the sample itself. It is preferred to control at least the temperature, the humidity and the CO<NUM>-content on its own. In hypoxia experiments, the O<NUM>-content is controlled by N<NUM>-input.

In order to control the above parameters, it is preferred to arrange sensors in the conduits <NUM> and/or in the sample chamber <NUM> and/or at the microscope stage <NUM> close to the sample <NUM>. In a preferred embodiment, at least some of the sensors are integrated into a conduit <NUM> for supplying incubation atmosphere to the sample chamber <NUM>.

As shown in <FIG>, the housing section <NUM> is bounded downward by a working surface <NUM> including an upper side of the microscope stage, in other words the microscope stage tabletop. This construction provides a user-friendly access to the working area for placing and manipulating the sample <NUM>. To the other sides, the housing section is bounded by the inner sides of the lid <NUM> and the backside of the housing section <NUM> itself.

For increasing the live span of the imaging optics <NUM> and in case of using immersion objectives, it is preferred to air condition and/or temperature-control the second housing section <NUM>. This can either be done by the same conditioning unit <NUM> and a correspondingly enlarged interface <NUM> or, more preferred, by a separate air conditioning unit.

In a preferred embodiment, the microscope <NUM> comprises a controlling unit <NUM> that controls functional components like the illumination optics <NUM>, the microscope stage <NUM> and the imaging optics <NUM> of the microscope <NUM> for controlling the microscopic examination/imaging of the sample <NUM>. Typically, a graphical user interface (GUI) is displayed on the display screen <NUM> for a user-friendly operation of the microscope <NUM>. The controlling unit <NUM> is further configured to control the operation of the incubation environment conditioning unit <NUM> when the conditioning unit <NUM> is connected to the interface <NUM>. Cables and lines for communication between the microscope controlling unit <NUM> and a conditioning unit controller (not shown) can be guided through one or more of the above openings/conduits of the interface <NUM>. In this case, the same GUI may display corresponding buttons for setting desired values for the incubation atmosphere parameters as mentioned above. After confirmation that the lid <NUM> is closed and the sample to be examined is placed on the table of the microscope stage <NUM>, generation of the user-defined incubation atmosphere automatically starts and, once the desired atmosphere is established and the sample <NUM> is in the right position, examination/imaging of the sample is carried out.

As already mentioned above, <FIG> shows an assembling process of equipping a microscope <NUM> with an incubation environment conditioning unit <NUM> resulting in a system <NUM>. Same reference signs as in <FIG> designate the same components. As shown in <FIG>, interface <NUM> also comprises mounting sites for fixing the conditioning unit <NUM> at its corners to the backside of the microscope <NUM>. The interface <NUM> further comprises openings <NUM> configured for receiving two conduits <NUM> of the conditioning unit <NUM>, the two conduits <NUM> providing respective ducts through which incubation atmosphere is fed into the sample chamber <NUM>. In this embodiment, the conduits <NUM> are connected to the conditioning unit <NUM> e.g. by welding or by a flanged connection. The assembling process shown in <FIG> is particularly suitable for retrofitting a microscope <NUM> with a conditioning unit <NUM>.

Claim 1:
An inverse transmitted-light microscope (<NUM>) for microscopic examination of a sample (<NUM>) comprising
a microscope housing (<NUM>) enclosing an illumination optics (<NUM>), a microscope stage (<NUM>) and an imaging optics (<NUM>),
an integrated sample chamber (<NUM>) located within the microscope housing (<NUM>) and formed by a separated first housing section (<NUM>) of said microscope housing (<NUM>),
wherein the first housing section (<NUM>) encloses, at least partly, the microscope stage (<NUM>) of the microscope and a transmitted-light illumination unit as the illumination optics (<NUM>) of the microscope,
the microscope housing (<NUM>) comprising a second housing section (<NUM>) that encloses, at least partly, the imaging optics (<NUM>) of the microscope, the second housing section being separated from the first housing section,
the first housing section (<NUM>) comprising a lid (<NUM>) for providing direct access to the microscope stage (<NUM>) for placing the sample (<NUM>) in the sample chamber (<NUM>),
wherein said first housing section (<NUM>) comprises an interface (<NUM>) for connection of an external incubation environment conditioning unit (<NUM>) to the sample chamber (<NUM>),
the interface (<NUM>) being configured to provide a connection between said external incubation environment conditioning unit (<NUM>) and the sample chamber (<NUM>), such that environmental conditions in the sample chamber (<NUM>) can be controlled when the external incubation environment conditioning unit (<NUM>) is connected to the interface (<NUM>),
characterised in that the interface (<NUM>) is arranged outside and apart from the lid (<NUM>).