GB2459897A

GB2459897A - Experiment Environmental Control Devices

Info

Publication number: GB2459897A
Application number: GB0808458A
Authority: GB
Inventors: Fergus Aeneus Murdoch Macdonald
Original assignee: RS BIOTECH LAB EQUIPMENT Ltd
Current assignee: RS BIOTECH LAB EQUIPMENT Ltd
Priority date: 2008-05-09
Filing date: 2008-05-09
Publication date: 2009-11-11
Also published as: GB0808458D0

Abstract

A device for controlling atmospheric conditions within a removable module (2) comprising a chamber (4) having a sealable opening and at least one chamber gas port (8) and at least one module (2) having a module gas port (10) which can be stored within the chamber (4) and is removable therefrom. Either the module gas port (10) and/or the chamber gas port (8) have connection means (12, 20) which, when the module (2) is within the chamber (4), are operable to form a sealed connection between the gas ports (8, 10) allowing gas transfer with the module (2). Preferably, one or each of the module gas port connection means and the chamber gas port connection means have a magnet (20) to enable their correct position relative to one another to form the sealed connection. The chamber may have a heating source and control means.

Description

EXPERIMENT ENVIRONMENTAL CONTROL DEVICES

The present invention relates to a device for controlling the environmental conditions in a module containing some sample forming part of an experiment.

The invention has applications in the fields of cell research and tissue growth. A researcher may wish to study growth rates and other behaviour of a sample of cells.

This is likely to require the control of certain conditions in the environment of the sample such as temperature, humidity, and gas mixture or atmosphere.

It is useful in many fields of cell research to be able to simulate within a laboratory the sorts of conditions cells may experience in a human or other mammal. Apparatus such as hypoxic incubators enables researchers to study cell samples at a predetermined temperature and relative humidity and to control the carbon dioxide and oxygen concentrations in the immediate environment of the sample.

Carbon dioxide incubators, such as the RS Biotech Galaxy R+, allow a user to control atmospheric conditions within the incubator. Parameters such as humidity, carbon dioxide concentration, oxygen concentration and temperature within the main chamber of the incubator can be monitored and controlled. The incubator also has a shelving assembly so that cell cultures can be placed in a space-efficient manner within the incubator.

Patent document US 6,593,136 Bi discloses a stack of sealable chambers which may be placed in an incubator and each have one or more ports which enable substances to be introduced into, or removed from, the chamber when the chamber is sealed by a lid. The apparatus and method disclosed therein enables the cell cultures in different chambers within the stack to be exposed to different atmospheres simultaneously thereby enabling the incubator into which the stack is placed to be utilised more efficiently. Setting the atmosphere within individual chambers within an incubator also removes the need for controlling the atmosphere within the incubator itself, therefore removing associated time and financial costs.

This method suffers from the disadvantage that whilst the ports enable substances to be introduced into, or removed from, the chambers themselves, the stack would either need to be outside of the incubator, or the incubator to be opened, in order to utilise these ports. Opening the incubator allows heat to escape which can disrupt the development of samples within the incubator. Even in incubators with quick heat recovery systems there is likely to be some level of disruption. If the stack is removed from the incubator each time substances are to be removed from or inserted into a chamber, there is likely to be significant physical disruption to the samples within the chambers of the stack. Under these circumstances, continued monitoring of the atmospheric conditions within any chambers of the stack is likely to cause significant disruption to the development of the cell cultures therein.

It may be desirable to place a chamber under a microscope for observation of the cell culture samples therein without the need for removing the samples from the sealed chamber. The stack of chambers disclosed in US 6,593,136 may be provided with transparent lids or walls to facilitate this observation. However, the stack of chambers is arranged in such a way that when one chamber is to be removed from an incubator, the entire stack would need to be removed, and disassembled to the point where the required chamber can be isolated and removed from the stack. This process is likely to be time-intensive for a researcher, and also cause physical disruption to the contents of every chamber in the stack. It will also cause fluctuation in the environmental conditions of each chamber (not just the one to be observed) as the stack would need to be removed from an incubator for disassembly, so the temperature may fluctuate.

It is desirable to provide a device or system enabling cell culture samples to be quickly and conveniently removed from an incubator and observed, possibly under a microscope, without allowing the atmosphere immediately surrounding the samples to diffuse away.

It is also desirable to provide a system or device enabling continued monitoring and alteration of the environmental and/or atmospheric conditions surrounding a cell culture sample to be studied whilst minimising fluctuations in any environmental conditions.

According to an embodiment of the present invention there is provided a device for controlling atmospheric conditions within a removable module, the device comprising a chamber having a sealable opening and at least one chamber gas port, and at least one module having a module gas port and being operable to be stored within the chamber and able to be removed from the chamber.

One or each of the module gas port and the chamber gas port have connection means which, when the module is stored within the chamber, are operable to form a sealed connection between the gas ports allowing gases to be inserted into, and removed from, the module. This device has the technical advantage that the connection between the gas ports allows gases to be inserted into, and removed from, the module without having to either open the sealable chamber opening or remove the module. Furthermore, should it be required that an observation of the contents of the module be made which necessitated the removal of the module from the chamber, the atmosphere within the module can be substantially maintained, and any fluctuation of environmental conditions within the chamber is minimised.

It is advantageous to provide a device having two or more independently removable modules, each containing a desired atmosphere which may differ between modules.

Preferably, a device embodying the present invention will comprise two or more removable modules each having a module gas port and being operable to be stored within the chamber and able to be removed from the chamber, the chamber having two or more such chamber gas ports. Such a device has the advantage that any of the modules can be removed and the contents observed, perhaps under a microscope, without either having to remove the cell culture samples from the atmosphere of the modules, or to remove any modules which are not to be observed. Furthermore, the removal of one module can be achieved easily and without causing significant fluctuation to the environmental conditions within the chamber, such as temperature. Another advantage of such a device is the ability to house a number of cell culture samples within a single incubator exposed to different atmospheric conditions, and also to enable continued monitoring and/or changing of said different atmospheric conditions whilst minimisirig impact on other variables in the system.

In use, it is likely that in addition to controlling the atmosphere within individual modules, it will be desirable to control the temperature in the chamber. Since much cell culture research aims to simulate mammalian conditions, the temperature in the chamber may desired to be warmer than the laboratory or room temperature. In a preferred embodiment, the chamber is an incubator having a heating source operable to heat the modules therein by convection of heated air within the chamber. However, embodiments of the invention in which the chamber is operable to be cooled to below the room or laboratory temperature are equally possible.

Desirably, each of the modules will be operable to slide into a position within the chamber that enables a sealed connection to be formed between the module's gas port, and a chamber gas port. Either of the module gas port or the chamber gas port or both may be provided with some form of connection means. Preferably, the connection means comprise a seal provided on one of the module or the chamber and operable to deform against the other of the module and the chamber form the sealed connection.

The chamber may house a plurality of modules concurrently. Preferably, the modules will not rest on each other, but will be supported by, for example, a shelf or some other support within the chamber. Such support may also be arranged so that it helps to align the module gas port with the chamber gas port to facilitate forming the sealed connection between the two. Preferred embodiments of the present invention include guiding means arranged to guide the module gas port of a module into correct alignment with a chamber gas port. Thereby the process of inserting a module into, or removing a module from, the chamber is made easier. Having some framework such as shelving within the chamber to support the modules also means that modules may be inserted into, or removed from, the chamber without causing significant physical disruption to the contents of any other modules.

Preferably, the guiding means are shelves and/or runners.

The modules may be arranged so that they have grooves allowing the runners to guide them into position in the chamber.

In embodiments of the present invention, it may be that the guiding means, or merely careful placement by a user,

S

is sufficient to achieve the correct positioning of a module within the chamber to form a sealed connection. In preferred embodiments, one or each of the module gas port connection means and the chamber gas port connection means have a magnet operable to draw a module gas port connection means and a chamber gas port connection means into the correct position relative to one another to form the sealed connection. Preferably, one or each of the magnets are ring magnets. The magnet serves both as an aid in positioning the module within the chamber, and also to improve the sealed connection by providing an attractive force between the module and the chamber.

When sealed with a module gas port, the chamber gas ports are operable to allow gas into and out of the module.

A gas storage unit such as a gas canister may reside outside of the chamber. Therefore it is advantageous for the chamber gas port to be arranged to allow gas from outside of the chamber into the module. Similarly, monitoring the atmospheric constituents of a module may require sensors or other apparatus residing outside of the chamber, hence it is advantageous for the chamber gas port to be arranged to allow gas from inside the module to be drawn outside of the chamber.

Preferably, the or each chamber gas port is provided in a wall of the chamber. For example, the chamber gas port(s) may be in a rear wall of the chamber (opposing the sealable opening), so that when a module is slid into the chamber module port-first, a connection is made which allows gas to be inserted into, or removed from, the module. Advantageously, this allows gas piping and apparatus associated with gas supply and sampling to be arranged outside of the chamber, or even separately to the device.

When performing cell culture experiments it may be desirable to monitor the atmospheric conditions within which the cell culture sample is stored. For example, reactions and biological processes occurring within a module may gradually alter the atmosphere within a module.

This effect will be particularly significant in small modules. Therefore, it may be desirable to monitor and quantify this gradual alteration. Preferred embodiments are equipped with at least one gas sensor, one or each sensor being operable to measure a concentration of a specific gas within a sample, and some form of sampling connection means arranged to connect a pump to a chamber gas port via one or each of the gas sensors, and a pump operable, when a sealed connection is formed between a module gas port and a chamber gas port, to draw a sample of gas from the module through at least one of the gas sensors. The device may be equipped with oxygen and carbon dioxide sensors, or certain embodiments may have other gas sensors. Or, an embodiment of the device may not have any sensors itself, but merely have connection means for connecting to an external sensor. This may be required for specialist experiments where concentrations of less typical gases need to be measured.

In embodiments of the present invention comprising a plurality of chamber gas ports it is desirable to provide an arrangement whereby a single set of gas sensors can be used to sample gases from a module connected to any of the chamber gas ports. For example, if three modules were in position within the chamber and each connected to a chamber gas port, it is advantageous to be able to monitor the atmosphere within all three modules by sampling them in turn. Preferably, devices embodying the present invention have sampling connection means operable to connect a pump via one or each of the gas sensors to a plurality of chamber gas ports, and the sampling connection means further comprise a sampling valve manifold system operable to allow a sample of gas to be drawn through at least one of the gas sensors from any module with a module gas port forming a sealed connection with a chamber gas port.

In addition to monitoring atmospheric conditions which a cell culture sample is exposed to, it may be desirable for a user to have some control over them. For example, if constant gas concentrations are required, then any gradual alteration in atmospheric constituents due to the reactions and biological processes will need to be corrected for. In other experiments, it may be that the effects of a gradual change in atmospheric constituents over time are to be analysed. Preferred embodiments of the present invention include a gas supply system comprising supply connection means to connect at least one gas storage unit to a chamber gas port, and supply control means to facilitate, when a sealed connection is formed between a module gas port and the chamber gas port, the supply of a predetermined dose of a specific gas to the module.

In embodiments of the present invention comprising a plurality of chamber gas ports it is more efficient to use a single gas supply to provide gas to any connected module (rather than to have separate gas supplies for each module) . Preferably, the supply connection means connect at least one gas storage unit to a plurality of chamber gas ports, and the supply control means is a supply valve manifold system operable to facilitate the supply of a predetermined dose of a specific gas to any module with a module gas port forming a sealed connection with a chamber gas port.

Gas sampling, supply, and monitoring may be done manually using sensors external to the device.

Alternatively, the device may be connected to a separate control mechanism operable to monitor and control levels of specific gases within a module. It is desirable for the device to be provided with some form of control means.

This control means may include a data connection for accepting instructions from, and providing measurements and status information to, an external computer unit or other mechanism running control software. The device may also have a user interface allowing the user to provide instructions, and a display for displaying information to a user. Preferred embodiments include control means having an instruction interface operable to accept and store instructions indicating desired atmospheric conditions within the module, including desired concentrations of specific gases, gas sampling system control means for instructing the sampling system to take a sample from a selected module and to receive the resulting measurements, and gas supply system control means for instructing the supply system to provide a dose of a specific gas to a chosen module. Such control means are operable, in accordance with measurements received from the sampling system, to calculate a dose of a specific gas to be provided to a selected module to match the gas concentrations within the module to those desired by the user. Furthermore, in embodiments in which the chamber is the chamber of an incubation apparatus, the instruction interface is operable to receive and store a value indicative of a desired temperature for the chamber, and the control means is operable to communicate with a temperature sensor within the chamber, and with the heating source, to maintain the desired temperature within the chamber.

Preferred features of the present invention will now be described, purely by way of example, with reference to the accompanying drawings, in which:-Figure 1 is a cross-sectional view of a device embodying the present invention in which a single module is in position forming a sealed connection with a chamber gas port; Figure 2 is a cross-sectional view of a chamber gas port and a module gas port; and Figure 3 is a cross-sectional view of a chamber gas port and a module gas port forming a sealed connection.

As shown in Figure 1 a device embodying the present invention comprises a chamber 4 and a removable module 2.

The device may also comprise further such removable modules 2.

Each module 2 has a sealable opening, but also has a glass base and top which allows the user to view the contents of the module under an inverted microscope. Each module 2 can be completely removed from the chamber 4 independently of any other modules 2 and without significant loss of the internal atmosphere. Each module 2 has a module gas port 10 for connection with a chamber gas port 8.

The chamber 4 has a sealable opening. The chamber also has a plurality of chamber gas ports 10. The chamber is equipped with shelves 16 which support the modules 2 when inside the chamber 4, and acetal runners which guide the modules 2 to roughly the correct position to enable a sealed connection to be made between the chamber gas port 8 and the module gas port 10. In embodiments in which the chamber 4 is the chamber of an incubator, the device also comprises a heat source.

The chamber gas ports 8 and the module gas ports 10 are equipped with magnets 20. These magnets 20 may be ring magnets made from neodymium material. In alternative embodiments, either the chamber gas ports 8 or the module gas ports 10 may be provided without magnets. It may be desirable to use electromagnets.

The chamber also comprises proximity switches, which may be magnet-operated reed switches.

Each chamber gas port 8 is equipped with connection means such as a seal 12. The seal is preferably a V-ring seal, but a different form of compressible circular seal could be used, such as an 0-ring. The seal 12 may be made of nitrile rubber (NBR) or some alternative sealing material, for example silicone or EP]JM rubber. The seal 12 could alternatively or additionally be provided on the module gas port 10.

In one embodiment, the chamber 4 is provided with a gas sampling system 40 comprising a pump, an oxygen sensor, and a carbon dioxide sensor. There is a sampling valve manifold system between the pump and the chamber gas ports 8 which allows each of the chamber gas ports to be connected a single set of sensors. The gas sampling system has a connection to the control means for communicating measurements and accepting instructions.

The gas supply system is connected to, for example, carbon dioxide (C02) and nitrogen gas (N2) storage units.

Both of the carbon dioxide and nitrogen gas supplies are connected to the chamber gas ports 8 via a supply valve manifold system which splits the supply for each gas into a separate feed for each module. Each gas supply valve manifold system is connected to the control means so that instructions can be provided regarding the volume of each 1].

gas required to be supplied to each module (via the chamber gas port) to match the desired atmospheric conditions.

The control means may be capable of executing software control code. Alternatively, the control means may be provided with a connection means for connecting to a computer or other control mechanism. Additionally, the control means may comprise an instruction interface including user buttons and a display.

In embodiments of the invention in which the chamber is the chamber of an incubator apparatus, the device also comprises a temperature sensor or sensors which are connected to the control means and provide information thereto regarding the temperature within the chamber.

Accordingly, in such embodiments the control means is connected to a heat source.

Each module 2 has a sealable opening which allows cell culture samples to be inserted into and taken out of the module 2. When the modules 2 are in a position within the chamber 4 that enables a sealable connection to be formed between the chamber gas port 8 and a module gas port 10, the atmosphere within the module can be programmed and controlled.

In an embodiment of the present invention, the chamber gas port 8 is mounted in the outer wall 4A of the chamber 4. The module gas port 10 is mounted into a wall 2A of the module 2. When the module is put into the chamber 4 in the correct way the module gas port 10 becomes close to a chamber gas port 8. Preferably, each of the chamber gas port 8 and the module gas port 10 have integrated magnets which draw the gas ports into the correct position relative to one another to form a sealed connection. This sealing may be facilitated by additional means, for example a nitrile rubber V-ring seal 12 on the chamber gas port 8 operable to deform against the module gas port 10 as the magnets 20 draw the ports into position thereby sealing the connection. A clearance fit 11 on the chamber gas port 8 allows the chamber gas port 8 to align itself exactly with the module gas port 10, and mitigates any error in alignment of the gas ports due to a stack up of tolerances with other components in the device. By being connectable in this way, the gas ports 8,10 can provide a sealed connection so that gas cannot leak from the modules 2 into the chamber 4, or be drawn into the modules 2 from the chamber 4. They also facilitate inserting gas into, or removing gas from, the removable modules 2.

The magnets 20 are preferably made from neodymium material. They are sufficiently strong that the ports attract at a distance of a few millimetres (eg. 2mm), but also allow a user to remove a module 2 from the chamber 4, thereby breaking the sealed connection, without the requirement for any tools or excessive force (no more than lOON approximately).

The module gas port 10 has an M12 thread to attach it to the module. Respective holes 8A, 1OA through each chamber gas port 8 and module gas port 10 with an inner diameter of around 1mm which are brought together when a sealable connection is formed and allow gas to be passed into, or drawn from, the connected module 2 via the gas ports, as shown by arrow A. The hole 8A of the chamber gas port 8 is connected to a conventional barb fitting 9 for connection to a gas sampling system and/or a gas supply system.

Each chamber gas port 8 has an associated proximity switch such as a magnet-operated reed switch to detect and notify the control means whether or not a module is forming a sealed connection with the particular chamber gas port 8.

Whilst a module is absent the software knows not to sample gas from that chamber gas port 8, or insert any gas to that chamber gas port 8.

A device embodying the present invention may comprise, for example, eight independently removable modules 2. Each module can be hermetically sealed, and the gas mixture within each can be programmed and controlled by the user.

The user can use the control means to specify different gas mixtures for different modules. This may be via user input buttons on an instruction interface, or it may be by using an external computer and a data connection to the device.

Typical gases which are controlled may include carbon dioxide by addition of carbon dioxide (C02), and oxygen (02) by purging with nitrogen (N2). The user can also use the control means to select and control the temperature of the chamber 4 in embodiments where the chamber is provided with a heat source. In such embodiments, the modules (when inside the chamber) are heated by air convection with a heating source heating the air surrounding the modules.

In one embodiment, the gas sampling system comprises carbon dioxide and oxygen sensors. However, connections can be made to other gas sensors, or sensors for other gases can be provided in devices embodying the invention.

The device determines how much oxygen and carbon dioxide is present in the modules 2 by use of a common sampling system. The gas sampling system is connected to the control means and is operable to receive instructions regarding which modules are to be sampled, for which gases, and when. The sensors can then provide readings for the concentration of a specific gas within a chosen module to the control means. The gas sampling system comprises a pump to draw gas from a module, via the sealed connection of the gas ports, through a gas sampling valve manifold system, and to a gas sensor. The gas sampling manifold system between the pump and the chamber gas ports 8 allows all chamber gas ports 8 to be connected to a common sampling system. The valve manifold system is operable to accept instructions from the control means so that the appropriate valve in the manifold can be opened in accordance with which connected module 2 is to be sampled.

Once a particular module has been sampled the control means is operable to compare the measured gas mixture therein with the gas mixture programmed by the user for that module at that time. The control means can calculate the amounts of each gas which can be supplied by the gas supply system to add to the module to achieve the gas mixture desired by the user. The control means can then instruct the gas supply system to open the appropriate valve in such a way that the correct amount of each gas is added to the module 2. The control means may re-sample the module 2 after adding the gases to check that the user defined gas mixture has been achieved, and if not to repeat the steps of calculating amounts of gases to add and adding gases accordingly. Desirably, a short time delay is left between subsequent samplings to allow time to purge the system of any residual gases from the previous sample. The connection to the gas storage units is split into separate feeds for each chamber gas port 8 via a gas supply valve manifold system. This gas supply valve manifold system is operable to accept instructions from the control means so that a valve can be opened to allow a required amount of a gas to be added to a specific module 2. Once the control means has achieved the desired gas mixture in a module 2 the control moves on to a next module 2 in the sequence.

Preferably, there will be a maximum time that the system Will spend on each module 2 so that the system does not get stuck on the same module indefinitely, for example if there was a problem with the gas supply. If the time out period is reached then the control means is operable to execute some form of alarm to notify the user to check the gas supply.

In any of the above aspects, the various features may be implemented in hardware, or as software modules running on one or more processors. Features of one aspect may be applied to any of the other aspects.

The invention also provides a computer program or a computer program product for carrying out any of the methods described herein, and a computer readable medium having stored thereon a program for carrying out any of the methods described herein. A computer program embodying the invention may be stored on a computer-readable medium, or it could, for example, be in the form of a signal such as a downloadable data signal provided from an Internet website, or it could be in any other form.

Claims

CLAIMS1. A device for controlling atmospheric conditions within a removable module, the device comprising: a chamber having a sealable opening and at least one chamber gas port; and at least one module having a module gas port and being operable to be stored within the chamber and to be removable therefrom; wherein one or each of the module gas port and the chamber gas port have connection means which, when the module is stored within the chamber, are operable to form a sealed connection between the gas ports allowing gases to be inserted into, and removed from, the module.
2. The device according to claim 1 having: two or more such modules each having such a module gas port and being operable to be stored within the chamber and to be removable therefrom independently of another module in the chamber; and the chamber having two or more such chamber gas ports.
3. The device according to claim 1 or 2, wherein the chamber is an incubator having a heating source operable to heat the modules therein by convection of heated air within the chamber.
4. The device according to any of the preceding claims, wherein the connection means comprise a seal provided on one of the module or the chamber and operable to deform against the other of the module and the chamber form the sealed connection.
5. The device according to any of the preceding claims, further comprising: guiding means arranged to guide the module gas port of the or each module into correct alignment with a chamber gas port.
6. The device according to claim 5, wherein the guiding means are shelves and/or runners.
7. The device according to any of the preceding claims, wherein one of the module gas port connection means and the chamber gas port connection means have a magnet operable to draw a module gas port connection means and a chamber gas port connection means into the correct position relative to one another to form the sealed connection.
8. The device according to any of the preceding claims, wherein each of the module gas port connection means and the chamber gas port connection means have a magnet operable to draw a module gas port connection means and a chamber gas port connection means into the correct position relative to one another to form the sealed connection.
9. The device according to claim 7 or 8, wherein one or each of the magnets are ring magnets.
10. The device according to any of the preceding claims, wherein the or each chamber gas port is provided in a wall of the chamber.
11. The device according to any of the preceding claims, further comprising a sampling system having: at least one gas sensor, one or each sensor being operable to measure a concentration of a specific gas within a sample; sampling connection means arranged to connect a pump to a chamber gas port via one or each of the gas sensors; and a pump operable, when a sealed connection is formed between a module gas port and a chamber gas port, to draw a sample of gas from the module through at least one of the gas sensors.
12. The device according to claim 11, wherein the sampling connection means are operable to connect a pump via one or each of the gas sensors to a plurality of chamber gas ports, and the sampling connection means further comprise a sampling valve manifold system operable to allow a sample of gas to be drawn through at least one of the gas sensors from any module with a module gas port forming a sealed connection with a chamber gas port.
13. The device according to any of the preceding claims, further comprising a gas supply system having: supply connection means to connect at least one gas storage unit to a chamber gas port; and supply control means to facilitate, when a sealed connection is formed between a module gas port and the chamber gas port, the supply of a predetermined dose of a specific gas to the module.
14. The device according to claim 13, wherein the supply control means is a valve or system of valves,
15. The device according to claim 13, wherein the supply connection means connect at least one gas storage unit to a plurality of chamber gas ports; and the supply control means is a suppiy valve manifold system operable to facilitate the supply of a predetermined dose of a specific gas to any module with a module gas port forming a sealed connection with a chamber gas port.
16. The device according to any of the preceding claims, further comprising: control means, the control means having: an instruction interface operable to accept and store instructions indicating desired atmospheric conditions within the module, including desired concentrations of specific gases; gas sampling system control means for instructing the sampling system to take a sample from a selected module and to receive the resulting measurements; and gas supply system control means for instructing the supply system to provide a dose of a specific gas to a chosen module; wherein the control means is operable, in accordance with measurements received from the sampling system, to calculate a dose of a specific gas to be provided to a selected module to match the gas concentrations within the module to those desired by the user.
17. The device according to claim 16, when read as appended to claim 3, wherein the instruction interface is operable to receive and store a value indicative of a desired temperature for the chamber, and the control means is operable to communicate with a temperature sensor within the chamber, and with the heating source, to maintain the desired temperature within the chamber.