WO2005095576A2

WO2005095576A2 - Controlled temperature water (or other fluid)-jacket co2 microscope stage incubator

Info

Publication number: WO2005095576A2
Application number: PCT/IT2005/000161
Authority: WO
Inventors: Luca Lanzaro; Sergio Caserta; Stefano Guido; Luigi Sabetta; Vincenzo Sibillo; Marino Simeone
Original assignee: High Tech Consulting S.R.L.
Priority date: 2004-04-02
Filing date: 2005-03-24
Publication date: 2005-10-13
Also published as: WO2005095576A3; ITNA20040016A1

Abstract

Controlled temperature water (or other fluid)-jacket CO2 Microscope Stage Incubator developed by “High Tech Consulting s.r.l.” is designed to maintain all the required environmental conditions for cell cultures (or other biological species) right on the microscope stage, thus allowing prolonged observations of cell (or biological) events. It consist of a chamber made of steel or aluminium or other materials, and it has dimensions so that it can fit right on the microscope stage. It enables to stably maintain the environmental conditions of temperature and/or humidity level and/or CO2 (or other gases) level required to study biological phenomena. Desired temperature conditions are provided by inner water (or different fluid) circulation and are guaranteed by the joint action of a PID software controller, with the thermocouple directly inserted inside the incubator. Humidity and CO2 levels are maintained by feeding a preconditioned air stream regulated by flow rate controllers. Holes are drilled into the chamber bottom to host samples for observation.

Description

"Controlled temperature water (or other fluid)-jacket C02 Microscope Stage Incubator"

DESCRIPTION

TECHNICAL FIELD

In many biological studies, the specimens under investigation, such as cell

cultures, need to be maintained at controlled environmental conditions, that typically involve a temperature of 37°C and a gas mixture of humid air with 5% of CO₂. Furthermore, it is often required to follow sample evolution with time. In such a case, the most desirable experimental route is to maintain the desired environmental conditions right on the microscope throughout the experiment to enable continuous image recording and data acquisition, rather than taking the samples in and out of a bench incubator for each observation.

BACKGROUND ART Different types of equipment have been developed to implement this experimental route, but none of these meets the standards of the invention that is here claimed. In particular, two kinds of instruments are currently used for live cell imaging under the microscope: a box surrounding the entire microscope (Takashi and Kikuo, 2003-JP2003116518) and a chamber fitting on the microscope stage (Focht, 1996-US5552321). In the former case, the desired thermal conditions are provided by warm air circulation into the chamber. The gas stream of humid air and CO₂ can be fed either in the entire box, resulting in potential damage of microscope mechanical parts and in water condensation on the box walls, or just in a small chamber placed on the microscope stage, thus resulting in water condensation on glass and plastic surfaces and medium evaporation from the sample under analysis, if the gas stream is fed at a temperature lower than the one inside the box surrounding the microscope. In addition, for this type of instrument, it is quite laborious and time-consuming to remove the incubation set-up when it is not needed, and it is also very difficult to manage with the specimen once it is inside the box.

On the other hand, in the case of the small chamber fitting on the microscope stage, the main disadvantage is that it is heated through electrical resistance, a method that cannot provide the required thermal accuracy and stability upon ambient temperature changes. In fact, due to 1) chamber low thermal inertia and 2) what is controlled is chamber -metal temperature rather than sample temperature (thus causing a delay in the thermal controller response), ambient temperature changes affect the controlled temperature jeopardizing specimen vitality. In addition, none of these systems is designed to be used with long

working distance objectives and immersion-objectives at the same time, and to allow one to host 35mm Petri-dishes as well as glass slides and multiwell plates. Furthermore, because in many of these systems only the base of the chamber is heated with electrical resistances, warm-air is also circulated inside the chamber to avoid thermal non-uniformity throughout the chamber, but this causes fast evaporation of the sample under analysis.

DISCLOSURE OF INVENTION AND BRIEF DESCRIPTION OF DRAWINGS

"Controlled temperature water (or different fluid)-jacket CO₂ Microscope Stage Incubator" (that in the following will be called incubating chamber) developed by High Tech Consulting s.r.l. is designed to maintain all the required environmental conditions suitable for cell cultures (or other biological specimens) right on the microscope stage, thus allowing prolonged observations of cell (or biological) events in any kind of inverted microscope (optical, confocal, electron, stereo and so on).

It is an object of the present invention to provide an incubating chamber that is designed to maintain all the required environmental conditions for cell cultures (or other biological species) right on the microscope stage, thus allowing prolonged observations of cell (or biological) events. It is also an object of the present invention to provide a precise control of specimen temperature during microscopic examination with enhanced thermal uniformity inside the entire instrument. It is another object of the present invention to avoid water condensation on glass, plastic or any other surface.

It is still another object of the present invention to minimise sample evaporation. It is also another object of the present invention to host several types of sample-lodgings.

It is another object of the present invention to provide an incubating chamber that can be used with long working distance objectives and immersion- objectives at the same time.

In a preferred embodiment, the incubating chamber is composed of two parts, a base and a cover, both of them being heated through inner controlled temperature water (or different fluid) circulation, provided by a water bath. In a different embodiment, the incubating chamber could be composed of three parts, base, side walls and cover, if required due to the size of the sample vessels.

The incubating chamber has a thermal accuracy within 0.1 °C despite changes of ambient temperature, and temperature control is guaranteed by the joint action of a PID software controller and a thermocouple directly inserted inside the incubator.

The incubating chamber is made of aluminium (or alternatively of steel or any other metal). It has rectangular shape and dimensions that allow to place it on inverted microscope stages. The incubating chamber dimensions and shape may vary depending on the brand of the microscope and/or of the microscope stage.

In the central zone of both the base and the cover of the incubating chamber correspondent holes are drawn to allow sample observation (i.e. Fig. 11 and Fig.13 -page 4 of the drawings) . Such holes are always closed with glass (or Plexiglas or any other transparent non disruptive material) in the cover so that light can pass, whereas in the base they are left empty if immersion objectives have to be used to analyse the specimen, or again closed with glass (or Plexiglas or any other transparent non disruptive material) if long working distance objectives have to be used to analyse the specimen. Shapes and dimensions of the holes in the central zone of the base of the incubating chamber, are especially designed according to the type of vessel (containing the specimen) that has to be hosted. Holes in the cover are designed to correspond to holes in the base, so that light can pass through , thus allowing observation of the entire hosted sample.

As already stated, the incubating chamber consists of two main parts: the base (that, in turn, can be composed of one or more pieces, without altering its operation principle) and the cover. One or both of the two parts are partially hollow, to allow circulation of temperature-controlled water (or different fluid). Fluid circulation is usually provided by a water bath (or a cryostat water bath if a temperature below ambient temperature is required). This heating (or cooling, when required) method allows, through heat conduction and heat radiation, to provide the desired thermal profile in the incubating chamber where specimens are hosted. Provided that the thermal profile could reach values both above and below the ambient temperature, in the following we will refer only to the above ambient temperature application of the present invention, submeaning that cooling function is as possible as well . The base comprises a central zone where specimen vessels are hosted and the desired gas stream is fed (as shown in Fig.8-page 2 of the drawings), and an inner zone where temperature controlled water (or other fluid) circulation takes place (as shown in Fig.9-page

2 of the drawings). The outside walls of the peripheral zone host three small holes (that don't have to be necessarily placed on the same base wall), one for water (or other fluid) inlet, one for water (or other fluid) outlet and a third hole for gas stream-inlet. Water-inlet hole can be placed in the middle or in the side- part of the base wall. Depending on water-inlet hole position, the design and the position of the inner channels, where water (or other fluid) circulates, would change without altering operation principle of the present invention. Alternatively, the incubating chamber could also be internally totally empty, thus resulting in a different distribution of the internally circulating fluid (Fig.

3 l-Pag.6 of the drawings).

In a preferred embodiment, controlled temperature water (or other fluid) circulation is as follows: it starts from the water bath, then it moves into the base of the incubating chamber through the channels drawn inside the inner zone of the base (i.e. Fig. 18-page 5 of the drawings). The water moves from the base to the cover of the incubating chamber(fro detail E to detail G-Fig. 34-page 8 of the drawings), again flowing through the channels drawn inside the cover (Fig. 24-page 5 of the drawings). From the cover (detail I-Fig.34- page 8 of the drawings), it then exits the chamber and goes back into the water bath, thus closing the water (or other fluid) circuit. In a different configuration, the controlled temperature water (or other fluid) circulation is as follows: coming from the water bath it moves into the base of the incubating chamber through the channels located inside the inner zone of the base. From the base it moves to the side walls of the incubating chamber, flowing through the channels inside the side walls (Fig. 5-page 2 of the drawings), it then flows into the channels inside the cover of the incubating chamber, and finally goes back to the water bath, thus closing the water (or other fluid) circuit. Controlled temperature water (or different fluid) circulation could change such above- mentioned direction without altering the operation principle and performances of the present invention. Connections between the water bath and the base of

the incubating chamber, as well as connections between the base and the cover of the incubating chamber, as well as connections between the cover of the incubating chamber and the water bath, as well as connections between base and side walls, as well as connections between side walls and cover are made through plastic (i.e. silicon or PNC) or non plastic tubes, that could be coated with thermally insulator pipes to avoid thermal losses of the heating fluid. As already mentioned, each sample is placed in a recess drilled into the base of the incubating chamber. Shape and dimension of the recess vary according to the type of sample vessel (Petri dish, glass slide, Petri-dish for immersion objectives, glass slide, chamber slide and multiwell plate) to be accommodated. The incubating chamber can be designed to host one or alternatively more than one (i.e. #four 35mm Petri-dishes, as shown in 13-page 4 of the drawings) of each sample vessel, or also different sample at the same time (i.e. #1 35mm Petri dish and #1 glass or chamber slide as shown in Fig. 31-pag.6 of the drawings). Each recess is closed by a glass plate (or Plexiglas or any other transparent material) if the specimen has to be analysed by using long working distance objectives, or is left open if the specimen has to be analysed by using immersion objectives. Thickness of the base metal layer where the sample vessels are accommodated varies according to the necessary working distance between the sample and the objective. Possible configurations include an incubating chamber with each recess closed by a glass plate (or Plexiglas or any other transparent material), an incubating chamber with each recess open and an incubating chamber with some recess closed by a glass plate (or Plexiglas or any other transparent material) and some other open. Sample vessels usually have circular or rectangular shape.

When the sample vessel has a circular shape, each recess is made by drilling, from the bottom of the base, two or more concentric cylindrical holes. Usually, the first cylindrical hole (starting from the bottom of the base) allows to host the vessel, while the second one is to leave some room for the vessel lid, thus enabling gas exchange with the air stream circulating in the incubating chamber (Fig. 12-page 4 of the drawings). The depth of the recess is designed to allow the correct working distances between the specimen and the objectives for observation under the microscope. Analogous recesses, but obviously with different shape, will be made when sample vessels have a rectangular form (Fig 30 and Fig. 32-page 6 of the drawings). Different types of recess could be made in the same base to host different types of sample vessels. Small vessels along the edge of the central zone of the incubating chamber base can be filled with water (meaning distilled water, or water and glycerine or any other liquid water solution) in order to minimise sample evaporation (Detail A-Page 3 of the drawings).

In the incubating chamber cover (which can be made of aluminium, steel or any other metal), recesses are drilled whose shape and dimension correspond to the ones in the base where sample vessels are accommodated (Fig.11 -page 4 of the drawings). Each recess in the cover can be closed by a glass, Plexiglas or any other transparent material plate. Controlled temperature water (or other fluid) circulating inside the cover, as well as into the side walls, comes from the inner channels of the base. Indeed, cover and base are linked through a plastic tube, going from the water-outlet hole, placed in the external side of the base, to the water inlet hole on the top of the cover. Water (or other fluid) passing into this tube, moves from the base to the cover, heating both, and then, from the water-outlet hole placed on the top of the cover, returns into the water bath. In a different embodiment the base and the side walls and the cover are linked through two plastic tubes, one going from the water-outlet hole, placed in the external side of the base, to the water inlet hole placed in the external side of the side walls and the other going from the water-outlet hole, placed in the external side of the side walls to the water inlet hole on the top of the cover. Water (or other fluid) circulating through these tubes, goes from the base to the

side walls and then to the cover, heating all these three parts, and then, from the water-outlet hole placed on the top of the cover, returns into the water bath. In the top of the cover a third hole to allow gas stream outlet is also present (Fig. 34-detail L-Page 8 of the drawings). In a different embodiment, the cover of the incubating chamber is not heated through water circulation, due to the absence of the inner channels where controlled temperature water (or other fluid) flows (Fig.29-Page 6 of the drawings).

Additional holes could be made, on the top of the cover or alternatively on the sides of the base or of the side walls, to allow perfusion of culture medium or other liquids.

The cover has overall dimensions so that it can be embedded, inserted or screwed to the base (or to the side walls) of the incubating chamber. The desired gas stream, that typically is composed of a mixture of air with 5% CO₂ but that could consist of different gases depending on the required conditions, is provided by gas sources and is a mixture of two or more gases, whose ratio is regulated trough gas flowmeters, connected to the gas sources by plastic (i.e. silicon or PNC) pipes. Microbiological (or similar) filters can be placed between the gas sources and the gas flowmeters to remove impurity or soil and to make the gas stream sterile. After mixing, the gas stream goes into a bubbling column to be humidified (Fig. 38-Page 12 of the drawings). The bubbling column consists of a glass cylinder filled with water. The gas stream enters in this column and gurgles into the water, thus being humidified by mass transfer. From the bubbling column the gas stream flows into the incubating chamber, entering from a gas inlet-hole placed on one of the external walls of the base (detail D in Fig. 31-Pag.6 of the drawings) and outgoing from a gas outlet hole placed on the top of the cover (alternatively, a gas stream outlet hole can also be placed on an external side of the base of the incubating chamber). The bubbling column itself is placed inside the water bath, so that the humidified gas stream enters into the incubating chamber at a temperature equal to or above the temperature in the incubating chamber. Before getting to the zone where sample vessels are accommodated, the gas stream goes into a tube placed inside the channel drawn in the inner zone of the base or the cover of the incubating chamber, where temperature-controlled water (or different fluid) circulates (Fig. 31, detail C-page 6 of the drawings). This pre-heating system is used to bring the gas stream at a temperature close to the one in the zone where the samples are accommodated. In a different embodiment, the gas stream can go directly from the bubbling column to the zone where the samples are accommodated, without passing in the pre-heating circuit of the incubating chamber.

In both configurations, the gas stream is continuously fed into the incubating chamber, thus resulting in a continuous replacement of the gas surrounding the specimen, so that gas conditions in the incubating chamber never change from the desired values.

Temperature control is achieved by the joint action of a PID controller via software and a thermocouple directly inserted inside one of the sample vessels (filled with water, or with water and glycerine, or with any other liquid having a heat capacity close to the heat capacity of the medium in the sample under analysis. As a matter of fact, thanks to the symmetry of the system, temperature in each sample vessel is the same. Sample temperature as measured by the thermocouple is read by a temperature meter communicating via serial (or USB) port with the PID controller software. In the same way, the PID controller software communicates with the water bath and regulates the temperature of the fluid inside the water bath based on the difference between sample temperature and the desired temperature (set- point temperature). The PID controller software will act to increase the temperature of the water bath fluid if the thermocouple is reading a temperature

lower than the set-point value, until the desired sample temperature is reached, or it will diminish the temperature of the water bath fluid if the thermocouple is reading a temperature above the set-point value, until the desired sample temperature is reached (feedback control). To avoid possible thermal shocks to the biological specimens placed in the incubating chamber in the case the latter has to be shortly opened, the PID controller software stops the feedback control and maintains the water bath temperature at the latest value before the incubating chamber was opened. The stopping of PID controller operation can be extended to a programmable duration after the incubating chamber has been closed again.

The present invention could also be used to control just one or two of the three parameters, i.e., temperature, humidity level and CO2 (or other gas) level, which usually need accurate control in long-term experiments involving biological specimens. The present invention has been strictly tested, also comparing (referring to cell proliferation and sample medium evaporation) it with the bench incubator. Test results are shown in Fig. 33, 36 and 37-pages 7, 10 and 11 of the drawings

BEST MODE FOR CARRYING OUT THE INVENTION

Preferred embodiments of the present invention has been described in detail herein with reference to the Figures, in which like numbers represent the same or similar elements. While this invention is described in terms of the best mode for achieving this invention's objectives, it will be appreciated by those skilled in the art that variations may be accomplished in view of these teachings without deviating from the spirit or scope of the present invention. Accordingly, the particular arrangements are illustrative only and not limiting as to the scope of the present invention which is given the full breadth of the appended claims and any and all equivalents thereof. Several possible little changes that do not vary the operation principle of the present invention have already be mentioned (i.e. the fact that the incubating chamber can be composed of two, base and cover, or three, base, side walls and cover, depending on the height of the sample vessels; the fact that shape and dimensions of every part of the incubating can vary depending on the microscope it has to be fitted on; the fact that the number and the position of the holes where fluids, controlled temperature liquid fluid and gas stream, ingoing and outgoing to and from channels inner to the incubating chamber can vary as well as the direction of the circulation of the controlled temperature water or other fluid).

Also other changes that do not vary the operation principle of the present invention are possible. For example, in a preferred embodiment of the present invention the thermocouple is directly inserted in one of the sample- vessel filled with water, or with water and glycerine, or with any other liquid having a heat capacity close to the heat capacity of the medium in the sample under analysis but alternatively, it could also be inserted inside a small container (again filled with water, or with water and glycerine, or with any other liquid having a heat capacity close to the heat capacity of the medium in the sample under analysis) which is placed in close proximity with the sample vessels. Furthermore, electrical resistance can be used to heat the bubbling column and the gas stream, instead of placing the bubbling column in the water bath. At the same way, electrical resistance, instead of thermally insulator pipes, can be used to heat the tubes that allow to the controlled temperature water (or other fluid) to move, thus avoiding thermal losses of the heating fluid. It has been described how, in order to accommodate different types of sample vessels, the geometry and shape of the holes drilled in the base of the incubating chamber and correspondingly in the cover of the incubating chamber, could vary without altering, in any different configuration, the operation principle of the present invention. Disclosed different embodiments can be used depending on different needs.

) For example, when it is required to analyse within the same experiment more than one sample, the embodiment shown in Fig. 11 -Page 4 of the drawings, could be preferred since it allows to host three different samples (we remember that the forth sample-lodgings is usually used to control the temperature). Otherwise when only one field of view has to be analysed, the embodiment shown in Fig. 31 -Page 6 of the drawings could be preferred. Furthermore, depending of the sample vessel that has to be used, different embodiments of the present invention could be preferred (i.e. Fig. 35-Page 9 of the drawings shows an embodiment designed to host a Multiwell plate). In the same way, depending on the type of objectives that have to be used, a specific embodiment could be preferred. Typical application of the present invention are time-lapse studies of cell events under microscope observations.

Claims

What is claimed is:

1) A "Controlled temperature water (or other fmid)-jacket CO₂ Microscope Stage Incubator" designed to maintain all the required environmental conditions for biological specimens (i.e. cell cultures) right on the microscope stage, thus allowing prolonged observations of biological events, wherein improvement comprises: the heating/cooling method based on inner circulation of controlled temperature water or other fluid, that is usually provided by a thermostatic or cryostat water bath; the direct control of specimen temperature (rather than the temperature of the surrounding air or metal); the fact that sample under analysis is heated/cooled all around with same heating/cooling method; the controlled temperature water (or different fluid) circuit inside the incubating chamber is especially designed to guarantee thermal uniformity all around the samples under analysis; the gas stream circuit inside the incubating chamber is especially designed to avoid water condensation on glass and plastic surfaces (that could occur if the gas stream entered in the incubating chamber at a temperature lower than the temperature in the incubating chamber); the bubbling column, where the desired gas stream is humidified, is maintained at a temperature above or close to the temperature in the incubating chamber, the temperature control obtained via software; the feature of the temperature control software that avoid thermal shocks to the biological specimens under analysis when the incubator is opened; the serial/USB communication between the meter reading the thermocouple output with the temperature control software, and the communication between the thermostatic or cryostat water bath and the temperature control software; the presence near the sample vessels of small

containers filled with water or a water liquid solution to minimise sample medium evaporation; the possibility to host within the same incubating chamber different types of sample vessels or one or more than one vessel of the same type; the possibility to use both long working distance objectives and immersion-objectives; the possibility to host within the same recess one or more than one type of sample vessel; the possibility to thermally insulate the tubes where the following fluids circulate: the controlled temperature water (or other fluid), the gas stream in and out from the incubating chamber, and the gas stream within the incubating chamber itself; the possibility to realise adapters to be applied on the bottom and/or on the lateral sides of the base of the incubating chamber, so

that this can be inserted in various models of microscope and microscope stages or in any other required instrument useful to directly analyse biological specimen; one or more than one of the following parameters can be maintained at controlled levels and conditions: temperature, humidity level and CO₂ level; the possibility to realise the present invention as made of two (base and cover) or three parts (base, side walls and cover); the possibility to add holes to allow fluid perfusion; . 2) A "Controlled temperature water (or other fluid)-jacket CO₂ Microscope Stage Incubator" where as claimed in claim 1 the heating/cooling method is obtained through inner circulation of controlled temperature water or other fluid. Such fluid generally is supplied from a circulating thermostatic or cryostat water bath. This heating (or cooling, when required) method allows, through heat conduction and heat radiation through metal walls wherein controlled temperature fluid circulates, to provide the desired thermal profile in the incubating chamber where specimens are hosted. Thanks to the thermal inertia of the water, the present invention guarantees a superior thermal accuracy and stability compared to electrical resistances.

3) A "Controlled temperature water (or other fluid)-jacket CO₂ Microscope Stage Incubator" where, as claimed in claim 1, control of the specimen temperature is achieved, thus ensuring that, even if ambient temperature is varying, the PID temperature control software acts to maintain the desired temperature of the specimen. A thermocouple is directly inserted in a vessel filled with a liquid having heat capacity very close to that of the sample medium. This method guarantees that the actual specimen temperature is controlled rather than the temperature of the metal walls or of the air in the incubating chamber. 4) A "Controlled temperature water (or other fluid)-jacket CO₂ Microscope Stage Incubator" where, as claimed in claim 1, specimen is heated/cooled from every side, i.e., both from the base and the cover of the incubating chamber, which are in turn heated/cooled through inner controlled temperature water (or different fluid) circulation. When applied to biological samples, this method is superior compared to warm air convective heat transfer, since sample medium evaporation is minimised.

5) A "Controlled temperature water (or other fluid)-jacket CO₂ Microscope Stage Incubator" where, as claimed in claim 1, the controlled temperature water (or different fluid) circuit inside the incubating chamber is especially designed to guarantee thermal uniformity all around the samples under analysis. As illustrated in Fig. 9-page 2 of the drawings, controlled temperature water (or other fluid) circulates in the middle of the base and of the cover as well as along their perimeter. This circuit, providing heating/cooling from every side through conductive heat transfer, guarantees a strongly enhanced thermal uniformity all around specimen vessel.

6) A "Controlled temperature water (or other fluid)-jacket CO₂ Microscope Stage Incubator" where, as claimed in claim 1, the desired gas stream circuit inside the incubating chamber is especially designed to avoid water condensation on glass and plastic surfaces, which would occur if gas stream entered in the incubating chamber at a lower temperature than the one in the incubating chamber. In fact, this especially designed circuit allows a double pre-heating of the gas stream before it enters in close contact with the specimens under analysis inside the incubating chamber. A first preheating of the gas stream is obtained by flow through the bubbling column (maintained at a temperature above the specimen temperature) and a second pre-heating of the gas stream is obtained by circulation in small channels surrounded by the controlled temperature water (or other fluid) flowing in the inner channels of the incubating chamber.

7) A "Controlled temperature water (or other fluid)-jacket CO₂ Microscope Stage Incubator" where, as claimed in claim 1, gas stream can be preheated by passing in the heated bubbling-column and/or circulating in the small channels surrounded by the controlled temperature water (or other fluid) circulating in the inner channels of the incubating chamber.

8) A "Controlled temperature water (or other fluid)-jacket CO₂ Microscope Stage Incubator" where, as claimed in claim 1, a bubbling column is used to humidify the gas stream before it enters in close contact with the specimen.

9) A "Controlled temperature water (or other fluid)-jacket CO₂ Microscope Stage Incubator" where, as claimed in claim 1, a bubbling column, where the desired gas stream is humidified and pre-heated, is maintained at a temperature above or close to the temperature in the incubating chamber.

10) A "Controlled temperature water (or different fluid)-jacket CO₂ Microscope Stage Incubator" where, as claimed in claim 1, a dedicated PID temperature control software acts to maintain the desired specimen temperature.

11) A "Controlled temperature water (or other fluid)-jacket CO₂ Microscope Stage Incubator" where, as claimed in claim 1, the temperature control software avoid thermal shocks to the specimen under analysis when the incubating chamber is opened. This is achieved by stopping the feedback control and maintaining the water bath at the last temperature it had before the incubating chamber was opened. The stopping of PID controller operation can be extended to a programmable duration after the incubating chamber has been closed again. 12) A "Controlled temperature water (or other fluid)-jacket CO₂ Microscope Stage Incubator" where, as claimed in claim 1, the PID temperature control software communicates via serial/USB communication both with the meter reading the temperature measured by the thermocouple inside the incubating chamber, and with the thermostatic or cryostat water bath. The PID controller software acts to increase the temperature of the fluid inside the water bath if the thermocouple is measuring a temperature lower than the set-point value, until the desired sample temperature is reached, or to diminish the temperature of the fluid inside the water bath if the thermocouple is measuring a temperature above the set-point value, until the desired sample temperature is reached 13) A "Controlled temperature water (or other fluid)-jacket CO2 Microscope Stage Incubator" where, as claimed in claim 1, small containers filled with water or a water liquid solution are placed close to the specimen vessels,

) thus water saturating, through matter transfer, the gas stream that surrounds the specimens, and allowing to minimise sample medium evaporation. 14) A "Controlled temperature water (or other fluid)-jacket CO2 Microscope Stage Incubator" where, as claimed in claim 1, different types of sample vessels (i.e. Petri-dishes, glass slides, glass chambers, multiwell plates) can be hosted. In order to accommodate different types of sample vessels, the geometry and shape of the holes drilled in the base of the incubating chamber and correspondingly in the cover of the incubating chamber, will vary without altering, in any different configuration, the operation principle and the innovations of the present invention. 15) A "Controlled temperature water (or other fiuid)-jacket CO₂ Microscope Stage Incubator" where, as claimed in claim 1, one or more than one sample-vessel (of the same type) can be hosted (i.e., as shown in Fig.8-page 2 of the drawings, # 4 35mm Petri-dishes can be hosted within the same incubating chamber). The possibility to host within the same incubating chamber more than one vessel allows to analyse different specimens within the same experiment. 16) A "Controlled temperature water (or other fluid)-jacket CO₂ Microscope Stage Incubator" where, as claimed in claim 1, different types of sample vessels can be hosted within the same incubating chamber (i.e., as shown in Fig.11 -page 4 of the drawings a 35mm Petri-dish and a glass-slide can be hosted within the same incubating chamber). 17) A "Controlled temperature water (or other fluid)-jacket CO2 Microscope ) Stage Incubator" where, as claimed in claim 1, specimens can be analysed both with long working distance (embodiment where all the holes accommodating sample vessels, in the base of the incubating chamber are closed by glass or any other transparent material plates) and immersion objectives (in the configuration where all the holes accommodating sample vessels in the base of the incubating chamber are left open to allow direct contact between the bottom glass of the specimen vessel and the objective). 18) A "Controlled temperature water (or other fluid)-jacket CO2 Microscope Stage Incubator" where, as claimed in claim 1, specimens can be analysed both with long working distance and immersion objectives in the same present invention embodiment (where both holes closed by glass or any other transparent material plates and empty holes are present in the same base). 19) A "Controlled temperature water (or other fluid)-jacket CO₂ Microscope Stage Incubator" where, as claimed in claim 1, thermal insulation is provided for the tubes where the following fluids circulate: the controlled temperature water (or other fluid), the gas stream in and out from the incubating chamber, and the gas stream within the incubating chamber itself. 20) A "Controlled temperature water (or other fiuid)-jacket CO₂ Microscope Stage Incubator" where, as claimed in claim 1, adapters to be applied on the bottom and/or on the lateral sides of the base of the incubating chamber, so that this can be inserted in various models of microscopes and

) microscope stages or in any other required instrument useful to directly analyse biological specimen are designed. 21) A "Controlled temperature water (or other fluid)-jacket CO₂ Microscope Stage Incubator" where, as claimed in claim 1, one or more than one of the following parameters can be maintained at controlled levels and conditions: temperature, humidity level and CO₂ level. 22) A "Controlled temperature water (or other fluid)-jacket CO₂ Microscope Stage Incubator" that, as claimed in claim 1, can consist of two parts (base and cover) in a preferred embodiment or of three parts (base, side walls and cover) in a different embodiment.

23) A "Controlled temperature water (or other fluid)-jacket CO₂ Microscope Stage Incubator" where, as claimed in claim 1, uniform environmental conditions, suitable for biological specimens, are maintained inside the incubating chamber, all around the samples. In particular, temperature, humidity level and CO₂ level conditions are the same throughout the chamber (all around the samples), thus creating homogeneous air conditions rather than localised micro-environments. 24) A "ControUed temperature water (or other fluid)-jacket CO₂ Microscope Stage Incubator" where, as claimed in claim 1, shape and dimensions of every part and the number and the position of the holes where fluids (controlled temperature liquid fluid and gas stream) in-going and outgoing to and from channels inner to the incubating chamber can vary without altering the principle of operation of the present invention. ) 25) A "Controlled temperature water (or other fluid)-jacket CO₂ Microscope Stage Incubator" where, as claimed in claim 1, additional holes can be drilled in every part of the chamber to allow every kind of fluid perfusion or tube insertion inside the incubating chamber. 26) A "Controlled temperature water (or other fluid)-jacket CO₂ Microscope Stage Incubator" where the cover of the incubating chamber can have the same length and width of the base and/or of the side walls, to be tightened onto it (i.e. with screws) or it can have length and width smaller that those of the base and/or the side walls so as to be supported by or embedded into one of them.

27) A "Controlled temperature water (or other f_uid)-jacket CO₂ Microscope Stage Incubator" where the cover can also be made of glass (or Plexiglas or any other transparent material) or without heating/cooling by controlled temperature water (or other fluid) inner circulation.

28) A "Controlled temperature water (or other fluid)-jacket CO2 Microscope Stage Incubator" that can be supported by or embedded into the microscope stage or alternatively be placed in any other place according to the function it is used for. 29) A "Controlled temperature water (or other fluid)-jacket CO₂ Microscope Stage Incubator" where the tubes where controlled temperature water (or other fluid) and/or gas stream in to and out from the incubating chamber as well as in the incubating chamber itself flow can be externally heated (i.e. through electrical resistances) in a different embodiment.