Description

The invention relates to a sample preparation apparatus comprising a vacuum chamber, at least one sample holding unit for holding a sample in the vacuum chamber, and at least one sublimation device arranged in the vacuum chamber at a distance from the at least one sample holding unit, wherein the sublimation device comprises a heat conducting body having at least one receiving section configured to receive a substance to be sublimated and at least one temperature conditioner for cooling/heating the receiving section of the heat conducting body to carry out a sublimation and deposition process on the sample in the vacuum chamber.

A sample preparation apparatus is known from EP 2 975 393 B1 , see for example the sample preparation apparatus shown in figure 5. In this sample preparation apparatus the matrix substance volume is not processed in an efficient manner in the sublimation and deposition process, i.e. the sublimation process is relatively time consuming and the sublimation process requires relatively much energy.

It is an object to provide a sample preparation apparatus for processing a substance to be sublimated in a more efficient manner.

This object is achieved with the sample preparation apparatus as defined in claim 1 .

The sample preparation apparatus comprises:

- a vacuum chamber;

- at least one sample holding unit for holding a sample in the vacuum chamber, and

- at least one sublimation device arranged in the vacuum chamber at a distance from the at least one sample holding unit.

The sublimation device of the apparatus comprises a heat conducting body having at least one receiving section configured to receive a substance to be sublimated and at least one temperature conditioner for cooling/heating the receiving section of the heat conducting body to carry out a sublimation and deposition process on the sample in the vacuum chamber.

The heat conducting body has an optimized thermal geometry to provide a sample preparation apparatus configured for processing the substance to be sublimated in a more efficient manner. The optimized thermal geometry of the heat conducting body is achieved by varying the thickness of the heat conducting body below the receiving section. By providing at least a first portion of the heat conducting body close to the at least one temperature conditioner thicker than at least a second portion of the heat conducting body further away from the at least one temperature conditioner, it is possible to obtain a relatively large surface area which can be heated or cooled in a relatively fast way. By means of the relatively large surface area to be heated or cooled by the heat conducting body and the at least one temperature conditioner, the sublimation device is configured to process relatively large quantities/volumes of substance in each sublimation process per time unit. The optimized thermal geometry of the heat conducting body further permits to use a relatively low number of temperature conditioners, as the amount of material of the heat conducting body to be cooled/heated by each temperature conditioner can be reduced significantly. In other words, the or each temperature conditioner is capable of cooling/heating the heat conducting body in a more efficient manner than a heat conducting body without varying thickness below the receiving section. In addition, the thermal geometry of the heat conducting body is configured to provide a uniform temperature distribution in the surface of the receiving section contacting directly or indirectly the substrate to be sublimated. The relatively low amount of material of the heat conducting body also provides an improved temperature controllability for the substance to be sublimated, because the heat conducting body is able to react in a faster manner for adapting the temperature conditions for the substance. Further, the temperature conditioner(s) for cooling/heating the heat conducting body will have in use a relatively low power consumption, because the amount of material in the heat conducting body to be heated by the temperature conditioner is relatively low. Normally, the temperature conditioner is configured as a heater element to heat the receiving section of the heat conducting body for the sublimation process, but it is also possible for substances having specific properties to cool the heat conducting body by means of the temperature conditioner before or after applying the substance to be sublimated. For cooling, it is for example possible to use a cooling medium flowing in the temperature conditioner. Hence, by providing the sublimation device of this disclosure a more efficient and improved sublimation process can be achieved in the sample preparation apparatus.

The apparatus disclosed in this disclosure is in particularly applicable to prepare a sample to conduct mass spectroscopy and mass spectroscopy imaging (MS imaging) using a matrix assisted laser desorption/ionization (MALDI) methods.

In an aspect of this disclosure, the sublimation device comprises a temperature sensor, wherein the temperature sensor is positioned closer to the second portion than to the at least one temperature conditioner. By positioning the temperature sensor closer to the second portion, it is possible by means of a temperature controller connected to the sensor to adjust the (heating) current in an accurate manner such that the desired temperature for the sublimation process can be adjusted in a fast manner, because the thinner second portion is more sensitive to a temperature change than the thicker first portion. The temperature sensor may also be arranged in the heat conducting body adjacent to or at least partly in the second portion of the heat conducting body.

In another aspect of this disclosure the receiving section of the heat conducting body is a basin for receiving the substance. By means of a basin a trained operator of the apparatus is able to prepare the composition of substance to be sublimated in situ and/or adapt the volume of the substance to be sublimated. Hence, it is possible to test and adapt if desired the composition of the substance to be sublimated. This is beneficial if it is desired to test the effects of certain components in the substance to be sublimated and in particular the effects of the vapor deposition on the sample. In other words, the basin of the heat conducting body provides a flexible sample preparation device, because the substance to be sublimated can be manually adapted and tailored by an operator. The basin is preferably made in the heat conducting body, such that the basin and the heat conducting body are made in one- piece.

In a different aspect, the receiving section of the heat conducting body is configured for supporting or holding a cartridge containing the substance. An advantage of using cartridges in the sample preparation apparatus is that the cartridges with the substance can be produced in an automated process. Hence, the operator does not need to compose the substance or select the volume of substance to be sublimated. Further, the risks of (human) errors in the composition of the substances to be sublimated can be reduced significantly. The composition of the substances on the cartridges can be made identical or almost identical, such that reproducibility of the sample preparation process increases significantly. This also makes it possible to deploy operators for the apparatus who do not require knowledge how to prepare a substance to be sublimated. Cartridges are further beneficial if the samples to be imaged for example in a matrix-assisted laser desorption ionization (MALDI) mass spectrometer (MS), should be produced in series. In such an in series process, the cartridges save a lot of time. The cartridge may be detachably positioned in the receiving section. This can be done by an operator by hand or by a machine in an automated process.

In still another aspect, the receiving section of the heat conducting body comprises a contact surface for transferring heat to the substance in the receiving section, wherein the contact surface has at least one portion having a rounded surface. Such a portion or portions of the contact surface having a rounded surface may for example provide a edge section having a rounded surface towards a central contact surface section in a receiving section. The central contact surface may be substantially flat. Such a rounded edge section facilitates the cleaning process of the receiving section after use compared to a receiving section with an edge section having corners. As a result the rounded edge section also reduces the risk of remaining contaminants in a subsequent sublimation process when a different substance is being used. In the heat conducting body the contact surface forming the receiving section is also forming, at least partially, the top side of the heat conducting body.

In the receiving section the contact surface may have a substantially rounded shape in general. The inventors have found out that such a round shape of the contact surface seen in a cross section view in the longitudinal direction and/or transverse direction, drastically increases the thermal contact between a cartridge and the contact surface as a heating/cooling surface of the heat conducting body. It has been observed that the free spaces between a relatively flat contact surface of the receiving section and a relatively flat contact surface of a cartridge are relatively large, which in particular during vacuum conditions have the result that the contact surface of the cartridge is not heated/cooled in an efficient manner at all by the heated/cooled contact surface. By providing a curved contact surface in the receiving section for receiving a cartridge the actual contact surface between the curved contact surface and the corresponding curved contact surface of the cartridge is increased drastically. By increasing this contact between the cartridge and the contact surface the heat transfer from the heat conducting body to the cartridge is increased. Thus, the sample preparation apparatus is able to process a substance to be sublimated provided on the cartridge in a more efficient manner. It is noticed, that the curvature of the contact surface may be relatively low, e.g. the curvature may follow a circle having a radius of curvature of at least two times the largest dimension of the contact surface.

The heat conducting body is made of a nickel-comprising material, preferably the heat conducting body is made of nickel. Nickel is not only corrosion resistant for a relative large number substances to be sublimated, in particular substances used in a sample preparation method for MALDI, such that the heat conducting body can relatively easily be cleaned, but also has positive thermal conductivity properties. Further, a heat conducting body made of nickel or a nickel comprising material is robust and design friendly for example to provide the rounded contact surface or curved (edge) portions thereof in the heat conducting body.

The invention will be explained in more detail below by means of the following figures which show as an example an embodiment of a sample preparation apparatus and as an example various embodiments of components of the sample preparation apparatus.

The figures show:

Figs. 1 a - 1 b - a perspective view, and a cross section view of a sample preparation apparatus;

Fig. 2a - 2b - a perspective view of a cartridge-type sublimation device for a sample preparation apparatus;

Fig. 3 - a perspective view of a basin-type sublimation device for a sample preparation apparatus; Fig. 4a - 4c show a top view, and two cross section views of the basin- type sublimation device as shown in figure 3.

For the sake of clarity, identical or similar parts in the following figures - even in mutually different embodiments - are in each case provided with the same reference numeral.

Figure 1 a, 1 b show a sample preparation apparatus 100 for depositing a chemical layer of one or more components from a substance, such as a MALDI matrix, onto a medium, for example a biological sample. The apparatus 100 comprises a vacuum-tight chamber 15 where sublimation conditions can be achieved for vapor depositing a chemical layer onto the medium, a vacuum pump (not shown) and fittings 1 for providing the vacuum chamber 15, a sublimation device 4 comprising a heat conducting body 32 and a receiving section in the form of a matrix tray 34 allowing a substance, such as a MALDI matrix, to be heated above their sublimation temperature at vacuum, a sample holding unit 8 having a medium/sample holder 3 configured to keep the medium/sample positioned at a set distance facing the matrix tray 3 and keeping the medium/sample at a desired temperature by a heating/cooling unit 2 of the sample holding unit 8 for deposition of the sublimated components of the substance, a venting system (not shown) allowing the introduction of air or a neutral gas to the vacuum chamber 15 and release the vacuum. The pressure of the chamber 15 is monitored in use by a vacuum gage (not shown) and can be automatically de pressurized and re-pressurized by opening and closing an electrically actuated valve (not shown).

The vacuum chamber 15 has a clam shell design having two pivotally connected vacuum chamber portions 15a, 15b for opening and closing the chamber 15 for inspection or specific cleaning purposes and a bulkhead viewing window 17 providing visualization of the process carried out in the apparatus 100.

The vacuum chamber 15 has connectors 5, 6 to be connected to an auxiliary liquid or gas reservoir, i.e. a first connector 5 allows the introduction of water or solvent vapor for further enhancement of the surface chemistry, including rehydration of the component layer atop the sample to enhance the co-crystallization of the components with the biological samples and a second connector 6 allowing the introduction of for example nitrogen. The apparatus 100 may comprise automated valves (not shown) in the connections 5, 6 between the vacuum chamber 15 and the liquid or gas reservoir to allow automated introduction of water or solvent vapor at a specific time point and under specific pressure condition.

As shown in figures 2a, 2b and 3 in more detail the sublimation device 4; 104 comprises a heat conducting body 32; 132 having at least one receiving section 34; 134 configured to receive a substance to be sublimated and two temperature conditioners 36; 136, for example two heater elements for heating the receiving section 34; 134 of the heat conducting body 32; 132 to carry out a sublimation and deposition process on the sample in the sample holding unit 8 in the vacuum chamber 15 shown in figures 1 a, 1 b. As clearly shown in figures 2a, 2b and 3 the thickness of the heat conducting body 32; 132 below the receiving section 34; 134 varies in that at least a first portion 32a; 132a of the heat conducting body 32; 132 close to one of the two temperature conditioners 36; 136 is thicker than at least a second portion 32b; 132b of the heat conducting body 32; 132 further away from the temperature conditioner(s) 36; 136. The second portion 32b; 132b of the heat conducting body 32; 132 comprises a recess 38; 138 provided on a bottom side of the heat conducting body 32; 132 opposite to the receiving section 34; 134. The recess 38; 138 extends over substantially the width dimension w (figure 2a) of the heat conducting body 32; 132. In the embodiments shown the recess 38, 138 is located centrally in the heat conducting body between the two temperature conditioners 36; 136 and at an equal distance to the two temperature conditioners 36; 136. It is also possible that the recess (not shown) is provided in a side wall as for example a hole surrounded by the heat conducting body 32; 132 to provide a second portion (not shown) of the heat conducting body. In other words, a variety of thermal geometries for the heat conducting body are possible, including designs wherein the thickness of the heat conducting body below the receiving section varies in a continuous decreasing fashion in a direction away from the temperature conditioner. It also possible to use a sublimation device 4; 104 having a single temperature conditioner 36; 136.

Each temperature conditioner 36; 136 is positioned in the heat conducting body 32; 132. The heat conducting body 32; 132 is provided with receipt holes for detachably attaching therein the temperature conditioners 36; 136. The sublimation device 4 differs from the sublimation device 104 in that the sublimation device 4 comprising a heat conducting body 32 having a receiving section 34 provided as a basin for receiving the substance to be sublimated, whereas the sublimation device 104 comprises a heat conducting body 132 with a receiving section 134 configured for holding/supporting a cartridge 150 containing the substance. Besides the receiving sections 34; 134 the sublimation devices 4; 104 are identical. In fact, the basin and the cartridge embodiment preferably both have a receiving section 34; 134 having a rectangular shape, wherein the receiving section 34; 134 has a length greater than its width, and the width is greater than its height. In the receiving section 34; 134 the height dimension h (figure 2a) is the smallest dimension, wherein the length dimension I of the receiving section 34; 134 is at least two times the width dimension w of the receiving section 34; 134.

The cartridge 150 may be a flexible foil tray onto which a matrix or chemical components are affixed. Instead of a flexible foil tray, a thin plate or a sheet may also be used.

In the sublimation device 104 the cartridges 150 can be detachably positioned in the receiving section 134, because the receiving section 134 is provided with slots 141 a, b at the longitudinal ends thereof for locking and holding the longitudinal ends of the elongated cartridge 150. The cartridge 150 is manually detachably positioned in the receiving section 134 of the heat conducting body 132. The matrix cartridges 150 can be disposable and the cartridges 150 allow standardization of sample preparation. The heat conducting body is designed to accept consumable matrix cartridges while maintaining uniform heat distribution. The heat transfer between the contact surface 144 and the contact surface of the cartridge 150 positioned on the contact surface 144 is improved drastically by providing a substantially round shape seen in a cross section view in the longitudinal direction I. The round shape seen in a cross section view in the longitudinal direction follows substantially a circle having a radius of curvature of at least two times the longitudinal dimension I of the I contact surface 144. This slightly curved contact surface 144 reduces or even eliminates gap(s) between the heat conducting body 132 and the cartridge 150. These gaps, in particular during vacuum conditions, have the result that the contact surface of the cartridge is not heated in an efficient manner at all. As seen in figure 2a, the receiving section 134 and the slots 141 a,b are configured such that after insertion of a cartridge 150 into the receiving section 134 of the heat conducting body 132 the shape of the cartridge 150, and in particular the contact surface of the cartridge 150 contacting the contact surface 144 corresponds/is identical to the (rounded) shape of the contact surface 144 which results in an efficient heat transfer between heat conducting body 132 and the cartridge 150 as the cartridge 150 is pushed against the contact surface 144. It is also observed that to increase the heat transfer between the contact surface and the cartridge, it is possible to provide a substantially round shape of the contact surface (not shown) seen in a cross section view in the transverse direction w.

The receiving section 34 of the basin also comprises a contact surface 44 for transferring heat to the substance (not shown) in the receiving section 34, wherein the contact surface 44 has a substantially flat central section 44a surrounded by an edge section 44b having a rounded surface between the central section and a top edge 44c of the basin. As already mentioned above a rounded edge section provides advantages from a cleaning perspective.

As shown in figures 4a-c the sublimation device 4; 104 further comprises a temperature sensor 70 (figure c) connected by a cable 71 to a connector 73 to be connected to a temperature controller (not shown) controlling the temperature conditioners 36; 136, wherein the temperature sensor 70 is positioned closer to the recess 38; 138 than to the at least one temperature conditioner 36; 136. More specific, the temperature sensor 70 is arranged in the heat conducting body 32; 132 adjacent to the recess 36; 136 of the heat conducting body 32; 132. By positioning the temperature sensor 70 in such a position it is possible to obtain a temperature by a single sensor 70 which provides a good representation of the temperature in the contact surface 44; 144. By using in the controller the measured temperature and the characteristics of the thermal conductivity of the heat conducting body 32; 132 heated or cooled by the temperature conditioners 36; 136, the controller is able to achieve or maintain the desired temperature of the contact surface 44; 144. The figures 3 and 4a-c further show that the heaters 36 are connected by cables 76 to connectors 78. The heat conducting body 32 is further provided with seals 81 to maintain a vacuum in the chamber 15 and an insulator 83 near the rear side wall 45 to maintain the heat inside the heat conducting body 32; 132.

The heat conducting body 32; 132 is made of a nickel-comprising material, preferably the heat conducting body 32; 132 is made of nickel. Nickel has excellent thermal conductivity properties and is corrosion resistant for a relative large number substances to be sublimated, in particular substances used in a sample preparation method for MALDI. Further, a heat conducting body made of nickel or a nickel-comprising material is robust and design friendly for example to provide the rounded contact surface or curved (edge) portions thereof in the heat conducting body.

The sample holding unit 8 and the sublimation device 4 are releasable attached to a side wall 45 of the vacuum chamber 15. A modular construction of the sample holding unit 8 and the sublimation device 4 has the advantage that they can be removed for repair, cleaning purposes or replacement by a similar one, or one of a different design, allowing ease of repair and upgradability. For example, both sample holding unit 8 and the sublimation device 4 can be replaced with another sample holding unit 8 and sublimation device 4 having a different size or a different configuration.

The rear side wall 45 of the vacuum chamber 15 comprises the vacuum connection 1 , a support for supporting the sample holding unit 8 in the vacuum chamber 15 and a support for supporting the sublimation device 4 in the vacuum chamber 15, and the first and second connectors 5, 6 as discussed above. In the vacuum chamber 15 all electrical connections are moved to the outside of the chamber 15, creating a safer, more robust and easier to clean chamber 15 of the apparatus 100. Further, this configuration provides an apparatus comprising more serviceability, because it makes the clam shell design having two pivotally portions possible which provide great access to the interior of the chamber 15 for cleaning, inspection and maintenance. In addition, the arrangement of the various functions in the rear wall 45 also reduced the time to create a vacuum atmosphere inside the apparatus 100. The sample holding unit 8 is arranged between the vacuum connection 1 and the sublimation device 4 in the chamber 15. The sublimation device 4 is arranged in the vacuum chamber 15 between the first and second connectors 5, 6 and the sample holding unit 8. Hence, seen in vertical direction the sample holding unit 8 and the sublimation device 4 are arranged between the vacuum connection 1 and the first and second connections 5, 6. This configuration of the apparatus 100 provides an excellent sublimation process and an excellent deposition process on the sample. Further, it is possible to provide on a single outer side of the apparatus, i.e. the rear side wall 45, all the connections for temperature control and vacuum control.

The sublimation device 4; 104 may additionally or instead of the curved contact surface 144 comprise a force applicator (not shown) applying a force against the cartridge 150 to increase the thermal contact between the receiving section and the cartridge and/or the sublimation device 4; 104 may comprise a thermal interface layer made of a thermal conductive material to be positioned between the cartridge 150 and the receiving section 134 to increase the thermal contact between the receiving section and the cartridge.

In the sublimation device 4; 104 it is also possible to position the temperature conditioners 36; 136 outside (not shown) the heat conducting body 32; 132 in contact with an outer surface of the heat conducting body.

In a process to prepare a sample using the sample preparation apparatus of this disclosure, the substance to be sublimated is pre-prepared as follows: a matrix powder is added to a solvent, for instance acetone, to obtain a mixture of the matrix powder and the solvent. The mixture is applied into the matrix tray, for example pipetted in the basin. The matrix tray is (pre-)heated and the solvent evaporates. During this process the matrix substance will stick to the surface of the matrix tray in an uniform manner, such that an optimal heat transfer can be obtained in the sublimation device for the sublimation process of the matrix substance for preparing a sample. By using the pre-preparation step using the solvent, excellent results are achieved in preparing the sample in a relatively short period of time.