EP2669011B1 - Image projection method and device for supporting manual MALDI sample preparation - Google Patents

Description

field of use

The invention relates to a method for supporting the manual preparation of samples on a sample carrier for ionization with matrix-assisted laser desorption and a corresponding assignment aid.

State of the art

Occupancy aids have become known in the prior art, in particular for use with microtitration plates.

The utility model DE 20 2007 018 535 U1 describes a pipetting aid for transparent microtitration plates, which are stored via an adapter in a base plate. The base plate includes light sources each associated with an opening in the adapter and a cavity of the transparent microtitration plate. A switching or control unit activates the light sources independently of each other and indicates by means of illumination through the adapter and the transparent plate, where a sample liquid is to be pipetted. The utility model DE 20 2005 017 946 U1 concerns a similar subject.

In contrast to microtitration plates, sample carriers for ionization with matrix-assisted laser desorption are usually opaque. This is due to their electrical conductivity, which serves to prevent static charges on the sample carrier, which can form during the laser desorption. Electrical conductivity is fundamentally undesirable for microtitration plates, because the cavities-in contrast to the flat sample samples with a largely aligned design on MALDI sample carriers-have a larger interaction surface with the filled sample liquid. This increased interaction area can - with existing conductivity and liquid samples - unwanted interfacial processes, such as the deposition of dissolved in the liquid charge carriers such as salts, or require chemical interface reactions.

The patent US 4,692,609 A describes, in a manner similar to the above utility model, a receptacle for a transparent microtitration plate, at the bottom of which a plurality of light sources are arranged so that they can illuminate a well of the plate from below to indicate to a user where to pipet. Alternatively The lighting can also be done on the front by a feasible light source, it is not disclosed how a feasible light source should be designed.

The publication WO 2007/038521 A1 shows a construction with telescopic arm, at the end of a light source is mounted. The arm can be extended by means of an actuator so that the light source can be positioned vertically over each cup of the microtitration plate for the purpose of illumination. This embodiment has the disadvantage that when starting a particular cup always the light source itself must be moved together with the holder, which places increased demands on the mechatronic actuators.

Other publications dealing with sample preparation on microtitration plates FR 2 649 511 . US 2005/0046847 A1 . WO 83/00047 A1 . WO 2007/071575 A1 and WO 2007/121324 A1 ,

The publication US 2002/0191864 A1 discloses the use of an image for identifying the sample areas occupied by a sample on the sample support and aligning the laser beam with those areas. A method for supporting the manual preparation of a MALDI sample carrier, however, is not disclosed.

The publication EP 1 763 061 A2 concerns, among other things, the monitoring of occupancy processes on MALDI sample carriers by means of an imaging workstation.

The publication DE 10 2004 020 885 A1 is concerned with the preparation of samples of microbial origin on MALDI slides with the aim of automating the transfer of biological material from agar plates to sample locations of MALDI slides. For this, agar plates are transported via a conveyor belt to a robot and placed on a 3D table. Image processing recognizes single colonies on the agar plate and positions a sample rod accordingly. A single sample rod is used only for a single transmission and then replaced. The uptake of biological material with the sample rod is accomplished by detaching the sample rod from a support and dropping it from a height of a few millimeters onto the colony. The contact with the colony thus produced is intended to guarantee that only biological material will adhere to the sample rod, but no agar will be transferred to the MALDI sample carrier. If too much agar is transferred to the MALDI sample carrier, the quality of the mass spectrometric identification is reduced because agar suppresses the signals of the characteristic protein ions. A fine sensor to control the contact is not provided. However, the sample rod vibrates and can be wetted with water before sampling, so that a sufficient Amount of biological material from a colony sticks to the sample rod and can be transferred to a sample site of a MALDI sample carrier.

An allocation aid which is suitable for flat and opaque MALDI sample carriers is described in the German patent application 10 2010 052 976.1 (Inventors: Markus Kostrzewa and Ulrich Weller) disclosed. The application relates in particular to the highlighting of samples to be occupied by front illumination or mechanical hands or shadow masks, see illustration 1 (Above: viewable beam, center: pointer, bottom: shadow mask). However, the mechanical variants almost always entail the risk of cross-contamination of sample locations if part of the sample to be applied remains attached to the pointer or shadow mask. On the other hand, because of the usually metallic surface of the MALDI sample carrier, a collimated light beam thrown onto the front side can lead to users blinding, disturbing light reflections, in particular if the angle of incidence is unfavorable. US2005102056 shows an image system for visual highlighting of a recording in which the process steps are automatically detected and the sample locations are automatically controlled.

Object of the invention

Thus, there remains a need to provide an improved placement aid for sample preparation on sample carriers of matrix assisted laser desorption and ionization.

Description of the invention

The invention proposes an allocation aid for the manual preparation of samples on a sample carrier for ionization with matrix-assisted laser desorption. It contains a holder for a sample carrier with several sample types, which is preferably adapted to standardized sample carriers for ionization with matrix-assisted laser desorption. Furthermore, a device is provided which throws a two-dimensional optical image, or a corresponding image sequence, onto the front side of the sample carrier having the sample location when the sample carrier is arranged in the receptacle, the image or the image sequence being designed such that highlighting a selected sample location, or group of selected sample locations, at least opposite non-selected sample locations adjacent thereto in a human visual manner. Furthermore, an interface for confirming the occupancy by hand or a device for the automated detection of a manual occupancy process are available. With a control system, a sample location, or a group of sample locations, can be selected and the device can be controlled accordingly.

The term two-dimensional image is to be understood in the context of the present disclosure. For example, it is possible to project two two-dimensional images one after the other onto the sample carrier in rapid succession, so that a viewer, possibly using an aid such as a pair of glasses, creates a three-dimensional image impression on the sample carrier front side. However, a component of such a "3D" image could also be a two-dimensional map.

The device preferably has a surface light modulator, a liquid crystal projector, or a liquid crystal on silicon projector. In this way, a very flexible image, or a very varied image sequence, can be generated on the sample carrier using conventional video projection methods. The front of the sample carrier thus acts as a "canvas" of the projected image. The design of the image with respect to color choice of individual pixels (pixels), brightness and / or image sequence are virtually unlimited.

Surface light modulators are particularly used in video projectors such as those sold by Texas Instruments, Inc. (Dallas, United States of America) under the name Digital Light Processing (DLP). Such a surface light modulator essentially consists of micromirror actuators arranged in the form of a matrix, that is to say tiltable reflecting surfaces of small edge length, which can be housed in very large numbers in a small space such as a microchip. The movement of the actuators is caused by the force of electrostatic fields. Each micromirror can be individually adjusted in its angle and usually has two stable end states, between which it can switch at a frequency of several kilohertz. By means of the switching frequency, the brightness of a picture element can be adjusted. The number of mirrors corresponds to the resolution of the projected image, wherein a mirror can represent one or more picture elements. Meanwhile, resolutions of up to 4160 by 2080 pixels are possible. In addition, very high-contrast images can be generated in a small area.

If a projection lamp emitting white light is used, whose light is reflected by the micromirrors, a color wheel can be switched in the light path in front of the area light modulator, on which color filters of the primary colors rotate (usually red, green and blue, partially but also more). In order to achieve better brightness values in white, the color wheel can also be added to a white sector. With the position of the color filter, the electronics changes the part of the image, the Modulator is reflected. Due to the rotational speed of the color wheel and the inertia of the human eye, the partial images are added to a colored image impression. High rotational speeds of the color wheel or the provision of multiple color segments ensure a smooth, seamless color representation in the projection.

In another variant, the color representation can be achieved by decomposing the white projection lamp light by means of dichroic mirrors into the three primary colors red, green and blue and their individual transmission to three different modulators. The respective partial reflection can then be added back to the complete color image in a dichroic prism which contains two crossed dichroic mirrors. Additional micromirror sets are required for this variant. In some embodiments, the color dispersion may also be caused by a dichroic prism.

In further embodiments, instead of a single white light source, it is also possible to use individual colored light sources, for example individual light-emitting LEDs (red, green, blue).

In various embodiments, the device may generate an image or image sequence, thereby producing a brightness and / or color contrast at least to adjacent non-selected sample locations at the selected sample location, or group of sample locations.

It is particularly preferred for the device to generate an image sequence that highlights a sample location or a group of sample locations in an eye-catching manner, for example by the image having a signal color (such as red, yellow or even green) at the location of the sample location to be highlighted whereas the other parts of the image or sequence of images contain muted colors (such as gray or brown) that usually recede behind the signal colors. Flickering or blinking effects can also be achieved with image sequences if, for example, a series of projected images has varying intensity and / or color ranges.

The inclusion of the assignment aid is adapted, preferably geometrically, to standardized sample carriers for ionization with matrix-assisted laser desorption. This adjustment can also be done with adapters that are used in a recording. In this way, sample carriers of different configurations or dimensions can be fitted in the receptacle. As a result, a flush and / or aligned arrangement of the sample carriers in the receptacle can be achieved. The standardization of the sample carriers is determined in particular by their geometric dimensions such as height, length, width or area, the Number of sample locations and / or their shape and / or their size or their (matrix) arrangement, in particular in rows and columns. It should be noted that sample carriers, which are used in time-of-flight mass spectrometers with axial ion injection and laser desorption, must be designed with as flat a front as possible, so that the simplest possible conditions exist for spanned in the space in front of the sample carrier front electrical fields. This facilitates the control of the area in phase space (spanned by place and momentum coordinates) occupied by the ions of interest arising during laser desorption. Cavities as incorporated in microtitration plates are not suitable for this purpose.

The operation of the device may include generating brightness and / or color contrast at least at adjacent non-selected sample locations at the selected sample location. For example, a selected sample location can be irradiated with intense yellow or red light, while the rest of the optical image has a rather low intensity gray tone.

The control system as part of the assignment aid can be provided with an interface for data input or data output. This is particularly useful when a user wants to enter or read in an occupancy plan of a sample carrier to be processed in the control system. The interface can also be used, for example, by manual input for confirmation of an assignment process made. In this way, a sequence of occupancy processes can be carried out reliably. In an extension, the interface may also have a remote communication function, for example for receiving sample origin data and / or corresponding identification mark, which can then be stored with the occupancy data and / or corresponding identification mark the occupied sample locations for the purpose of assignment. The remote communication function may also include transmitting corresponding data. The remote communication function can be set up with known remote communication means such as a radio, Bluetooth, infrared or other interface.

The control system may further comprise a memory for the allocation and recording of identification marks of samples and sample locations. There, the assignments made are safe and can be retrieved as often as desired for later evaluation or review.

The allocation aid can be stationary in one embodiment. Then it is preferably in an arrangement of a breeding plate support on which, for example, Petri dishes arrange for sampling, and arranged a sample loading station for a mass spectrometer with a laser desorption so that the most time-saving transfer of samples from a rearing plate in the rearing plate support on a sample carrier in the assignment aid and from there to the loading station is possible.

In a further variant, the assignment aid can also be designed to be portable. As a portable handset, for example, the assignment aid can be worn by a user such as a painter's palette on or on the hand. In this case, the placement aid preferably has a holding device such as a handle, adapted to the fingers of a human hand blind holes, or a tether, with which it is festschnallbar on an arm of a user. However, the portability can also be achieved in that the placement aid designed in the manner of a belly shop, so for example provided with at least one shoulder or neck strap, so that it can be worn by a user in front of the abdomen or chest. This variant has the advantage that the user has both hands free. Due to the portability, the handling of the assignment aid is more flexible, in particular is no longer locally limited.

Together with the formation of the assignment aid as a portable device, in particular handheld device, a docking station can be provided, which is preferably stationary and has a receptacle for the assignment assistance. A user can, if he needs the necessary freedom, dedicate the worn on the body or in the hand portable assignment aid in the recording and to other work in which the assignment aid is not required to devote. In the docking station, the assignment aid can transfer data of an executed assignment sequence to a stationary computer located in the station. It would also be possible and useful to make an electrical connection to recharge any accumulators of the occupancy aid that provide the energy for their operation.

If an automated detection device is present, it preferably comprises a scattered light sensor. The scattered-light sensor is preferably arranged above the receptacle and serves, in particular, to detect changes in the scattered-light behavior which indicate a manual occupying process on the front side of a sample carrier located in the receptacle, in particular spatially resolved. In addition to the variant of spatially resolved detection, it is also possible to search in a targeted manner for a scattered light signal from the sample location provided (and highlighted) for the next pending occupancy procedure. In this second variant, the temporal correlation or synchronization of the visual highlighting with the detection of a scattered light event an important parameter. The scattered light can basically originate from the optical image and / or from a separately generated light beam which is thrown onto the sample carrier (possibly onto the highlighted sample location on the sample carrier). For the spatially resolved detection of a manual occupancy, a selected sample location can be illuminated individually prior to occupancy, and the scattered light emanating therefrom can be measured with an integrally measuring scattered light sensor. The scattered light measurement can be automatically repeated after confirmation of manual occupancy by the user or at intervals. From the differences in the scattered light intensities or their absence can be concluded that a correct or incorrect occupancy of the selected sample location. For a group of selected sample locations, the scattered light measurement can be performed one at a time for each of the different sample locations of the group individually. The light for the illumination of the individual selected sample locations is preferably generated by the device which throws the two-dimensional optical image onto the sample carrier, but can also be generated by a corresponding second device, in particular in the infrared spectral range which is not visually discernible to the user , An image sequence can be helpful in the evaluation of the scattered light signal by means of frequency filters.

In addition or as an alternative to the scattered light sensor, a camera with image recognition can also be used for an automatic detection of the occupancy.

Especially on the metallic shining surfaces of a MALDI sample carrier, changes in the scattered light behavior can be detected very reliably. If the sensor is aligned with a sample location to be occupied, the occupation process initially expects a strongly varying scattered light signal if the user moves his pipette or his vaccum stamp, for example, through the light cone of the two-dimensional optical image or the light beam of the separate light source. If the vaccination stamp is withdrawn, the changes in the scattered light behavior at the freshly occupied sample location result from the deposited sample (or not at all if the assignment was unsuccessful or took place in the wrong place).

If more than just the sample location intended for occupancy is monitored by the sensor, an incorrect occupancy may also be detected if, in fact, a scattered light change appears at a sample location other than that intended for the next occupancy. It is also possible to confirm an occupancy by hand via changes in the scattered light behavior of a part of the surface of the sample carrier. So can on the Front of the sample carrier, alternatively also in the edge area of the receptacle, a certain area for the occupancy confirmation must be designated, in which no sample locations are and which is monitored with the scattered light sensor. After occupancy, the user can travel with the vaccination stamp over the designated area and thus generate a temporary flare change signal that causes a connected processor to proceed to the next sample location of an occupancy sequence. The term border area is to be understood broadly and is not only intended to describe areas of the image itself, but may also include (mostly marginal) areas of the sample carrier.

In other embodiments, the image, or image sequence, may be divided into an area highlighting the selected sample location, or the selected sample locations, and an area displaying information to the user. The display area may include, for example, a text display with information about the sample to be applied.

The invention further discloses a method for assisting the manual preparation of samples on a planar sample support for matrix-assisted laser desorption ionization. First, a sample carrier with multiple sample locations is provided. Then a selection criterion is defined, or several selection criteria are defined according to which or after which an assignment sequence is to take place. After the one or more selection criteria, a set of sample locations is selected. A two-dimensional optical image, or sequence thereof, is thrown onto the front surface of the planar sample support having the sample locations, the image, or image sequence, being arranged such that a selected sample location, or a group of selected sample locations, at least towards adjacent non-selected sample locations in a manner visually perceptible to a human. Then, a sample or substance for preparing a sample (e.g., a solution containing a MALDI substance) is manually deposited on the highlighted sample site. The occupancy is confirmed by hand and / or automatically detected by a sensorium. If the set contains further unprocessed sample locations, or groups of sample locations, the highlighting and manual picking steps may be repeated with the next sample location of the set, or the next group of sample locations. Otherwise, the assignment sequence would be temporarily terminated.

A user who wishes to carry out a manual preparation of a sample carrier is assisted by the visualization that is visually recognizable by means of the image or the image sequence therein, which is a nutrient medium - for example agar plate, bouillon or blood culture - to deposit the sample taken or processed from it at the right place. The risk of occupancy errors, which is essentially due to the fact that, as a rule, the transferred sample material is barely perceptible visually due to its small amount, can be reduced in this way.

The emphasis should in particular be reversible, that is activatable and deactivatable, and can be reversed again, for example by selectively switching the projected image on or off (or even changed). The work of a specialist should also be facilitated in particular by the fact that the selection and highlighting are done (semi-) automatically with electronically supported means. Low process cost can be achieved if the highlighting of the selected sample location is limited to the immediately adjacent non-selected sample locations, for example by occupying the sample site to be highlighted with a region of the image of light color or high light intensity, whereas those immediately adjacent are not highlighted Sample locations are covered by a region of the image with dark color or low light intensity. The highlighting effect can be enhanced by increasing the amount of non-selected sample locations, in the extreme case such that the selected sample location is emphasized over all other non-selected sample locations. In this case, the image or sequence of images is essentially thrown on the entire front side of the sample carrier.

In the following, MALDI is given as the preferred type of ionization in which ions are formed during laser-induced desorption. However, it is understood that in the present case only laser desorption is important for transferring the analyte substances - thus proteins or protein chains - into the gas phase. The type of ionization can be freely selected depending on the application. The laser desorption can be carried out, for example, with a chemical ionization ( laser desorption chemical ionization - LDCI). But also other types of ionization can be used. The term ionization with matrix-assisted laser desorption is correspondingly broad.

The sample location can then be selected to be unoccupied. The method offers some flexibility in different stages of an occupancy sequence. It is also possible to make a geometric range setting, for example in the way that only every nth - for example, every second - sample location must be documented. This can be useful if the risk of cross contamination by outgassing of a sample and transition of the outgassed sample particles in the gas phase to another sample location with a small spatial distance the occupied sample is increased. In a variant, the selection can be made by an electronic-based technical control system, for example by all unoccupied sample locations are occupied, alternatively by a user of the method.

Multiple sample locations may be selected and the highlighting performed repeatedly in an assignment process, with each repeat highlighting a different selected sample location, or a different group of selected sample locations. The method is therefore particularly suitable for sequential processing of various samples, which originate from different colonies on a rearing plate and are to be placed on a sample carrier. Preferably, in such a sequential processing, the use of a monitoring and control system that supports the user of the method in the selection of the samples to be transmitted.

A process is also proposed for manually preparing a sample on a sample carrier for matrix-assisted laser desorption ionization in which the sample and the sample locations are each provided with identification marks in which a sample location is selected and highlighted according to a method described above, the sample is applied to the selected sample location and the tags are associated and stored. In this way it can be understood after the assignment of the sample carrier and check which samples were transferred from which source to a specific sample location. This allows for subsequent process control and may, for example, indicate an error if a sample of specific origin has been deposited on two sample locations, although only one sample location was provided for each sample of the corresponding origin.

The assignment and storage can be carried out jointly or separately in a combined method step. The assignment can be carried out, for example, before the actual allocation process, the storage after the completion of the allocation process. A certain chronological sequence of the assignment and storage during the procedure is basically not mandatory. Preferably, however, the identifiers are assigned and stored after the allocation process, since such a misallocation or incorrect allocation can be more easily detected.

As samples, especially those of microbial origin are suitable. Preferably, it is understood to mean the microorganisms themselves in their untreated form, as grown in or on a nutrient medium.

The identification mark of the sample can be derived from an identification of the sample vessel - for example a Petri dish - from which the sample originates. This ensures a high level of safety in sample tracking. It is likewise possible to generate or supplement an identification mark by a camera recording the sample source, in particular the planar nutrient medium in a Petri dish, and determining the coordinates of the sample source location in the recording by means of image evaluation and assigning it to the sample. In addition or as an alternative to an optical imaging of the planar nutrient medium, the sample source of origin can also be identified by measuring the change in capacitance on the two-dimensional nutrient medium before the sample is taken after the sample has been taken.

In one variant, the sample origin data and / or identification marks can be transmitted via remote communication means for sample preparation instrumentation to be stored there after occupying a sample location on a sample carrier together with the occupancy coordinates and / or identification mark of the sample carrier or sample location. In this way, a particularly detailed sample tracking is possible.

Brief description of the pictures

• Abbildung 1 drei Belegungshilfen in schematischer Darstellung, wie sie in der deutschen Patentanmeldung 10 2010 052 976.1 offenbart werden,
• Abbildung 2a-c ein schematischer Aufbau einer Belegungshilfe nach Prinzipien der Erfindung,
• Abbildung 3 eine detailliertere (schematische) Darstellung eines Projektionsverfahrens,
• Abbildung 4 ein Beispiel eines projizierten Bildes,
• Abbildung 5A-C ein Beispiel einer projizierten Bildfolge und
• Abbildung 6 eine Flussdiagramm-Darstellung von Verfahren gemäß Prinzipien der Erfindung.

In the following, the invention will be described with reference to embodiments in conjunction with the accompanying drawings. In the drawing show:

• illustration 1 three assignment aids in a schematic representation, as disclosed in German patent application 10 2010 052 976.1,
• Figure 2a-c a schematic structure of a placement aid according to principles of the invention,
• Figure 3 a more detailed (schematic) representation of a projection method,
• Figure 4 an example of a projected image,
• Figure 5A-C an example of a projected image sequence and
• Figure 6 a flowchart representation of methods according to principles of the invention.

Preferred embodiments

The Figure 2a shows schematically the structure of an assignment aid 2 according to the principles of the invention. A base plate 4 contains a receptacle 6 whose internal dimensions preferably correspond to the standardized outer dimensions of an LDI sample carrier 8 (in particular MALDI sample holder) are adapted. In certain cases, adapters (not shown) may be used which set a required spatial configuration.

In the Figure 2a a sample carrier 8 is arranged in the receptacle 6. In the bottom and / or side area of the receptacle 6, a sensor (not shown) can be integrated, which detects the presence of a sample carrier and forwards a corresponding information signal to a control system 10, for example, a built-in microprocessor microprocessor. The sensor may for example consist of a simple push button, which is actuated when inserting the sample holder 8 in the receptacle 6. But there are also other, in particular non-contact sensor variants (ultrasonic proximity sensor, light barrier, ...) conceivable.

A recording of the assignment aid can also be designed as a frame (not shown). A frame that fixes the sample carrier on the narrow sides has the advantage that both the front side and the rear side of the sample carrier are accessible to a measuring or examination instrument (possibly a sensorium). This facilitates the handling of the assignment aid, especially if it is portable.

On one side of the base plate 4, an arm or carrier 12 rises in the height at which an imaging device 14 is arranged. The imaging device 14 may be formed, for example, in the manner of a video projector, as will be explained in more detail below. The imaging device 14 is arranged and aligned so that it can throw a two-dimensional optical image 16, or a corresponding image sequence, onto the front side of a sample carrier 8 located in the receptacle 6. The imaging device 14 communicates with and is controlled by the guidance system 10, for example to indicate which image is to be projected to highlight a sample location, or a group of sample locations. The imaging device 14 preferably includes various optics that cause the image or image sequence, while being laterally thrown at a certain angle onto the sample carrier front, to be displayed undistorted.

The imaging device 14 is arranged to allow a user to transfer a microbiological sample, for example cells of a microbial colony that had been grown on an agar plate, to a sample location on the sample carrier 8 largely unhindered with a vaccination stamp 18 or similar transfer device ,

It is possible that the control system 10 has an interface (not shown) by means of which a user manually confirms a manual occupancy of a sample location can. The term confirming by hand is to be understood here broadly and may also include entering identification data of the next sample to be prepared, for example by scanning a barcode on an agar plate.

Also not shown is a variant in which the control system 10 is equipped with a sensor for the automated detection of occupancy operations, and thus the completion of occupancy of a sample location is automatically detected and reported to the control system 10. Of course, the automated detection can also include the detection of incorrect allocations, that is, if a sample has been deposited on a different than the intended sample location.

Examples of such a sensorium are in the German patent application 10 2010 052 975.3 the patent applicant, which is hereby incorporated by reference into the present disclosure. For example, at a sample location, the sample amount may be probed or the occupancy state of the sample site may be determined by changing at least one of the following chemo-physical properties: resonant frequency of a piezoelectric material, density, geometric dimension, transit time of ultrasound or electromagnetic waves, electrical capacitance, electrical Resistance, inductance, permittivity, magnetizability, light scattering, light absorption, light reflection or luminescence. However, variants with light barrier beams crossing over the sample locations, whereby a monitoring network is built up, are also conceivable.

The control system 10 can detect certain configuration data of the sample carrier 8 via a further telecommunication connection to the sample carrier 8, for example the number, arrangement and position of the individual sample locations. In one example, a microchip attached to the sample carrier 8 containing the corresponding configuration data may be read out. Alternatively, the guidance system 10 may also include, or communicate with, a camera and an optical image recognition system (not shown) that receives the front of the sample carrier 8 and locates identifiable features of the sample locations for application of sample material. These recognizable features can be designed as markings, for example annular framing, on the front side.

The communication with the device 14 also enables the guidance system 10 in this example to (de-) activate a video projector for generating an optical image on the sample carrier front, to change the image and, if necessary, to select different image formats. An interface can be used to record the configuration data, the selection of an image (or a sequence of images) as well as the (de-) activation of the projector in some embodiments also be done by hand.

A user of the assignment aid can in a semi-automatic embodiment of the control system 10 enter the occupancy state of the sample carrier 8 or read in, for example via an interface. He can simultaneously specify the criterion according to which the sample locations are to be selected. This can be, for example, a blank state. The control system 10 then checks which of the sample locations are eligible for occupancy, selects one of them (optionally also a group) to highlight the corresponding sample location, selects the image to be projected accordingly, or generates it, and activates the video projector. An image or image sequence is then thrown onto the front side of the sample carrier 8, where a sample site and optionally the surrounding area of the sample carrier front side is distinguished in a human visually perceptible manner from the other areas of the sample carrier with non-selected sample locations.

The highlighting effect may be enhanced by a visual impact enhancement design of the sample support material, for example by incorporating particles into the sample support material 8 which will produce a glitter or color effect when illuminated. A kind of light primer with white particles may also be useful to better accentuate color differences in the various picture elements.

Supported by this highlighting, the user can apply his sample to the correct sample location, and then, for example, confirm the assignment by hand via the interface. This can then lead to the deactivation of the emphasis, in this example, the turning off of the projection, or to the change of the displayed image. In other embodiments, a sensor for automatic detection of manual occupancy operations may also be used.

In order not to irritate the user at work, the sample carrier front side may be provided with an anti-glare coating. This can prevent dazzling reflections of light that might occur during the projection of the image or the sequence of images. Basically, however, the risk of glare when using a projector for imaging on the sample carrier in contrast to bundled light rays is low.

The control system 10 can be provided with a memory (not shown) for the allocation and recording of identification marks of samples and sample locations. This information can optionally be entered or read by a user via the interface; alternatively also by automatic data transmission.

According to a further embodiment, a scattered light sensor 19 can also be arranged on the support 12 (as in FIG Figure 2b indicated), which monitors the front side of the sample carrier 8 spatially resolved to detect changes in the scattered light behavior and to be able to spatially assign an area on the sample carrier, for example a sample location. The spatial resolution can be achieved, for example, with a c harge-coupled device (CCD) and corresponding upstream optics. The light scattered on the surface of the sample carrier 8 and then detected may originate from the projector of the imaging device 14 or also from a separate light source (not shown). However, the scattered light sensor (19) can also measure the integral (not spatially resolved) scattered light that originates from a sample location when it is individually illuminated by the imaging device 14 or the separate light source.

On the sample carrier 8, a specific area 21 (FIG. Figure 2c ) to confirm a successful occupancy process. After the application of the sample, the user can wipe the area 21 with the vaccination stamp and thus trigger a scattered light pulse which indicates the completion of an assignment process and thus leads to the continuation of an assignment sequence. This is an example of an interface for confirming an occupancy. It is understood that the area 21 should be placed on a side of the sample carrier from which a user does not access the sample locations to avoid unnecessary false signals. In alternative embodiments, the area may not be arranged on the sample carrier itself, but in an edge region of the receptacle.

The Figure 3 shows in more detail an embodiment of an assignment aid 2 * according to principles of the invention.

The highlighting device in this example has a surface light modulator disposed in a housing 20. The housing 20 is supported by a carrier or holder (not shown here for simplicity of illustration). Area light modulators are just one example of a video projection technique. Likewise, liquid crystal projectors or liquid crystal on silicon projectors can be used. Such projectors have the advantage that they can generate a very flexible image 16, or a very varied image sequence, on the sample carrier 8. The design of the image 16 with respect to color choice of individual pixels (pixels), brightness and / or image sequence are virtually unlimited.

Schematically illustrated in the housing 20 is a micromirror actuator 22 onto which light is projected by a suitable projection lamp 24 via an imaging optic 26A. From the micromirror 22, the image reaches the front of a sample carrier 8 via a further imaging optic 26B. Micromirror actuators 22 can be accommodated in very large numbers in a small space such as a microchip. Each micromirror 22 can be individually adjusted in its angle and usually has two stable end states, between which it can switch at a frequency of several kilohertz. By means of the switching frequency, the brightness of a picture element can be adjusted. The number of mirrors corresponds to the resolution of the projected image 16, wherein a mirror can represent one or more picture elements. There are resolutions up to 4160 by 2080 picture elements and thus very high-contrast images in a small area possible. In practice, however, a resolution of 480 by 320 pixels can provide satisfactory results. Of course, even lower resolutions can be chosen, if the specific application permits it.

To generate a colored image, a color wheel 28 is switched in this example in the light path in front of the Mikrospiegelaktuator 22, on the color filter of the primary colors (usually red, green and blue, but sometimes even more) are rotated. In order to achieve better brightness values in the white, the color wheel 28 can also be added to a white sector. With the position of the color filter, the electronics change the partial image, which is reflected by the modulator 22. Due to the rotational speed of the color wheel 28 and the inertia of the human eye, the partial images are added to a colored image impression. High rotational speeds of the color wheel 28 or the provision of multiple color segments ensure a smooth, seamless color representation in the projection. In some embodiments, the color dispersion may also be caused by a dichroic prism.

In another, not shown variant, the color representation can be achieved by decomposition of the projection lamp light by means of dichroic mirrors in the three primary colors red, green and blue and their individual forwarding to three different modulators. The respective partial reflection can then be added to the complete color image, for example in a dichroic prism which contains two crossed dichroic mirrors.

Of course, it is also possible to use individual colored light sources, for example individual light LEDs (red, green, blue), instead of a single white light source.

The Figure 4 shows a simple example of a projected image highlighting one sample location on a sample support over another. The sample carrier in this example has 9 × 9 sample locations in matrix arrangement (columns A to I and rows 1 to 9). The projected image in this case covers the entire area of the slide front. In some embodiments, only partial areas of the sample carrier can serve as the "canvas" of the image. In other variants, the image or image sequence extends beyond the edges of the sample carrier. At the location of the sample location G4, the optical image has a high brightness and / or color contrast to the other sample locations on the sample carrier. A color contrast can be achieved, for example, with the color yellow compared to a faint gray (hatched). For example, a brightness contrast would result if the intensity of white light on the selected sample location G4 is higher (ten times higher in an example) than in the surrounding areas. The image to be projected can be generated automatically by a control system according to the acquired configuration data of the sample carrier. Alternatively, it can also be specified by a user.

With a color coding of the highlighting, a user can also be displayed as to whether a selected sample location with a sample or with which substance a selected sample location is to be assigned in a next method step. Alternatively, a particular color could also indicate the occupancy status of the selected sample site. For example, a bright white could represent a blank sample location, yellow for a microbial sample site, red for a digestion or extractant, and green for a matrix solution. The versatility of the present method has no limits in this regard.

Figures 5A, 5B and 5C show an embodiment in which an image sequence is thrown onto a sample carrier front. The image sequence comprises two pairs of arrows, each arranged perpendicular to one another (each pointing in opposite directions), which point with their tips to a selected sample location D5. In the sequence of images, the arrows from an outermost position with each successive image of the image sequence can continue to move to the location of the sample location D5 until the arrowheads seemingly touch the outer contours of the sample location D5. It is understood that also a single image as in Figure 5C can be used without animation to highlight the sample location D5.

The Figure 6 shows a flow chart representation of a preferred sequence of a method according to the invention: A sample carrier for ionization with matrix-assisted laser desorption with multiple sample locations is provided. It can be to be a MALDI sample carrier that does not need to be transparent. It can be a flat metallic plate or a plate made of a conductive plastic or a doped semiconductor, such as silicon. Furthermore, a Petri dish is provided which contains a flat nutrient medium on which colonies of microorganisms have grown. However, pellets obtained by centrifugation or filtration can serve as sample sources. The Petri dish mentioned here by way of example can be provided with a barcode as identifier, which is read in, for example optically scanned, in an optional method step. In addition or as an alternative, it would also be conceivable (although with a greater outlay) to use an RFID chip as the carrier of an identification tag, which could be read out by radio. The arrangement of the colonies on the nutrient medium can be recorded with a camera and evaluated on the exact positioning of the individual colonies, for example on the XY coordinates of the individual colonies on the flat nutrient medium. With this information, the identification mark of the nutrient medium carrier, in particular the Petri dish, can be supplemented by sample or colony and thus specified in more detail.

Next, a selection criterion - or several selection criteria - can be defined according to which the assignment sequence is to take place. Criteria for the selection can be for example: a selection according to the counting method (for example assignment of every nth [unoccupied] sample location), random selection, consideration of an exclusion list with already prepared sample locations. The sequence in which the sample locations fulfilling the criteria and thus selected can be specified in principle as desired, for example, it can follow a numbering of the sample types in question on the sample carrier from smaller to larger numbers.

An optical image or a sequence of images is then projected onto the sample carrier, which is configured such that the first selected sample location-in one variant, also several sample locations-is highlighted relative to other sample locations. This can now be manually occupied by a specialist. Optionally, an identification tag of the highlighted sample location can be read in between these steps in order to enable later assignment to the sample source location. At the end of the assignment process, the emphasis can be ended, in the case of a video projection, for example, this can be turned off. Alternatively, the projected image can also be changed. Optionally, the identifiers can then be assigned to each other and stored on a suitable storage medium, in particular an electronic memory. If more than one sample site met the selection criteria, all other selected sample locations can now be iteratively processed until none of the selected sample locations is left. It is understood that a further, not explicitly shown criterion for terminating the iterations is that there are no more samples for transfer to the sample carrier.

In summary, the invention proposes: An improved placement aid for manual sample preparation, in particular on level MALDI sample carriers, comprises a receptacle for a sample carrier with a plurality of sample locations, which is adapted to standardized sample carriers for ionization with matrix-assisted laser desorption, a device which a two-dimensional optical image, or image sequence, onto which the specimen-bearing front side of the specimen carrier is thrown, the image or image sequence being arranged such that a selected specimen location, or a group of selected specimen venues, at least opposite thereto highlighting non-selected sample locations in a manner humanely perceivable to a human, an interface for confirming occupancy by hand and / or a device for automatically detecting a manual occupancy procedure, and a guidance system including a sample location, or G group of sample locations, selects and controls the device accordingly. Also disclosed is a method for assisting a manual occupancy of sample locations.

Claims (15)

1. Deposition aid (2, 2*) for manual preparation of samples on a sample support (8) for ionization using matrix-assisted laser desorption, the aid comprising:

   - a base plate (4) containing a holder (6) for a sample support (8) having several sample sites;

   - an arm or carrier (12) which rises on one side of the base plate (4) in an upward direction and on which an imaging device (14) of the type of a video projector is disposed, wherein the imaging device (14) projects a two-dimensional optical image (16) or a corresponding image sequence from the side at a certain angle onto the front side, having the sample sites, of a sample support (8) arranged in the holder (6), and contains various optics that ensure that the image (16) or the image sequence is illustrated without distortion, and wherein the image (16) or the image sequence is designed such that a selected sample site or a group of selected sample sites is highlighted at least with respect to neighbouring not-selected sample sites in a manner which can be perceived by the human eye;

   - an interface for confirming manual deposition and/or a device for automated detection of a manual deposition process; and

   - a control system (10) which selects a sample site or a group of sample sites and correspondingly controls the device (14).
2. Deposition aid according to claim 1, characterized in that the device (14) comprises a spatial light modulator, a liquid crystal projector or a liquid-crystal-on-silicon projector.
3. Deposition aid according to claim 1 or 2, characterized in that the device (14) generates an image (16) or an image sequence, thereby generating a brightness and/or colour contrast at least with respect to neighbouring not-selected sample sites at the selected sample site or group of sample sites.
4. Deposition aid according to any one of the claims 1 to 3, characterized in that the device (14) generates an image sequence which visually highlights a sample site or a group of sample sites.
5. Deposition aid according to any one of the claims 1 to 4, characterized in that the two-dimensional optical image (16) or the image sequence substantially covers the overall front side of the sample support (8).
6. Deposition aid according to any one of the claims 1 to 5, characterized in that the control system (10) has an interface for data input or data output.
7. Deposition aid according to any one of the claims 1 to 6, characterized in that the control system (10) comprises a memory for the assignment and acquisition of identification tags of samples and sample sites.
8. Deposition aid according to any one of the claims 1 to 7, characterized in that adapter pieces are provided in the holder (6) for fitting in standardized sample supports (8) for ionization using matrix-assisted laser desorption.
9. Deposition aid according to any one of the claims 1 to 8, characterized in that the device for automated detection comprises a scattered light sensor (19) which is designed to detect changes in the scattered light behaviour on the front side of a sample support (8) located in the holder, which changes are indicative of a manual deposition process.
10. Deposition aid according to any one of the claims 1 to 9, characterized in that the interface for confirming the deposition comprises a scattered light sensor (19) which is directed to an area (21) in the boundary region of a sample support (8) arranged in the holder, and detects manually produced changes in the scattered light behaviour in this area (21).
11. Deposition aid according to any one of the claims 1 to 10, characterized in that the image (16) or the image sequence is divided into an area that highlights the selected sample site or the selected sample sites, and into an area that displays information to the user.
12. Method for supporting manual preparation of samples on a sample support for ionization using matrix-assisted laser desorption by using a deposition aid according to any one of the claims 1 to 11, in which:

    (a) a sample support with several sample sites is provided in the holder,

    (b) at least one selection criterion is defined according to which a deposition sequence shall be performed,

    (c) an amount of sample sites is selected in accordance with the at least one selection criterion,

    (d) a two-dimensional optical image or a corresponding image sequence is projected by the video projector onto the front side of the sample support having the sample sites, wherein the image or the image sequence is designed such that a selected sample site or a group of selected sample sites is highlighted at least with respect to neighbouring not-selected sample sites in a manner which can be perceived by the human eye,

    (e) a sample is manually deposited on the highlighted sample site, the performed manual deposition is confirmed and/or automatically detected by sensors, and

    (f) if the amount contains further unprocessed sample sites or groups of sample sites, steps (d) and (e) are repeated with the subsequent sample site of the amount, or the subsequent group of sample sites.
13. Method according to claim 12, characterized in that the sample site is selected based on the fact that it is not occupied.
14. Method for manual preparation of a sample on a sample support for ionization using matrix-assisted laser desorption, wherein the sample is provided with an identification tag, in which a sample site, which is also provided with an identification tag, is selected and highlighted in accordance with a method according to claim 12 or 13, the sample is disposed on the selected sample site, and the identification tags are allocated to one another and stored.
15. Method according to claim 14, characterized in that the identification tag of the sample is derived from a tag of the sample container from which the sample is taken.

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