Image projection method and apparatus for supporting manual MALDI sample preparation

Abstract

An improved deposition aid for manual sample preparation, particularly on flat MALDI sample supports, comprises a holder for a sample support with several sample sites, which is adapted to standardized sample supports for ionization with matrix-assisted laser desorption and a device which projects a two-dimensional optical image, or a suitable sequence of images, onto the sample sites. The image, or sequence of images, is constructed such that a selected sample site or group of selected sample sites is highlighted in a way which can be perceived by the human eye, at least with respect to neighboring, not-selected sample sites. The deposition aid also includes an interface for confirming the manual deposition and/or a device for the automatic detection of a manual deposition process; and a guidance system which selects a sample site or group of sample sites, and controls the device accordingly.

Claims

1. A method for assisting manual preparation of samples on a sample support for subsequent ionization via matrix-assisted laser desorption, comprising: (a) providing a sample support having several sample sites; (b) defining at least one selection criterion according to which a deposition sequence is to be conducted; (c) selecting a sample site according to the at least one selection criterion; (d) projecting onto the sample sites from above at a non-orthogonal angle relative to a plane of the sample support at least one two-dimensional optical image, having a resolution of between 480 by 320 pixels and 4160 by 2080 pixels, that is constructed so that the selected sample site is highlighted with respect to neighboring, not-selected sample sites in a manner which can be perceived by the human eye, wherein the two-dimensional optical image is conditioned by optics, including at least one of a lens and mirror, such that the two-dimensional optical image is displayed without distortion; (e) manually depositing a sample on the highlighted sample site; (f) performing at least one of manually confirming the completed deposition and automatically detecting the completed deposition by a sensor; and (g) when unprocessed sample sites remain, repeating the steps (d) to (f) with another sample site selected according to the at least one selection criterion.

2. The method of claim 1, wherein in step (c) a sample site is selected when not containing a sample.

3. The method of claim 1, further comprising providing a sample with a sample identification tag, providing the selected sample site with a sample site identification tag, and assigning the sample identification tag and the site identification tag to each other and storing the assignment in a memory.

4. The method of claim 3, wherein the sample identification tag is read-in from a labeling of a sample vessel from which the sample originates.

5. A method for assisting manual preparation of samples on a sample support for subsequent ionization via matrix-assisted laser desorption, comprising: (a) providing a sample support having several sample sites; (b) defining at least one selection criterion according to which a deposition sequence is to be conducted; (c) selecting a sample site according to the at least one selection criterion; (d) projecting onto the sample sites from above at a non-orthogonal angle relative to a plane of the sample support a time sequence of non-identical two-dimensional optical images, which generates the impression of an animated image, that is constructed so that the selected sample site is highlighted with respect to neighboring, not-selected sample sites in a manner which can be perceived by the human eye, wherein the time sequence of non-identical two-dimensional optical images is conditioned by optics, including at least one of a lens and mirror, such that the time sequence of two-dimensional optical images is displayed without distortion; (e) manually depositing a sample on the highlighted sample site; (f) performing at least one of manually confirming the completed deposition and automatically detecting the completed deposition by a sensor; and (g) when unprocessed sample sites remain, repeating the steps (d) to (f) with another sample site selected according to the at least one selection criterion.

6. The method of claim 1, wherein the at least one two-dimensional optical image in step (d) is generated by one of a spatial light modulator, a liquid crystal projector and a liquid-crystal-on-silicon projector.

7. The method of claim 1, wherein the at least one two-dimensional optical image in step (d) comprises at least one of a brightness contrast and a color contrast at the selected sample site in order to highlight the selected sample site.

8. The method of claim 7, wherein the selected sample site is highlighted using signal colors.

9. A method for assisting manual preparation of samples on a sample support for subsequent ionization via matrix-assisted laser desorption, comprising: (a) providing a sample support having several sample sites; (b) defining at least one selection criterion according to which a deposition sequence is to be conducted; (c) selecting a sample site according to the at least one selection criterion; (d) projecting onto the sample sites from above at a non-orthogonal angle relative to a plane of the sample support at least one two-dimensional optical image that is constructed so that the selected sample site is highlighted with respect to neighboring, not-selected sample sites in a manner which can be perceived by the human eye, wherein the two-dimensional optical image is conditioned by optics, including at least one of a lens and mirror, such that the two-dimensional optical image is displayed without distortion, wherein the said projecting generates a time sequence of images which generates an impression of an animated image and highlights a group of sample sites with respect to neighboring, not-selected sample sites in a manner which can be perceived by the human eye; (e) manually depositing a sample on the highlighted sample site; (f) performing at least one of manually confirming the completed deposition and automatically detecting the completed deposition by a sensor; and (g) when unprocessed sample sites remain, repeating the steps (d) to (f) with another sample site selected according to the at least one selection criterion.

10. The method of claim 1 wherein the at least one two-dimensional optical image is sized to cover all of the sample sites simultaneously.

11. The method of claim 1, wherein the sensor in step (f) comprises a scattered light sensor that detects changes in scattered light behavior on the sample support, which changes are indicative of a manual deposition process.

12. The method of claim 11, wherein the scattered light sensor comprises a charge coupled device (CCD).

13. A method for assisting manual preparation of samples on a sample support for subsequent ionization via matrix-assisted laser desorption, comprising: (a) providing a sample support having several sample sites; (b) defining at least one selection criterion according to which a deposition sequence is to be conducted; (c) selecting a sample site according to the at least one selection criterion; (d) projecting onto the sample sites from above at a non-orthogonal angle relative to a plane of the sample support at least one two-dimensional optical image that is constructed so that the selected sample site is highlighted with respect to neighboring, not-selected sample sites in a manner which can be perceived by the human eye, wherein the two-dimensional optical image is conditioned by optics, including at least one of a lens and mirror, such that the two-dimensional optical image is displayed without distortion; (e) manually depositing a sample on the highlighted sample site; (f) automatically detecting the completed deposition by a sensor comprising a scattered light sensor, wherein one of a peripheral area on the sample support not comprising any sample sites and a peripheral area on a holder for the sample support is assigned for the deposition detection in that said assigned area is monitored with the scattered light sensor for manually produced changes in scattered light; and (g) when unprocessed sample sites remain, repeating the steps (d) to (f) with another sample site selected according to the at least one selection criterion.

14. The method of claim 1, wherein the sensor in step (f) comprises a camera with image recognition function.

15. A method for assisting manual preparation of samples on a sample support for subsequent ionization via matrix-assisted laser desorption, comprising: (a) providing a sample support having several sample sites; (b) defining at least one selection criterion according to which a deposition sequence is to be conducted; (c) selecting a sample site according to the at least one selection criterion; (d) projecting onto the sample sites from above at a non-orthogonal angle relative to a plane of the sample support at least one two-dimensional optical image that is constructed so that the selected sample site is highlighted with respect to neighboring, not-selected sample sites in a manner which can be perceived by the human eye, wherein the two-dimensional optical image is conditioned by optics, including at least one of a lens and mirror, such that the two-dimensional optical image is displayed without distortion, wherein projecting the at least one two-dimensional image comprising projecting an image that is divided into an area which highlights a selected sample site and an area which displays information to a user; (e) manually depositing a sample on the highlighted sample site; (f) performing at least one of manually confirming the completed deposition and automatically detecting the completed deposition by a sensor; and (g) when unprocessed sample sites remain, repeating the steps (d) to (f) with another sample site selected according to the at least one selection criterion.

16. The method of claim 1, wherein the steps (d) to (f) are repeated with subsequently depositing different types of samples.

17. The method of claim 16, wherein the different sample types are one of a microbial sample, a digestion or extraction substance, and a matrix solution.

18. A method for assisting manual preparation of samples on a sample support for subsequent ionization via matrix-assisted laser desorption, comprising: (a) providing a sample support having several sample sites; (b) defining at least one selection criterion according to which a deposition sequence is to be conducted; (c) selecting a sample site according to the at least one selection criterion; (d) projecting onto the sample sites from above at a non-orthogonal angle relative to a plane of the sample support at least one two-dimensional optical image, wherein the image comprises at least one arrow that points toward the selected sample site, that is constructed so that the selected sample site is highlighted with respect to neighboring, not-selected sample sites in a manner which can be perceived by the human eye, wherein the two-dimensional optical image is conditioned by optics, including at least one of a lens and mirror, such that the two-dimensional optical image is displayed without distortion; (e) manually depositing a sample on the highlighted sample site; (f) performing at least one of manually confirming the completed deposition and automatically detecting the completed deposition by a sensor; and (g) when unprocessed sample sites remain, repeating the steps (d) to (f) with another sample site selected according to the at least one selection criterion.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) In the following, the invention is described with the aid of example embodiments in conjunction with the attached drawings. The drawings comprise:

(2) FIG. 1A-C illustrating a schematic design of a deposition aid according to principles of the invention;

(3) FIG. 2 illustrating a more detailed (schematic) representation of a projection method;

(4) FIG. 3 illustrating an example of a projected image;

(5) FIG. 4A-C illustrating an example of a projected sequence of images; and

(6) FIG. 5 illustrating a flowchart representing methods according to principles of the invention.

DETAILED DESCRIPTION

(7) FIG. 1A is a schematic representation of the design of a deposition aid 2 according to the principles of the invention. A base plate 4 contains a holder 6, whose internal dimensions are preferably adapted to the standardized external dimensions of an LDI sample support 8 (in particular a MALDI sample support). In certain cases, adapter pieces (not shown here) can be used to adjust the holder 6 to a required spatial configuration.

(8) In FIG. 1A, a sample support 8 is located in the holder 6. A sensor (not shown), can be integrated in the base and/or side area of the holder to detect the presence of a sample support and transmit an appropriate information signal to a guidance system 10, for example, a microprocessor integrated into the design. The sensor can consist of a simple pushbutton, for example, which is activated when the sample support 8 is inserted into the holder 6. Other, particularly non-contact sensor versions (ultrasonic proximity sensor, light barrier, . . . ) are also conceivable, however.

(9) A holder in the deposition aid can also take the form of a frame (not shown). A frame which fixes the sample support at the narrow sides has the advantage that both the front and back of the sample support are accessible to measuring and inspection instruments (possibly a sensor arrangement). This facilitates the handling of the deposition aid, particularly if it is portable.

(10) On one side of the base plate 4 is a vertical arm or support 12 on which an imaging device 14 is located. The imaging device 14 can be designed like a video projector, for example, as is explained further below. The imaging device 14 is positioned and aligned in such a way that it can project a two-dimensional visual image 16, or a suitable sequence of images, onto the front of a sample support 8 which is located in the holder 6. The imaging device 14 communicates with the guidance system 10 and is controlled by it, for example in order to specify which image is to be projected so as to highlight a sample site or group of sample sites. The imaging device 14 preferably contains a range of optics which ensures that the image, or sequence of images, is displayed without any distortion in spite of being projected onto the front of the sample support sideways at a certain angle.

(11) The imaging device 14 is positioned in such a way that a user can transfer a microbiological sample, for example cells from a microbial colony cultured on an agar plate, to a sample site on the sample support 8 with an inoculation instrument 18 or similar transfer device largely unhindered.

(12) It is possible for the guidance system 10 to have an interface (not shown) with which a user can manually confirm that a sample site has been manually deposited. The term manually confirm is here to be understood in a broad sense and may also comprise the input of identification data of the next sample to be prepared, for example by scanning a bar code on an agar plate.

(13) Also not shown here is a variant where the guidance system 10 is equipped with a sensor arrangement for the automated detection of deposition processes, and thus the completion of a sample site deposition is automatically recognized and reported to the guidance system 10. The automated detection can, of course, also include the detection of erroneous deposition, that is, if a sample has been deposited on a different sample site to the one intended.

(14) Examples for such a sensor arrangement are described in the international patent application WO 2012/072467 A2 assigned to Bruker Daltonik GmbH, which is hereby incorporated by reference in its entirety into the present disclosure. For example, the quantity of sample at a sample site can be probed, or the deposition state of the sample site can be determined, by means of a change in at least one of the following chemophysical properties: resonance frequency of a piezoelectric material, density, geometrical dimension, propagation time of ultrasonic or electromagnetic waves, electrical capacitance, electrical resistance, inductance, permittivity, magnetizability, light scattering, light absorption, light reflection or luminescence. Variants with light barrier beams which intersect above the sample sites, thereby forming a monitoring grid, are also conceivable.

(15) A further telecommunication connection to the sample support 8 can be provided to enable the guidance system 10 to acquire certain configuration data of the sample support 8, such as the number, arrangement and position of the individual sample sites. In one example, a microchip which is mounted on the sample support 8 and which contains the appropriate configuration data can be read out. As an alternative, the guidance system 10 can also be equipped with a camera and an optical image recognition system (not shown here), or can communicate with these; the camera images the front of the sample support 8 so that detectable features of the sample sites can be located for the depositing of sample material. These detectable features can take the form of markings, for example circular outlines, on the front of the sample support.

(16) Communication with the device 14 also allows the guidance system 10 in this example to (de-)activate a video projector in order to generate an optical image on the front of the sample support, to change the image, and to select different image formats where necessary. The acquisition of the configuration data, the selection of an image (or sequence of images) as well as the (de-)activation of the projector can also be done manually via an interface in some example embodiments.

(17) In a semi-automatic embodiment, a user of the deposition aid can input the deposition state of the sample support 8 into the guidance system 10, via an interface, for example. At the same time, the user can specify the criterion according to which the sample sites are to be selected. This can be an empty state, for example. The guidance system 10 then checks which of the sample sites is suitable for deposition, selects one of them (or possibly a group) in order to highlight the appropriate sample site, selects the image to be projected accordingly, or generates it, and activates the video projector. An image, or sequence of images, is then projected onto the front of the sample support 8, where a sample site, and possibly the surrounding area on the front of the sample support, is highlighted in a way visible to the human eye with respect to the other areas of the sample support with not-selected sample sites.

(18) The highlighting effect can be amplified by designing the sample support material so that it enhances the visual effect, for example by incorporating particles which glitter or create a color effect when illuminated into the material of the sample support 8. A type of bright primer with white particles can be useful in order to make color differences in the different pixels stand out better.

(19) Supported by this highlighting, the user can deposit the sample onto the correct sample site, and then manually confirm that deposition has taken place via the interface, for example. This can then lead to the deactivation of the highlighting, that is, in this example to the projection being switched off, or to the image shown being changed. In other embodiments, a sensor arrangement for the automatic detection of manual deposition processes can be used.

(20) The front of the sample support can be given an antiglare coating so that users are not irritated as they work. This can prevent dazzling light reflections which could occur as the image or sequence of images is projected. However, the risk of dazzling when a projector is used for generating an image on the sample support is essentially small, in contrast to bundled light beams.

(21) The guidance system 10 can be provided with a memory (not shown) for the assignment and acquisition of identification tags of samples and sample sites. If required, this information can be entered by a user via the interface or it can be read in; alternatively via automated data transmission.

(22) According to a further embodiment, it is also possible for a scattered light sensor 19 to be installed on the support 12 (as indicated in FIG. 1B); this sensor monitors the front of the sample support 8 with spatial resolution in order to detect changes in the scattered light behavior and to assign these changes to an area on the sample support, for example a sample site. The spatial resolution can be achieved with a charge-coupled device (CCD) and appropriate upstream optics, for example. The light which is scattered on the surface of the sample support 8 and then detected can originate from the projector of the imaging device 14 or from a separate light source (not shown). The scattered light sensor 19 can also measure the integrated (not spatially resolved) scattered light which originates from a sample site if the sample site is illuminated individually by the imaging device 14 or the separate light source.

(23) Moreover, a specific area 21 (FIG. 1C) can be identified on the sample support 8 as confirmation of a completed deposition process. After the sample has been deposited, the user can swipe the inoculation instrument across area 21 and thus trigger a scattered light pulse which indicates the conclusion of a deposition process and thus leads to the continuation of a deposition sequence. This is an example of an interface for confirming a deposition. Of course, in order to avoid unnecessary erroneous signals, the area 21 should be located on a side of the sample support from which a user does not access the sample sites. In alternative embodiments, the area can be arranged on part of the periphery of the holder, not on the sample support itself.

(24) FIG. 2 shows in somewhat more detail an example embodiment of a deposition aid 2* according to principles of the invention.

(25) In this example, the highlighting device has a spatial light modulator, which is located in a housing 20. The housing 20 is supported by a support or holder (not shown here in order to simplify the illustration). Spatial light modulators are only one example of a video projection technique. Liquid crystal projectors or liquid crystal on silicon projectors can also be used. Such projectors have the advantage that they can generate a very flexible image 16, or a very versatile sequence of images, on the sample support 8. There are virtually no limits to the design of the image 16 in terms of the color selection for the individual pixels, brightness and/or image sequence.

(26) Schematically represented in the housing 20 is a micromirror actuator 22, onto which light is projected by a suitable projection lamp 24 via imaging optics 26A. The image passes from the micromirror 22 via further imaging optics 26B onto the front of a sample support 8. Micromirror actuators 22 can be accommodated in very large numbers on a small space such as a microchip. The angle of each micromirror 22 can be changed individually, and each micromirror usually has two stable final states between which it can change with a frequency of several kilohertz. The brightness of a pixel can be set with the aid of the switching frequency. The number of mirrors corresponds to the resolution of the projected image 16, where one mirror can represent one or more pixels. Resolutions of up to 4160 by 2080 pixels, and thus very high-contrast images, are possible on a small area. In practice, however, a resolution of 480 by 320 pixels can also provide satisfactory results. It is, of course, possible to select even lower resolutions if the particular application allows this.

(27) In order to generate a colored image, in this example a color wheel 28, on which filters of the primary colors (usually red, green and blue, but sometimes others also) are rotated, is inserted into the light path in front of the micromirror actuator 22. In order to achieve better brightness values for white, a white segment can also be added to the color wheel 28. According to the position of the color filter, the electronics change the partial image which is reflected by the modulator 22. The rotational speed of the color wheel 28 and the inertia of the human eye mean that the partial images are added together to give the impression of a colored image. A smooth, transitionless color representation in the projection is ensured by the color wheel 28 rotating at high speeds or by providing several color segments. In several embodiments, the color dispersion can also be brought about by a dichroic prism.

(28) In another variant (not shown) the color representation is achieved by splitting the light of the projection lamp into the three primary colors red, green and blue by means of dichroic mirrors, and transmitting them individually to three different modulators. The respective partial reflections can then be added together in a dichroic prism, which contains two crossed dichroic mirrors, to form a complete color image again, for example.

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

(30) FIG. 3 shows a simple example of a projected image which highlights one sample site on a sample support with respect to others. In this example, the sample support has 99 sample sites in a matrix arrangement (columns A to I and rows 1 to 9). The projected image covers the whole area of the front of the sample support in this case. In some embodiments, only partial areas of the sample support may act as the projection screen for the image. In other variants, the image or sequence of images extends beyond the edges of the sample support. At the location of the sample site G4, the optical image has a high brightness and/or color contrast compared to the other sample sites on the sample support. A color contrast can be achieved by using yellow against light gray (hatched), for example. A brightness contrast would result, for example, if the intensity of white light on the selected sample site G4 is higher (in one example ten times higher) than in the surrounding areas. The image to be projected can be generated autonomously by a guidance system in accordance with the acquired configuration data of the sample support. Alternatively, it can be specified by a user.

(31) By color coding the highlighting, it is possible to indicate to a user whether a selected sample site is to be deposited with a sample, or with which substance a selected sample site is to be deposited, in a next deposition step. Alternatively, a specific color could indicate the deposition state of the selected sample site. A bright white could represent an empty sample site, for example, yellow a sample site which is deposited with a microbial sample, red a digestion or extraction substance, and green a matrix solution. There are virtually no limits to the variability of the present method in this respect.

(32) FIGS. 4A, 4B and 4C show an example embodiment where a sequence of images is projected onto the front of a sample support. The image sequence comprises two pairs of opposing arrows, perpendicular to each other, with the tips of all the arrows pointing to a selected sample site D5. In the image sequence, the arrows can move inwards, nearer and nearer to the location of the sample site D5, with each subsequent image of the image sequence, until the arrow tips appear to touch the external outlines of the sample site D5. Of course, it is also possible to use a single image, such as in FIG. 4C, without any animation to highlight the sample site D5.

(33) FIG. 5 shows an exemplary sequence of steps of a method according to the invention as a flow diagram: a sample support for ionization with matrix-assisted laser desorption with several sample sites is provided. This can be a MALDI sample support, which does not need to be transparent. It can be a flat metal plate or a plate made of a conductive plastic or a doped semiconductor, such as silicon. Moreover, a Petri dish is provided which contains a flat nutrient medium, on which colonies of microorganisms have been cultured. Pellets obtained by centrifugation or filtration can also serve as sources of samples. The Petri dish mentioned here by way of example can be equipped with a barcode as an identification tag, which is read in with an optional method step, by optical scanning, for example. Additionally or alternatively, an RFID chip carrying an identification tag, which could be read out via wireless communication, would be a possibility (albeit being more complex/costly). The arrangement of the colonies on the nutrient medium can be photographed with a camera and evaluated with regard to the exact positioning of the individual colonies, using the XY-coordinates of the individual colonies on the flat nutrient medium, for example. With this information, the identification tag of the nutrient medium carrier, particularly the Petri dish, can be supplemented sample-by-sample or colony-by-colony, and thus specified in more detail.

(34) Next, a selection criterion or criteria can be defined, according to which the deposition sequence is to be carried out. Possible criteria for the selection can be, for example: a selection according to the numbering (for example deposition of every nth [empty] sample site), random selection, or selection using an exclusion list of already prepared sample sites. The sequence of deposition in the sample sites which fulfill the criteria and are therefore selected can, in principle, be specified at will. For example, it can follow a sequential numbering of the relevant sample sites on the sample support from lower numbers to higher numbers.

(35) An optical image, or sequence of images, is now projected onto the sample support in such a way that the first selected sample siteor in another variant, several sample sitesis highlighted with respect to other sample sites. The selected site(s) can now be deposited manually by a technician. Optionally, an identification tag of the highlighted sample site can be entered between these steps in order to allow subsequent tracing back to the sample's site of origin. At the conclusion of the deposition process, the highlighting can be ended; in the case of a video projection, this can be switched off, for example. Alternatively, the image projected can be changed. Optionally, the identification tags can then be assigned to each other and stored on a suitable storage medium, particularly an electronic memory. If more than one sample site fulfills the selection criteria, it is now possible to iteratively process all the other selected sample sites until none of the selected sample sites remains. It goes without saying that a further, not explicitly stated, criterion for the termination of the iteration consists in there being no more samples to be transferred to the sample support.

(36) While the invention has been shown and described with reference to a number of embodiments thereof, it will be recognized by those skilled in the art that various changes in form and detail may be made herein without departing from the spirit and scope of the invention as defined by the appended claims.