WO2015104419A1 - Procédé d'analyse d'un échantillon au moyen d'un microscope - Google Patents

Procédé d'analyse d'un échantillon au moyen d'un microscope Download PDF

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Publication number
WO2015104419A1
WO2015104419A1 PCT/EP2015/050447 EP2015050447W WO2015104419A1 WO 2015104419 A1 WO2015104419 A1 WO 2015104419A1 EP 2015050447 W EP2015050447 W EP 2015050447W WO 2015104419 A1 WO2015104419 A1 WO 2015104419A1
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WO
WIPO (PCT)
Prior art keywords
sample
carrier elements
identification
microscope
slide
Prior art date
Application number
PCT/EP2015/050447
Other languages
German (de)
English (en)
Inventor
Falk Schlaudraff
Christian Woditschka
Original Assignee
Leica Microsystems Cms Gmbh
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Leica Microsystems Cms Gmbh filed Critical Leica Microsystems Cms Gmbh
Publication of WO2015104419A1 publication Critical patent/WO2015104419A1/fr

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/286—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q involving mechanical work, e.g. chopping, disintegrating, compacting, homogenising
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N35/00584—Control arrangements for automatic analysers
    • G01N35/00722—Communications; Identification
    • G01N35/00732—Identification of carriers, materials or components in automatic analysers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/286—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q involving mechanical work, e.g. chopping, disintegrating, compacting, homogenising
    • G01N2001/2873—Cutting or cleaving
    • G01N2001/2886—Laser cutting, e.g. tissue catapult
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N35/00584—Control arrangements for automatic analysers
    • G01N35/00722—Communications; Identification
    • G01N35/00732—Identification of carriers, materials or components in automatic analysers
    • G01N2035/00792—Type of components bearing the codes, other than sample carriers
    • G01N2035/00801—Holders for sample carriers, e.g. trays, caroussel, racks
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N35/00584—Control arrangements for automatic analysers
    • G01N35/00722—Communications; Identification
    • G01N35/00732—Identification of carriers, materials or components in automatic analysers
    • G01N2035/00821—Identification of carriers, materials or components in automatic analysers nature of coded information
    • G01N2035/00831—Identification of carriers, materials or components in automatic analysers nature of coded information identification of the sample, e.g. patient identity, place of sampling

Definitions

  • the present invention relates to a method for analyzing a sample with a microscope, and to a microscope for analyzing a sample.
  • sample carrier elements In order to analyze or process biological samples or preparations with a microscope, different preparation carrier elements are used.
  • the sample is applied in particular to a slide.
  • the slide can be placed in the microscope and the sample analyzed.
  • Several such slides can in turn be applied to a sample holder.
  • certain cells, tissue areas or the like of the sample can be selected and cut out or "dissected out” for further analysis with the aid of a focused laser beam as a so-called dissectate.
  • the sample is usually on one
  • the dissected out drops under the influence of gravity in a suitable container.
  • a method for laser microdissection is known, for example, from DE 10 2004 051 508 A1.
  • a desired cutting line of an object, which is to be cut out of the sample determined based on the object contour and the object is cut along this desired cutting line by means of a relative movement between a laser beam and the sample.
  • the slide carrier elements can also be marked digitally.
  • an object carrier device for receiving an object to be examined by a microscope or to be processed with a laboratory analysis system is known.
  • the slide has a radio frequency identification (RFID) tag. On this a the slide and / or the object identifying information is stored.
  • RFID tags are to overcome the disadvantage of limited and non-changeable amounts of data that can be stored on a barcode.
  • a sample holder is known, which is connected to a transponder, which in turn is readable and writable. As a result, information about the samples on the sample holder can be stored and read out.
  • preparation carrier elements Although an identification of preparation carrier elements is possible by means of these methods, the preparation carrier elements must be actively marked thereby, for example by manual labeling or by storing information on the RFID tag. A (partially) automatic or fully automatic assignment of different preparation carrier elements to a sample is not or at least hardly possible.
  • preparation carrier elements which are used to analyze the sample, each provided with an identification mark.
  • Identification labels of the individual preparation carrier elements are detected by a detection device.
  • memory entries are detected in a computing unit. These memory entries are in each case assigned to the individual preparation carrier elements.
  • the memory entries in the computing unit are linked together. Memory entries belonging to the same sample (and thus preparation carrier elements) are assigned to one another and can be identified automatically.
  • preparation carrier elements is to be understood as meaning elements on which the sample or parts of the sample can be applied or introduced.
  • preparation carrier elements are slides, Petri dishes, multiwell containers or collecting containers for microdissection.
  • the invention makes possible a (partially) automatic, in particular fully automatic, assignment of the preparation carrier elements to the sample and to one another. If the sample is to be analyzed and if an identification tag of a specimen carrier element is detected, memory entries which are assigned to this specimen carrier element are automatically assigned in the arithmetic unit to the sample to be analyzed or linked thereto. All memory entries which are assigned to corresponding preparation carrier elements whose identification markings are detected during the analysis of the particular sample are linked together (in particular automatically). Thus, in particular all the preparation carrier elements which are used for the analysis of the particular sample are linked to each other and thus to the sample. This linkage is in particular fully automatic in the arithmetic unit, without intervention or intervention of a user.
  • every possible preparation carrier element that can be used for analyzing the sample is stored a priori in the arithmetic unit.
  • At least one memory entry is stored in the arithmetic unit for each possible carrier element.
  • This memory entry contains in particular useful information about the specimen carrier element.
  • the preparation carrier elements already have one a priori Identification mark on.
  • the individual preparation carrier elements are uniquely characterized or identified by the respective identifier.
  • This identification marking is also stored in particular in the corresponding memory entry assigned to the preparation carrier element or points (in the form of a pointer) to the memory entry.
  • each specimen carrier element can thus be unambiguously assigned to its corresponding memory entry.
  • the individual memory entries of all specimen carrier elements whose identification markings are detected in the course of the analysis can be automatically linked to one another and thus, for example, matching specimen carrier elements or specimen carrier elements that are suitable for a particular specimen can be determined or identified.
  • the sample is applied to a plurality of slides or several parts of the sample are applied to different slides.
  • the invention can be deposited in a simple manner that these slides belong to the same sample or the same patient.
  • Such preparation carrier elements can also be sample holders, on each of which several slides are applied.
  • both the sample holder and the individual slides each have an identification tag.
  • all slides on the sample holder and the sample holder can be linked to each other via their memory entries in the arithmetic unit. Different slides on the sample holder may belong to the same or different samples. The invention thus makes it possible to automatically deposit in a simple manner which slides belong to which sample.
  • the invention is suitable for a microscope with a slide scanner.
  • a plurality of preparation carrier elements especially on slides, are introduced.
  • up to 400 slides can be introduced in such a slide scanner.
  • These preparation carrier elements are automatically loaded one after the other into the microscope and analyzed.
  • the identification marking of each individual preparation carrier element can be detected in a simple manner when this preparation carrier element is loaded into the microscope.
  • this preparation carrier element is linked to the respective sample or all preparation carrier elements with which the same sample (or different parts of the same sample) are analyzed are linked to one another via the corresponding memory entries. Subsequently, the sample is automatically analyzed.
  • a fully automatic analysis of the sample is possible.
  • the preparation carrier elements can already be provided in a manufacturing process with the respective identification marking.
  • the associated memory entries of the individual preparation carrier elements can be supplied, for example, with the preparation carrier elements on a corresponding data carrier.
  • the data carrier for example CD, DVD, USB stick, flash memory
  • the data carrier can be connected to the corresponding arithmetic unit and the memory entries can be transferred to the arithmetic unit from the data carrier.
  • the specimen carrier elements are provided with the corresponding identification markings.
  • the arithmetic unit is designed in particular as a part of the microscope.
  • the arithmetic unit can be designed as a module which can be connected to the microscope.
  • the arithmetic unit is designed as part of a computer or control unit.
  • the arithmetic unit comprises a suitable database.
  • the corresponding memory entries of the individual specimen carrier elements are stored in this database as a function of their identification markings.
  • the analysis of the sample is carried out in particular by means of a control device and / or a control software for controlling the microscope.
  • This control unit or this control software is in particular with the computing unit in communication.
  • the control software can be executed in particular also on the arithmetic unit itself.
  • the arithmetic unit is used in particular for the analysis of the sample. For example, by means of the arithmetic unit an enlarged image of the sample or a region of the sample which is generated by the microscope can be displayed and / or stored as an image or video file. This enlarged image is forwarded in particular by the control unit or the control software for controlling the microscope to the arithmetic unit.
  • the microscope has at least one expedient detection device for detecting the identification markings.
  • this detection device is connected to the arithmetic unit and / or the control unit or the control software or can communicate with the same. If the detection device detects a specific identification tag, the control device or the control software is in particular provided with the respective allocated memory entries.
  • a laser microdissection of the sample is carried out in the course of the analysis of the sample.
  • the microscope has a suitable laser microdissection device.
  • the microscope and the laser microdissection device together form a laser microdissection system.
  • the laser microdissection device has a device for generating a laser beam, for example an infrared or ultraviolet laser beam. From the sample, a dissectate can be isolated by means of this laser beam, which preferably falls under the influence of gravity into a suitable collecting container.
  • the microscope has an incident light device in the beam path of which the laser beam is coupled.
  • the laser beam is focused by the respective microscope objective used on the sample, which rests for example on a motor-automatically movable microscope stage.
  • a cutting line is produced, for example, by moving the microscope stage during cutting in order to move the sample relative to the stationary laser beam.
  • the laser microdissection device can also have a laser deflection or laser scanning device, which is set up to move the laser beam or its point of impingement over a stationary sample.
  • a slide is advantageously used as a first slide carrier element for analyzing the sample.
  • the slide is provided with a first identification mark.
  • a collection container is used as a second sample carrier member for analyzing the sample and is provided with a second identification tag.
  • the first identifier of the slide and the second identifier of the collection container are detected. Based on the detected first identifier of the slide a memory entry, which is associated with the slide, detected in the arithmetic unit, so found and read. Based on the detected second identifier of the collection container, a memory entry, which is associated with the collecting container, detected in the arithmetic unit.
  • the storage entries of the slide and the collection container are linked together. In this way, the correct collection container for a slide can be automatically determined and used for analysis.
  • a selected object is cut out of the sample by means of the laser microdissection and transported as a dissectate into the collecting container.
  • All the preparation carrier elements which are used in the course of the laser microdissection of the sample are linked to one another in the arithmetic unit.
  • the dissectate in the corresponding collecting container belongs to the sample on the corresponding microscope slide.
  • each individual of these combined preparation carrier elements can also be provided with its own identification mark.
  • a plurality of microscope slides are preferably combined to form a sample holder as a higher-level preparation carrier element and provided with a first, higher-order identifier.
  • a plurality of collecting containers are combined to form an overall holder as a higher-level preparation carrier element and provided with a second, upper-level identification marking.
  • an automatic or fully automatic laser microdissection can be carried out in a simple manner, both from one and several different samples.
  • a method for (automatic) laser microdissection is known, for example, from DE 10 2004 051 508 A1 by the same Applicant.
  • a desired cutting line of an object which is to be cut out of a preparation or a sample is marked and cut out by means of a relative movement between a laser beam and the sample.
  • An electronic image of at least one image section of the sample is taken, the image section is processed by image analysis, wherein at least one object to be cut out is automatically determined.
  • the desired cutting line around the at least one object to be cut out is automatically determined.
  • the automatically determined desired cutting line is automatically imaged by a mathematical transformation on a laser cutting line and this laser cutting line is converted into a relative movement between the laser beam and the preparation, whereby a laser cut is produced.
  • the present invention makes it possible to carry out such an automatic laser microdissection on any number of samples fully automatically.
  • object carriers and collecting containers as well as optionally further preparation carrier elements which are used for the laser microdissection of a special sample are linked to this.
  • the arithmetic unit or a suitable control device can in particular carry out a method for (automatic) laser microdissection analogous to DE 10 2004 051 508 A1. Subsequently (especially automatically) new slides, collection containers, etc. are introduced, which belong to another sample. According to the invention, these new preparation carrier elements are linked to the new sample. An automatic laser microdissection of this new sample can then be performed.
  • the invention is also suitable for a laser Mikrodissetationsvorraum which is operated independently of collecting containers.
  • a sample catcher can be used for a laser microdissection device, for example a capillary catcher.
  • a capillary curtain is described, for example, in DE 10 2013 209 455 A1 of the same Applicant.
  • a collection chamber for receiving a dissectate communicates with a capillary line for transporting the dissectate from the collection area. After this forwarding, the dissectate can be sent for analysis.
  • the invention makes it possible to automatically operate such a laser microdissection device with a sample catcher, in particular a capillary catcher.
  • the capillary catcher and attached equipment or -ports or a device for controlling this capillary can be in particular with the arithmetic unit in communication or in communication.
  • the memory entries which are assigned to the individual preparation carrier elements preferably each contain a dimension, a position, coordinates and / or a dimension of the respective preparation carrier element and / or the sample itself.
  • the memory entries thus contain, in particular, information which the particular preparation carrier element and / or the Identify sample or describe its physical extent.
  • the memory entries contain data or information relating to the type and dimensions of the slide, the sample holder, the collection container and / or the total holder.
  • the memory entries contain position data of the sample or the sample surface on the respective slide and optionally on the respective sample holder.
  • the memory entries contain certain control steps or program steps according to which the analysis or the laser microdissection of the sample is carried out.
  • the sample can be automatically aligned relative to the microscope in a desired convenient manner and the analysis or laser microdissection can be performed automatically according to the stored control steps or program steps.
  • the memory entries that are assigned to the parent preparation carrier elements each contain a position and / or number of identification labels on the respective subordinate preparation carrier element.
  • the specimen carrier element is designed, for example, as a larger unit, which is composed of several specimen carrier elements (in particular a specimen holder comprising a plurality of specimen slides or a total holder comprising a plurality of trapping containers)
  • the memory entries may contain in particular at which position or at which coordinates the identification markings of the respective ones Carrier elements are located or at which position the individual specimen carrier elements or the samples thereon are or which dimensions they have.
  • the memory entries of the individual preparation carrier elements in the arithmetic unit each contain a storage location at which analysis data, which are acquired during the analysis of the sample, are stored.
  • this analysis data is used to document the analysis.
  • these analysis data may also contain results of the analysis.
  • these stored analysis data are uniquely assigned to the corresponding sample.
  • all analysis data can be stored to allow for later evaluation or to be able to trace the analysis at a later date.
  • the identification tag the analysis data can be uniquely assigned to the respective sample and clearly traced.
  • images and / or videos which are acquired during the analysis of the sample are stored as analysis data at the storage location.
  • images of the sample which are generated by the microscope, stored as an image or video.
  • the process of cutting can be stored in particular as a video.
  • images before and after the laser microdissection can be stored as analysis data.
  • a date and / or time is stored as an analysis file, in particular for which the analysis was or was started or ended.
  • parameter values are stored as an analysis file. These parameter values are adjusted in particular during the analysis of the data on the microscope or the laser microdissection device.
  • Such parameter values may be, for example, set values with respect to an illumination light of the microscope, with respect to a camera and / or with respect to the laser beam of the laser microdissection device (intensity, wavelength).
  • Such parameter values can be used in particular for a later evaluation or in order to be able to follow the analysis at a later time.
  • the storage location at which the analysis data is stored is identical for all slide support elements used to analyze the sample. This ensures that the same analysis data is not stored separately for all specimen carrier elements, which would unnecessarily require storage space. Furthermore, it is thus ensured that all analysis data of a sample are stored in the same storage location.
  • This analysis data of a particular sample can be linked to all the memory entries of the individual sample carrier elements used to analyze that particular sample.
  • the preparation carrier elements which are used to analyze the sample respectively provided with a barcode as an identification mark.
  • the barcodes of the individual preparation carrier elements are preferably detected by means of a barcode scanner.
  • the preparation carrier elements which are used for analyzing the sample each provided with a QR code ("Quick Response Code") as identification identification. QR code is less error prone.
  • the QR codes of the individual preparation carrier elements are preferably detected by means of a QR code scanner.
  • This bar code scanner or QR code scanner is designed in particular as a part of the microscope.
  • the bar code scanner or the QR code scanner can also be designed as a module that can be flexibly arranged at a suitable position of the microscope.
  • the microscope has a plurality of bar code scanners or a plurality of QR code scanners, which can each be arranged in particular at appropriate positions.
  • the bar code scanner or the QR code scanner can detect the bar codes or the QR codes of the preparation carrier elements without contact.
  • the bar code scanner or the QR code scanner can be arranged to save space on the microscope.
  • the bar codes or QR codes can be arranged to save space on the specimen carrier elements.
  • FIG. 1 schematically shows a preferred embodiment of a microscope according to the invention.
  • a preferred embodiment of a microscope according to the invention for analyzing a sample is shown schematically and designated 100.
  • the microscope 100 has an expedient microscope body 110.
  • Such a microscope body 1 10 is well known and will not be explicitly explained at this point.
  • the microscope 100 has a laser microdissection device 120, by means of which a laser beam is generated, coupled into the incident light device of the microscope 100 and can be moved on a sample.
  • a laser microdissection device 120 is also well known and will not be explained explicitly at this point.
  • a sample holder 140 is formed as a (higher-level) preparation carrier element. On the sample holder 140 a plurality of slides 141 and 142 are arranged as further (subordinate) preparation carrier elements. The sample holder 140 and the slides 141 and 142 each have an identification mark 140a, 141a, 142a. The identification marks 140a, 141a, 142a are formed as barcodes. These barcodes 140a, 141a, 142a uniquely characterize the respective specimen carrier element.
  • sample section 171 and 172 On the slide 141 and 142 are each a part (sample section) 171 and 172 of the same sample.
  • certain objects are cut out of the parts 171 and 171 of the sample by means of a laser beam generated by the laser microdissection device 200. These objects are collected in catch containers 151, 152 and 153.
  • the collecting container 151, 152 and 153 are arranged on a total holder 150.
  • the total holder 150 and the individual collecting containers 151, 152 and 153 are designed as further preparation carrier elements.
  • Both the total holder 150 and the individual collecting containers 151, 152 and 153 each have an identification marking designed as barcode 150a, 151a, 152a or 153a.
  • the identification markings 140a, 141a, 142a and 150a, 151a, 152a, 153a may also be designed as QR codes.
  • the sample holder 140 and thus the individual slides 141 and 142 are loaded into the microscope 100. This can be done manually by a user or in particular also automatically. Furthermore, the total holder 150 and thus the individual collection containers 151, 152 and 153 are loaded into the microscope 100. This can likewise be done by a user or in particular automatically.
  • the microscope 100 further has three detection devices designed as bar code scanners 161, 162, 163 for detecting the bar codes.
  • the detection devices 161, 162, 163 can also be designed as QR code scanners for detecting QR codes.
  • the bar code scanner 161, 162, 163 are expediently arranged on the microscope body 1 10.
  • the bar code scanners 161, 162, 163 are freely pivotable and / or freely rotatable.
  • the barcode scanners can also be designed as modules that can be flexibly adapted to the current application on the microscope body 110.
  • a computing unit 130 is configured as a computer with a database. For each of the specimen carrier elements 140, 141, 142, 150, 151, 152, 153, a corresponding memory entry is present in this arithmetic unit 130. In preparation for the laser microdissection, a database of the computing unit 130 is created at least with all important information about the sample including its origin.
  • the barcode 140a is automatically detected by at least one barcode scanner 161, 162, 163.
  • the corresponding memory entry here contains information about the number and positioning of the bar codes 141 a, 142 a of the slide 141, 142 on the sample holder 140. Based on this information, the bar codes 141 a, 142 a of these slides can be scanned / read. For example, these barcodes are linked to memory entries that contain important information about the particular sample on the slide.
  • a corresponding memory in the database for recording the application information and the analysis data can be reserved.
  • the total holder 150 is loaded into the system.
  • the barcode 150a of the total holder 150 is scanned / detected by at least one barcode scanner 161, 162, 163 and the barcodes 151a, 152a, 153a of the individual receptacles 151, 152, 153 are scanned / detected on the basis of the positions determined by the barcode 150a.
  • the associated memory entries of the slides and the receptacles are linked together to automatically assign each slide to the right or the collection container.
  • the memory entries each contain data or information relating to the type and dimensions of the slides 141 and 142, the sample holder 140, the collection containers 151, 152, 153 and the total holder 150.
  • the memory entries contain specific control steps or program steps according to which the Laser microdissection of the sample is performed.

Abstract

La présente invention concerne un procédé d'analyse d'un échantillon au moyen d'un microscope, ainsi qu'un microscope d'analyse d'échantillons. Des éléments porte-préparation (140, 141, 142, 150, 151, 152, 153) utilisés pour analyser l'échantillon sont munis chacun d'une marque d'identification (140a, 141a, 142a, 150a, 151a, 152a, 153a). Les marques d'identification (140a, 141a, 142a, 150a, 151a, 152a, 153a) des éléments porte-préparation (140, 141, 142, 150, 151, 152, 153) individuels sont détectées par un dispositif de détection (161, 162, 163). Au moyen des marques d'identification (140a, 141a, 142a, 150a, 151a, 152a, 153a) détectées pour les éléments porte-préparation (140, 141, 142, 150, 151, 152, 153) individuels, des enregistrements mémoire associés aux éléments porte-préparation (140, 141, 142, 150, 151, 152, 153) individuels sont acquis par une unité de calcul (130) et, pour déterminer les éléments porte-préparation à utiliser pour l'analyse de l'échantillon, ces enregistrements mémoire sont chaînés les uns aux autres dans l'unité de calcul (130).
PCT/EP2015/050447 2014-01-13 2015-01-13 Procédé d'analyse d'un échantillon au moyen d'un microscope WO2015104419A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE102014200448.9 2014-01-13
DE102014200448.9A DE102014200448A1 (de) 2014-01-13 2014-01-13 Verfahren zum Analysieren einer Probe mit einem Mikroskop

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WO2015104419A1 true WO2015104419A1 (fr) 2015-07-16

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