Laser Microdissection and Pressure Catapulting (LMPC) is a well-known non-contact method to isola... more Laser Microdissection and Pressure Catapulting (LMPC) is a well-known non-contact method to isolate and collect specific cells from complex animal or plant tissues or living cultured cells. Thus, it is enabling pure and homogeneous sample preparation resulting in an eminent increase in the specificity of versatile downstream molecular analyses, like e.g., different PCR, nucleic acid and protein microarray analysis.An important innovation is the integration of computer-assisted recognition methods by object-oriented image processing. Here we show the automated recognition of sample material by applying object-oriented image processing on parameters like, e.g., density, area, neighborhood and shape. Coupled with this kind of software modules, the LMPC system is able to detect, isolate and finally capture the specimen of interest in a fully automated manner.Differential analysis of individual tissues and single cells will eliminate the averaging effect and allow the discovery of detailed differences between various cell types.
The understanding of the molecular mechanisms of cellular metabolism and proliferation necessitat... more The understanding of the molecular mechanisms of cellular metabolism and proliferation necessitates accurate identification, isolation, and finally characterization of a specific cell or a population of cells and subsequently their subsets of biomolecules. For the simultaneous analysis of thousands of molecular parameters within a single experiment, as realized by DNA, RNA, and protein microarray technologies, a defined number of homogeneous cells derived from a distinct morphological origin is required. Sample preparation is therefore a very crucial step for high-resolution downstream applications. Laser microdissection and laser pressure catapulting (LMPC) enables such pure and homogeneous sample preparation, resulting in an eminent increase in the specificity of molecular analyses. For microdissection, the force of focused laser light is used to excise selected cells or large tissue areas from object slides or from living cell culture down to a resolution of individual single cells and subcellular components like organelles or chromosomes, respectively. After microdissection this sample is directly catapulted into an appropriate collection device. As the entire process works without any mechanical contact, it enables pure sample retrieval from morphologically defined origin without cross contamination. Wherever homogenous samples are required for subsequent analysis of, e.g., cell areas, single cells, or chromosomes, the PALM MicroBeam system is an indispensable tool. The integration of image analysis platforms fully automates screening, identification, and finally subsequent high-throughput sample handling. These samples can be directly linked into versatile downstream applications, such as single-cell mRNA-extraction, different PCR methods, microarray techniques, and many others. Acceleration in sample generation vastly increases the throughput in molecular laboratories and leads to an increasing knowledge about differentially regulated mRNAs and expressed proteins, providing new insights into cellular mechanisms and therefore enabling the development of systems for tumor biomarker identification, early detection of disease-causing alterations, therapeutic targeting and/or patient-tailored therapy.
The hsp70/hsc70-associating protein Hap46 of human origin, also called BAG-1M (Bcl-2-associated a... more The hsp70/hsc70-associating protein Hap46 of human origin, also called BAG-1M (Bcl-2-associated athanogene 1), has been characterized previously as a DNA binding protein, which is able to stimulate transcription. By use of in vitro assays we now show that Hap46-mediated transcriptional activation can occur from linearized as well as from supercoiled circular DNA and does not require the presence of a transcription promoter. Accordingly, we observed no preferential binding of Hap46 to overlapping DNA fragments covering the sequence of the cytomegalovirus (CMV) early promoter, thus suggesting non-specific binding. The C-terminal deletion variant Hap46ΔC47, which is unable to associate with hsp70/hsc70 molecular chaperones, produced greatly diminished effects on transcription, indicating a significant involvement of hsp70/hsc70 chaperones but not an absolute requirement. In contrast, deletion of the acidic hexarepeat region, as in variant Hap46Δ12-62, did not disturb transcriptional stimulation. While full-length Hap46 readily formed complexes with a series of structurally unrelated transcription factors, variant Hap46ΔC47 proved incapable of doing so. Together these data suggest that transcriptional stimulation is a major biological activity of Hap46 and point to involvement of hsp70/hsc70 molecular chaperones in transcription in concert with Hap46, thus providing a link between hsp70/hsc70 molecular chaperones and components of the transcription machinery.
Handbook of immunohistochemistry and in situ hybridization of human carcinomas, 2005
The understanding of the molecular mechanisms of cellular metabolism and proliferation necessitat... more The understanding of the molecular mechanisms of cellular metabolism and proliferation necessitates accurate identification, isolation, and finally characterization of a specific cell or a population of cells and subsequently their subsets of biomolecules. For the simultaneous analysis of thousands of molecular parameters within a single experiment, as realized by DNA, RNA, and protein microarray technologies, a defined number of homogeneous cells derived from a distinct morphological origin is required. Sample preparation is therefore a very crucial step for high-resolution downstream applications. Laser microdissection and laser pressure catapulting (LMPC) enables such pure and homogeneous sample preparation, resulting in an eminent increase in the specificity of molecular analyses. For microdissection, the force of focused laser light is used to excise selected cells or large tissue areas from object slides or from living cell culture down to a resolution of individual single cells and subcellular components like organelles or chromosomes, respectively. After microdissection this sample is directly catapulted into an appropriate collection device. As the entire process works without any mechanical contact, it enables pure sample retrieval from morphologically defined origin without cross contamination. Wherever homogenous samples are required for subsequent analysis of, e.g., cell areas, single cells, or chromosomes, the PALM MicroBeam system is an indispensable tool. The integration of image analysis platforms fully automates screening, identification, and finally subsequent high-throughput sample handling. These samples can be directly linked into versatile downstream applications, such as single-cell mRNA-extraction, different PCR methods, microarray techniques, and many others. Acceleration in sample generation vastly increases the throughput in molecular laboratories and leads to an increasing knowledge about differentially regulated mRNAs and expressed proteins, providing new insights into cellular mechanisms and therefore enabling the development of systems for tumor biomarker identification, early detection of disease-causing alterations, therapeutic targeting and/or patient-tailored therapy.
Emerging correlative light and electron microscopy (CLEM) approaches enable the study of neuronal... more Emerging correlative light and electron microscopy (CLEM) approaches enable the study of neuronal structure-function relations at unprecedented depth and precision. However methodological hurdles in CLEM are the re-localization of a rather small target volume within a large tissue volume, changing sample orientation during the transition between different microscopy modalities, and structural distortions or preparation artefacts caused by artificial fiducial markers, such as polymer beads and near-infrared branding [1], which might obscure or even disrupt the structure under investigation.
In order to generate an analysis arrangement (3) having a plurality of discrete, separate measure... more In order to generate an analysis arrangement (3) having a plurality of discrete, separate measurement areas (16), preferably in the form of a micro-array (5) on supports (4), is proposed as "spotting" methods which (of a carrier 1 to irradiate) by the "spotting" to be transmitted (sample 15) targeted with the laser beam of a laser light source (9) to pass through the thus caused momentum transfer the Prioben (15) with high precision and without contact to the support (4) of the analysis arrangement (3) for to transfer produce the desired measurement ranges (16), where they can be subjected to a subsequent biological, biochemical or chemical analysis. The inventive "spotting" method is also suitable for non-contact transmission of very small amounts of an analyte-specific reagent in or on the measurement regions (16) of the analysis arrangement (3).
In the context of a laser induced transport process of an object from a support (2) to a collecti... more In the context of a laser induced transport process of an object from a support (2) to a collecting device (3) is proposed in the collecting device (3) a collecting medium (3A) to be used, which is in a liquid state. Preferably, the object in front of the laser-induced transport process by laser irradiation from one located on the carrier (2) surrounding mass is separated. After the laser induced transport process the selected object in this way and separated together with the collecting medium (3A) at a location, z. As a container for further processing, transferred. For this purpose a liquid handling system is preferably provided which allows for the handling of the collection medium (3A) with the contained object with high reliability and high throughput.
There is provided for receiving a dissolved out of a biological mass by means of laser radiation ... more There is provided for receiving a dissolved out of a biological mass by means of laser radiation biological object, a receiving element (1), wherein the receiving element has a receiving surface for receiving the object, wherein the receiving surface, an adhesive (4) to improve the adhesion of the respective object to the receiving surface having. The adhesive (4) suppresses the occurrence of acting on the object electrostatic forces in the receiving element (1), without damage to the object dissolvable and / or can receive means for further processing of the object. As the adhesive (4), in particular, a hydrogel such as agarose is suitable. Such a receiving element (1) is particularly suitable for collecting from the biological mass (7) catapulted objects.
There is provided a biological object receiving unit comprising a plate-shaped member (1) having ... more There is provided a biological object receiving unit comprising a plate-shaped member (1) having a plurality of openings (2). These openings are arranged in particular corresponding to the openings of a microtiter or multiwell plate. As a result, a simple introduction of biological objects, for example by means of laser catapulting in the receiving element and from there into the microtiter or multiwell plate is possible. In addition, a device for heat treatment of biological objects located in the receiving unit is provided.
Vorrichtung und Verfahren zur Laser-Mikrodissektion, wobei zur moglichst optimalen Einstellung ei... more Vorrichtung und Verfahren zur Laser-Mikrodissektion, wobei zur moglichst optimalen Einstellung eines Laserparameters einer Laserlichtquelle (4) eine Probe-Schnittlinie (22a-22c) mit dem Laserstrahl erzeugt und ein Bild von der Probe-Schnittlinie aufgenommen wird. Das Bild der Probe-Schnittlinie (22a-22c) wird mit einer Referenz-Schnittlinie (21) verglichen, um abhangig von dem Vergleichsergebnis entweder den Wert des Laserparameters zu verandern oder einen Laser-Mikrodissektionsvorgang mit dem eingestellten Wert des Laserparameters durchzufuhren.
A process for recovering biological objects (5), wherein a receiving unit is placed in a laser Mi... more A process for recovering biological objects (5), wherein a receiving unit is placed in a laser Mikrodissektionsvorrichtung, wherein the receiving unit (a plate-shaped element (1) having a plurality of a first side of the plate-shaped member (1) to a second side of the plate-shaped element (1) passing openings (2) for receiving biological objects (5) and a recording medium 3) (in the openings 2) or a cover means (4) with a coupling agent for the openings (2) for retaining biological objects comprises; wherein the openings (2) corresponding container openings of containers (10A) of an associated multi container member (10) are arranged, wherein cut with a laser beam biological specimens from a biological mass (6), wherein the biological objects (5) of the receiving unit are placed with a specific laser pulse in openings (2), wherein a reaction involving at least one biological specimen of biological objects (5) is carried out in the receiving unit, whereafter the receiving unit with ...
L'invention concerne une unite de logement pour des objets biologiques, qui presente un eleme... more L'invention concerne une unite de logement pour des objets biologiques, qui presente un element en forme de plaque (1) comportant une pluralite d'ouvertures (2). Ces ouvertures sont disposees en particulier de facon a correspondre aux ouvertures d'une plaque de microtitration ou d'une plaque multipuits. Ceci permet d'introduire facilement les objets biologiques, par exemple par catapultage laser, dans l'element de logement puis dans la plaque de microtitration ou la plaque multipuits. L'invention concerne egalement un dispositif pour le traitement thermique des objets biologiques se trouvant dans l'unite de logement.
Laser Microdissection and Pressure Catapulting (LMPC) is a well-known non-contact method to isola... more Laser Microdissection and Pressure Catapulting (LMPC) is a well-known non-contact method to isolate and collect specific cells from complex animal or plant tissues or living cultured cells. Thus, it is enabling pure and homogeneous sample preparation resulting in an eminent increase in the specificity of versatile downstream molecular analyses, like e.g., different PCR, nucleic acid and protein microarray analysis.An important innovation is the integration of computer-assisted recognition methods by object-oriented image processing. Here we show the automated recognition of sample material by applying object-oriented image processing on parameters like, e.g., density, area, neighborhood and shape. Coupled with this kind of software modules, the LMPC system is able to detect, isolate and finally capture the specimen of interest in a fully automated manner.Differential analysis of individual tissues and single cells will eliminate the averaging effect and allow the discovery of detailed differences between various cell types.
The understanding of the molecular mechanisms of cellular metabolism and proliferation necessitat... more The understanding of the molecular mechanisms of cellular metabolism and proliferation necessitates accurate identification, isolation, and finally characterization of a specific cell or a population of cells and subsequently their subsets of biomolecules. For the simultaneous analysis of thousands of molecular parameters within a single experiment, as realized by DNA, RNA, and protein microarray technologies, a defined number of homogeneous cells derived from a distinct morphological origin is required. Sample preparation is therefore a very crucial step for high-resolution downstream applications. Laser microdissection and laser pressure catapulting (LMPC) enables such pure and homogeneous sample preparation, resulting in an eminent increase in the specificity of molecular analyses. For microdissection, the force of focused laser light is used to excise selected cells or large tissue areas from object slides or from living cell culture down to a resolution of individual single cells and subcellular components like organelles or chromosomes, respectively. After microdissection this sample is directly catapulted into an appropriate collection device. As the entire process works without any mechanical contact, it enables pure sample retrieval from morphologically defined origin without cross contamination. Wherever homogenous samples are required for subsequent analysis of, e.g., cell areas, single cells, or chromosomes, the PALM MicroBeam system is an indispensable tool. The integration of image analysis platforms fully automates screening, identification, and finally subsequent high-throughput sample handling. These samples can be directly linked into versatile downstream applications, such as single-cell mRNA-extraction, different PCR methods, microarray techniques, and many others. Acceleration in sample generation vastly increases the throughput in molecular laboratories and leads to an increasing knowledge about differentially regulated mRNAs and expressed proteins, providing new insights into cellular mechanisms and therefore enabling the development of systems for tumor biomarker identification, early detection of disease-causing alterations, therapeutic targeting and/or patient-tailored therapy.
The hsp70/hsc70-associating protein Hap46 of human origin, also called BAG-1M (Bcl-2-associated a... more The hsp70/hsc70-associating protein Hap46 of human origin, also called BAG-1M (Bcl-2-associated athanogene 1), has been characterized previously as a DNA binding protein, which is able to stimulate transcription. By use of in vitro assays we now show that Hap46-mediated transcriptional activation can occur from linearized as well as from supercoiled circular DNA and does not require the presence of a transcription promoter. Accordingly, we observed no preferential binding of Hap46 to overlapping DNA fragments covering the sequence of the cytomegalovirus (CMV) early promoter, thus suggesting non-specific binding. The C-terminal deletion variant Hap46ΔC47, which is unable to associate with hsp70/hsc70 molecular chaperones, produced greatly diminished effects on transcription, indicating a significant involvement of hsp70/hsc70 chaperones but not an absolute requirement. In contrast, deletion of the acidic hexarepeat region, as in variant Hap46Δ12-62, did not disturb transcriptional stimulation. While full-length Hap46 readily formed complexes with a series of structurally unrelated transcription factors, variant Hap46ΔC47 proved incapable of doing so. Together these data suggest that transcriptional stimulation is a major biological activity of Hap46 and point to involvement of hsp70/hsc70 molecular chaperones in transcription in concert with Hap46, thus providing a link between hsp70/hsc70 molecular chaperones and components of the transcription machinery.
Handbook of immunohistochemistry and in situ hybridization of human carcinomas, 2005
The understanding of the molecular mechanisms of cellular metabolism and proliferation necessitat... more The understanding of the molecular mechanisms of cellular metabolism and proliferation necessitates accurate identification, isolation, and finally characterization of a specific cell or a population of cells and subsequently their subsets of biomolecules. For the simultaneous analysis of thousands of molecular parameters within a single experiment, as realized by DNA, RNA, and protein microarray technologies, a defined number of homogeneous cells derived from a distinct morphological origin is required. Sample preparation is therefore a very crucial step for high-resolution downstream applications. Laser microdissection and laser pressure catapulting (LMPC) enables such pure and homogeneous sample preparation, resulting in an eminent increase in the specificity of molecular analyses. For microdissection, the force of focused laser light is used to excise selected cells or large tissue areas from object slides or from living cell culture down to a resolution of individual single cells and subcellular components like organelles or chromosomes, respectively. After microdissection this sample is directly catapulted into an appropriate collection device. As the entire process works without any mechanical contact, it enables pure sample retrieval from morphologically defined origin without cross contamination. Wherever homogenous samples are required for subsequent analysis of, e.g., cell areas, single cells, or chromosomes, the PALM MicroBeam system is an indispensable tool. The integration of image analysis platforms fully automates screening, identification, and finally subsequent high-throughput sample handling. These samples can be directly linked into versatile downstream applications, such as single-cell mRNA-extraction, different PCR methods, microarray techniques, and many others. Acceleration in sample generation vastly increases the throughput in molecular laboratories and leads to an increasing knowledge about differentially regulated mRNAs and expressed proteins, providing new insights into cellular mechanisms and therefore enabling the development of systems for tumor biomarker identification, early detection of disease-causing alterations, therapeutic targeting and/or patient-tailored therapy.
Emerging correlative light and electron microscopy (CLEM) approaches enable the study of neuronal... more Emerging correlative light and electron microscopy (CLEM) approaches enable the study of neuronal structure-function relations at unprecedented depth and precision. However methodological hurdles in CLEM are the re-localization of a rather small target volume within a large tissue volume, changing sample orientation during the transition between different microscopy modalities, and structural distortions or preparation artefacts caused by artificial fiducial markers, such as polymer beads and near-infrared branding [1], which might obscure or even disrupt the structure under investigation.
In order to generate an analysis arrangement (3) having a plurality of discrete, separate measure... more In order to generate an analysis arrangement (3) having a plurality of discrete, separate measurement areas (16), preferably in the form of a micro-array (5) on supports (4), is proposed as "spotting" methods which (of a carrier 1 to irradiate) by the "spotting" to be transmitted (sample 15) targeted with the laser beam of a laser light source (9) to pass through the thus caused momentum transfer the Prioben (15) with high precision and without contact to the support (4) of the analysis arrangement (3) for to transfer produce the desired measurement ranges (16), where they can be subjected to a subsequent biological, biochemical or chemical analysis. The inventive "spotting" method is also suitable for non-contact transmission of very small amounts of an analyte-specific reagent in or on the measurement regions (16) of the analysis arrangement (3).
In the context of a laser induced transport process of an object from a support (2) to a collecti... more In the context of a laser induced transport process of an object from a support (2) to a collecting device (3) is proposed in the collecting device (3) a collecting medium (3A) to be used, which is in a liquid state. Preferably, the object in front of the laser-induced transport process by laser irradiation from one located on the carrier (2) surrounding mass is separated. After the laser induced transport process the selected object in this way and separated together with the collecting medium (3A) at a location, z. As a container for further processing, transferred. For this purpose a liquid handling system is preferably provided which allows for the handling of the collection medium (3A) with the contained object with high reliability and high throughput.
There is provided for receiving a dissolved out of a biological mass by means of laser radiation ... more There is provided for receiving a dissolved out of a biological mass by means of laser radiation biological object, a receiving element (1), wherein the receiving element has a receiving surface for receiving the object, wherein the receiving surface, an adhesive (4) to improve the adhesion of the respective object to the receiving surface having. The adhesive (4) suppresses the occurrence of acting on the object electrostatic forces in the receiving element (1), without damage to the object dissolvable and / or can receive means for further processing of the object. As the adhesive (4), in particular, a hydrogel such as agarose is suitable. Such a receiving element (1) is particularly suitable for collecting from the biological mass (7) catapulted objects.
There is provided a biological object receiving unit comprising a plate-shaped member (1) having ... more There is provided a biological object receiving unit comprising a plate-shaped member (1) having a plurality of openings (2). These openings are arranged in particular corresponding to the openings of a microtiter or multiwell plate. As a result, a simple introduction of biological objects, for example by means of laser catapulting in the receiving element and from there into the microtiter or multiwell plate is possible. In addition, a device for heat treatment of biological objects located in the receiving unit is provided.
Vorrichtung und Verfahren zur Laser-Mikrodissektion, wobei zur moglichst optimalen Einstellung ei... more Vorrichtung und Verfahren zur Laser-Mikrodissektion, wobei zur moglichst optimalen Einstellung eines Laserparameters einer Laserlichtquelle (4) eine Probe-Schnittlinie (22a-22c) mit dem Laserstrahl erzeugt und ein Bild von der Probe-Schnittlinie aufgenommen wird. Das Bild der Probe-Schnittlinie (22a-22c) wird mit einer Referenz-Schnittlinie (21) verglichen, um abhangig von dem Vergleichsergebnis entweder den Wert des Laserparameters zu verandern oder einen Laser-Mikrodissektionsvorgang mit dem eingestellten Wert des Laserparameters durchzufuhren.
A process for recovering biological objects (5), wherein a receiving unit is placed in a laser Mi... more A process for recovering biological objects (5), wherein a receiving unit is placed in a laser Mikrodissektionsvorrichtung, wherein the receiving unit (a plate-shaped element (1) having a plurality of a first side of the plate-shaped member (1) to a second side of the plate-shaped element (1) passing openings (2) for receiving biological objects (5) and a recording medium 3) (in the openings 2) or a cover means (4) with a coupling agent for the openings (2) for retaining biological objects comprises; wherein the openings (2) corresponding container openings of containers (10A) of an associated multi container member (10) are arranged, wherein cut with a laser beam biological specimens from a biological mass (6), wherein the biological objects (5) of the receiving unit are placed with a specific laser pulse in openings (2), wherein a reaction involving at least one biological specimen of biological objects (5) is carried out in the receiving unit, whereafter the receiving unit with ...
L'invention concerne une unite de logement pour des objets biologiques, qui presente un eleme... more L'invention concerne une unite de logement pour des objets biologiques, qui presente un element en forme de plaque (1) comportant une pluralite d'ouvertures (2). Ces ouvertures sont disposees en particulier de facon a correspondre aux ouvertures d'une plaque de microtitration ou d'une plaque multipuits. Ceci permet d'introduire facilement les objets biologiques, par exemple par catapultage laser, dans l'element de logement puis dans la plaque de microtitration ou la plaque multipuits. L'invention concerne egalement un dispositif pour le traitement thermique des objets biologiques se trouvant dans l'unite de logement.
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