公开(公告)号：EP1200821A4

公开(公告)日：2005-02-09

申请号：EP00948887

申请日：2000-07-21

Applicant: UNIV CALIFORNIA

Inventor： ALLBRITTON NANCY L ,  SIMS CHRISTOPHER E ,  BERNS MICHAEL W ,  MEREDITH GAVIN D ,  KRASIEVA TATIANA B ,  TROMBERG BRUCE J

IPC: G01N27/447 ,  C12M1/00 ,  C12M1/33 ,  C12N13/00 ,  C12Q1/00 ,  C12Q1/02 ,  C12Q1/48 ,  G01N33/48 ,  G01N33/483 ,  G01N33/50

CPC classification number: C12M41/46 ,  C12M35/02 ,  C12M47/06 ,  C12Q1/00 ,  C12Q1/485 ,  G01N27/44721 ,  G01N27/44743 ,  G01N33/50 ,  G01N33/5017

Abstract: The activity of intracellular chemical reactions of molecules is measured by the use of fluorescently labeled substrate molecules (114) that undergo a change in electrophoretic mobility upon chemical reaction such as that catalyzed by an enzyme (112). Specificity is achieved by using labeled substrate molecules (114) that can be acted upon only by specific enzymes (112). Thus, the activity of a specific enzyme (112) or class of enzymes can be determined. Measurements are made with the intracellular presence of such substrate molecules (114), at some time of interest, typically after exposure of the cell (46) to a stimulus (118) that activates a particular enzymatic pathway. To ensure accuracy, measurements must be made in a timely manner so as to minimize chemical reactions occurring subsequent to the time of interest. Fast controllable laser lysis is used to obtain the contents of a single cell (46) into which reporter substrate molecules (114) have been introduced. The cell contents are then subjected to capillary eletrophoresis (22) and enzymatic activity is determined by comparing amounts of substrate molecules (114) to amounts of enzymatically altered substrate molecules (128) which are separated by the electrophoresis and identified by the presence of a fluorescent label.

公开(公告)号：AU6232000A

公开(公告)日：2001-02-13

申请号：AU6232000

申请日：2000-07-21

Applicant: UNIV CALIFORNIA

Inventor： ALLBRITTON NANCY L ,  SIMS CHRISTOPHER E ,  BERNS MICHAEL W ,  MEREDITH GAVIN D ,  KRASIEVA TATIANA B ,  TROMBERG BRUCE J

IPC: G01N27/447 ,  C12M1/00 ,  C12M1/33 ,  C12N13/00 ,  C12Q1/00 ,  C12Q1/02 ,  C12Q1/48 ,  G01N33/48 ,  G01N33/483 ,  G01N33/50

Abstract: The activity of intracellular chemical reactions of molecules is measured by the use of fluorescently labeled substrate molecules that undergo a change in electrophoretic mobility upon chemical reaction such as that catalyzed by an enzyme. Specificity is achieved by using labeled substrate molecules that can be acted upon only by specific enzymes. Thus the activity of a specific enzyme or class of enzymes can be determined. Measurements are made with the intracellular presence of such substrate molecules, at some time of interest, typically after exposure of the cell to a stimulus that activates a particular enzymatic pathway. To ensure accuracy, measurements must be made in a timely manner so as to minimize chemical reactions occurring subsequent to the time of interest. Fast controllable laser lysis is used to obtain the contents of a single cell into which reporter substrate molecules have been introduced. The cell contents are then subjected to capillary electrophoresis and enzymatic activity is determined by comparing amounts of substrate molecules to amounts of enzymatically altered substrate molecules which are separated by the electrophoresis and identified by the presence of a fluorescent label.

公开(公告)号：AU2002360720A1

公开(公告)日：2003-11-03

申请号：AU2002360720

申请日：2002-12-19

Applicant: UNIV CALIFORNIA

Inventor： DVORNIKOV ALEXANDER S ,  TROMBERG BRUCE J ,  BERNS MICHAEL W ,  KRASIEVA TATIANA B

IPC: G02B21/12 ,  G02B21/00 ,  G02B21/06 ,  G02B21/26

Abstract: An illuminator and a reflectance microscope or system utilizing the illuminator for eliminating the need of a special light source, a reflected light vertical illuminator, and condenser lenses. The system may utilize an ordinary light source. The illuminator includes embedded chromophoric and diffusion properties. The illuminator further has a size and a shape to enable proximate positioning relative to the specimen to be observed. The illuminator further has an opening or aperture through which the specimen may be viewed. As such, the opening of the illuminator permits placement of the illuminator between the objective lens and the specimen. This positioning enables reflectance type or dark field microscopy with a simple and durable illuminator without complex optics. A method of using the reflectance microscope includes illuminating a specimen by the illuminator on a same side of the specimen as is the objective lens relative to a plane of the specimen normal to the optical path.

公开(公告)号：AU2889199A

公开(公告)日：1999-09-20

申请号：AU2889199

申请日：1999-03-03

Applicant: UNIV CALIFORNIA

Inventor： ALLBRITTON NANCY L ,  SIMS CHRISTOPHER E ,  BERNS MICHAEL W ,  MEREDITH GAVIN D ,  KRASIEVA TATIANA B ,  TROMBERG BRUCE J

IPC: G01N33/48 ,  C12M1/33 ,  C12N13/00 ,  G01N27/447 ,  G01N33/483 ,  G01N27/26

Abstract: Fast lysis of a single cell or cellular component thereof is performed by generating a shock wave in a medium in which the cell or cellular component thereof is positioned. The cell or cellular component thereof is either positioned by laser tweezers or cultured as an adhered cell or cellular component thereof to minimize manipulation trauma. The disclosed method completely lyses a single cell or cellular component thereof in a controllable manner in milliseconds or less followed immediately by the loading of the cellular contents into a capillary for analyte separation and detection. The cell or cellular component thereof is adjacent the inlet of an electrophoretic column through which a gravity siphon flow of the medium is maintained. The lysed contents of the cell or cellular component thereof enter the electrophoretic column in less than 33 msec, are separated and analyzed by laser induced fluorescence. The method takes advantage of the shock wave produced by a highly focused laser pulse which is created in a medium adjacent to the cell or cellular component thereof. In the illustrated embodiment the laser pulse is focused in the glass substrate at or near a glass-to-buffer interface of a cell chamber in which the cell or cellular component thereof to be lysed has been cultured.

公开(公告)号：IS5608A

公开(公告)日：2000-08-29

申请号：IS5608

申请日：2000-08-29

Applicant: UNIV CALIFORNIA

Inventor： ALLBRITTON NANCY L ,  SIMS CHRISTOPHER E ,  BERNS MICHAEL W ,  MEREDITH GAVIN D ,  KRASIEVA TATIANA B ,  TROMBERG BRUCE J

IPC: G01N33/48 ,  C12M1/33 ,  C12N13/00 ,  G01N27/447 ,  G01N33/483 ,  G01N27/26

公开(公告)号：CA2322857A1

公开(公告)日：1999-09-10

申请号：CA2322857

申请日：1999-03-03

Applicant: UNIV CALIFORNIA

Inventor： TROMBERG BRUCE J ,  BERNS MICHAEL W ,  SIMS CHRISTOPHER E ,  ALLBRITTON NANCY L ,  KRASIEVA TATIANA B ,  MEREDITH GAVIN D

IPC: G01N33/48 ,  C12M1/33 ,  C12N13/00 ,  G01N27/447 ,  G01N33/483 ,  C12M3/00 ,  G01N27/26 ,  C12M1/42

Abstract: Fast lysis of a single cell (46) or cellular component thereof is performed by generating a shock wave in a medium in which the cell (46) or cellular component thereof is positioned. The cell (46) or cellular component thereof is either positioned by laser tweezers or cultured as an adhered cell or cellular component thereof to minimize manipulation trauma. The disclosed method completely lyses a single cell (46) or cellular component thereof in a controllable manner in milliseconds or less followed immediately by the loading of the cellular contents into a capillary (22) for analyte separation and detection. The cell (46) or cellular component thereof is adjacent the inlet of an electrophoretic column (22) through which a gravity siphon flow of the medium is maintained. The lysed contents of the cell (46) or cellular component thereof enter the electrophoretic column (22) in less than 33 msec, and are separated and analyzed by a fluorescence detector (42). The method takes advantage of the shock wave produced by a highly focused laser pulse from a Nd:YAG laser (18) which is created in a medium adjacent to the cell (46) or cellular component thereof. In the illustrated embodiment, the laser pulse is focused in the slide (28) at or near a glass-to-buffer interface of a cell chamber in which the cell (46) or cellular component thereof to be lysed has been cultured.