PALM RoboSoftware. Quick Software Guide. MicroBeam Version (EN)

Transcription

1 PALM RoboSoftware Quick Software Guide MicroBeam Version (EN)

2 This document is delivered only to persons who are trained and authorized by P.A.L.M. Microlaser Technologies AG. No part of this document may be reproduced or distributed in any form or by any means without the prior written consent of P.A.L.M. Microlaser Technologies AG. The information in this document is subject to change without notice. P.A.L.M. Microlaser Technologies AG assumes no responsibility for any errors that may appear. Printed January 2003 The following technologies have been patented by P.A.L.M. Microlaser Technologies: - Laser catapult technology (Laser Pressure Catapulting LPC) Patents: US 5,998,129, EP B1 and others. - Three-dimensional laser beam positioning system Patents: US 5,689,109, EP B1 and others. - Additional patents pending. P.A.L.M. Microlaser Technologies AG Am Neuland Bernried / Germany Phone: +49-(0) Fax: +49-(0) info@palm-microlaser.com P.A.L.M. Microlaser Technologies AG, All rights reserved. Quick Software Guide MB (EN) 2

3 List of contents Chapter Page 1 Program layout Tool Bar Image Processing Tool Archive Tools Fluorescence Tool Lens Tool Graphic Tools Cut Tools/Start laser Menus Laser Tools Speed Tools Status Bar Element List Shortcuts Laser functions - an overview Quick Software Guide MB (EN) 3

4 Program layout In this quick guide the functions of the PALM RoboSoftware are described in a short way. 1 Program layout The site of each toolbar on the screen can be changed: with the cursor on the white thick stroke and while pressing the left mouse button you can move it. Via menu item "View >Menus >..." each toolbar and each tool can be hidden or shown. Menus (page 9) Tool Bar (page 5) Image Processing Tool (page 5) Archive Tools (page 6) Fluorescence Tool (page 6) Lens Tool (page 7) Laser Tools (page 14) Speed Tools (page 15) Start Laser (page 8) UV-Laser status (page 8) Graphic Tools (page 7) Status Bar (page 15) Cut Tools (page 8) Quick Software Guide MB (EN) 4

5 Tool Bar 2 Tool Bar The Tool Bar contains the following tools: Save Elements Stop Continue To save all elements in a new file. Before saving elements a reference position has to be determined. The reference position is necessary, when the slide is reinserted with a previously examined sample to ensure a correct positioning of the elements (see menu "Move > Define Reference-Position" page 11). The elements are saved in the default directory (see "Setup > PALM Robo..." page 13). To stop all laser functions and movements immediately (in case of emergency). Delete last To continue with the last not completed function after a "stop". Delete all First Element Last Element To delete the last drawn element. To delete all elements, including hidden elements. Previous Element Next Element About The stage is moved so that the desired element is centered on the screen. To get informations about the program (version number and manufacturer). Element List To show the "Element List". See also page 16. Stage To switch to stage mode. In stage mode you move the stage with mouse. To exit stage mode click left mouse button once. 3D-1 3D-2 Only active in PALM CombiSystem. 3 Image Processing Tool 1) The Image Processing Tool contains the following: Image Processing software To start the Image Processing software. 1) Only available in systems equipped with Image Processing software. Contact PALM for further information. The PALM CombiSystem combines the cutting abilities of MicroBeam with the trapping abilities of Optical Tweezers. Contact PALM for extending your PALM MicroBeam System. Check-Point Point of origin To position the stage to the defined check-point. If no check-point is defined no positioning occurs. To move the stage from check-point back to the point of origin. Quick Software Guide MB (EN) 5

6 Archive Tools 4 Archive Tools The Archive Tools contain the following tools: New Image folder 5 Fluorescence Tool The Fluorescence Tool contains the following tools: Shutter To select or create a folder for saving images. Save Image To save the actual image under the default name with an image number added in the default directory (see "Setup > PALM Robo..." page 13). The image numbers will be increased automatically. You can save the image with or without the drawn elements. Open Imaging If your system is equipped with a Ludl filter wheel: Opens or closes the fluorescence shutter to activate or deactivate the fluorescence beam. If your system is equipped with a Zeiss filter wheel and a motorized microscope Zeiss Axiovert 200M: Opens or closes the microscope shutter to activate or deactivate the fluorescence beam. Timer Allows to set a timer to close the shutter automatically. To start an image handling software to display stored pictures and to edit them. PALMPicture To start PALMPicture to display and organize stored pictures and organize folders. To select a set of fluorescence settings defined via menu item "Setup > Fluorescence..." (see page 13). Quick Software Guide MB (EN) 6

7 Lens Tool 6 Lens Tool The Lens Tools contain the following tools: Microscope window 7 Graphic Tools Display functions Graphic on/off To open the Microscope Window (only active when your system is equipped with motorized Zeiss Microscope Axiovert 200M). All functions of the microscope are controlled by this window. To show or hide all drawn elements. Newly drawn elements are shown automatically, even if the display of elements is switched off (all the other elements are hidden). Number on/off To show or hide the numbers of the elements. Element areas To select the required magnification on the microscope. To display the size of an element area in µm². For a correct display of your drawn figure elements and for correct laser functions it is important, that the setting of this menu matches with the set lens on the microscope. Measure, flag, comment, edit elements Ruler To measure. You measure with mouse moving while pressing the left mouse button. Flag, comment To add a flag into the image with or without a comment. Click with mouse to add a flag. Click with mouse and draw a rectangle to add a flag with a comment. You can change the comment with "Change" in the dialog "View > Element List" (see "Element List", page 16). To add a comment to an element, see "Element List", page 16. Eraser To delete parts of an element. If you divide elements into parts, each part will become a separate element with its own number. You can connect separate elements of type "line" with freehand tool. Quick Software Guide MB (EN) 7

8 Cut Tools/Start laser Select elements, create elements Pointer AutoCircle To select a part of an element, a whole element or several elements, which then can be changed (e.g. right mouse button > "Change"), moved or deleted. Freehand To mark areas for cutting and subsequent catapulting. These elements are used for the AutoCircle function. Change element attributes Lines To draw freehand lines. To draw a line press left mouse button and move the mouse. Line To determine the thickness of lines, dots and the ruler or eraser size. The thickness of selected elements will be changed. Colors To draw straight lines. You start drawing with the first mouse click, after another mouse click you can change the direction, with a double-click you finish the element. If the quadratic attribute is selected, you draw horizontal and vertical lines. To determine a display-color for each element. The color of the drawn elements will not be changed. Color Palette On/Off Rectangle To draw rectangles. If the quadratic attribute is selected, you draw quadratic elements. If the centric attribute is selected, you draw the elements from their center. Circle To draw ellipses. If the quadratic attribute is selected, you can only draw circles. If the centric attribute is selected, you draw the elements from their center. To show or hide the color palette. With the color palette you can select the color for the next drawn elements. The selected color is saved as a default for all elements of the actually set element type. 8 Cut Tools/Start laser To select the UV-Laser function for the next command "Start Laser function". The number states the total number of laser functions to be performed for each element. For an overview of the Laser functions see page 19. or Start laser function (UV). Dot To mark single cells for catapulting. These dots are used for the Laser Pressure Catapulting function (LPC). When the UV-Laser is on, the grey ball changes its color to blue. Quick Software Guide MB (EN) 8

9 Menus 9 Menus Menu "File" To prepare the software for drawing new elements. Existing elements can be saved previously. To start an image handling software to display stored pictures and to edit them. To open elements from a file: To start PALMPicture to display and organize stored pictures and organize folders. Insert the slide into the stage with exactly the same position as it had at last using. Open the elements with "Open Elements...". Define the reference position. To import the element properties shown in the element list (Color, Nr., Type etc.) and the coordinates of the anchor points of the elements from a text file. To save the drawn elements in a file. The file can be called up any time with PALM RoboSoftware. Before saving elements a reference position has to be determined. This is necessary, when the slide is reinserted with a previously examined sample to ensure a correct positioning of the elements (see menu "Move > Define Reference-Position" page 11). To export the element properties shown in the element list (Color, Nr., Type etc.) and the coordinates of the anchor points of the drawn elements into a text file. The text file can be opened with PALM RoboSoftware and any software that can handle *.txt files. To open the Image Processing Software. Only available in systems equipped with this software. Contact PALM for further informations. To open one of the last four files with saved elements. To quit PALM RoboSoftware. To select or create a document folder for saving pictures. To save an image as a file (with or without the drawn elements). The file can be called up any time. The folder for saving can be changed. Quick Software Guide MB (EN) 9

10 Menus Menu "Edit" Menu "View" To restore the last deleted element. To select or deselect all elements. To select whole figures (elements), if actually only a part of the figure is selected. To show and change the properties of the selected element. If more than one element is selected, the properties of the element with the lowest number are shown. To show or hide Tool Bar and Status Bar (see "Program layout", page 4). To show or hide the various tools (see "Program layout", page 4). To show or hide the Color Palette. To renumber the remaining elements after deletion of elements. To show or hide Element List (see "Element List", page 16). To copy the selected elements to clipboard. To paste the elements from clipboard. To open the Microscope Window (only active when your system is equipped with motorized Zeiss Microscope Axiovert 200M). To delete the selected elements or the selected parts of them. To delete the last drawn element. To show or hide all drawn elements. Newly drawn elements are shown automatically, even if the display of elements is switched off (all the other elements are hidden). To delete all elements, including hidden elements. To show or hide the numbers of the elements. To show or hide the scroll rectangle (dashed frame). To show or hide the screen center mark (red reticule). To show or hide the Reference Mark (cyan colores double cone). Quick Software Guide MB (EN) 10

11 Menus Menu "Move" To stop the UV-Laser function and the movement of the stage immediately in case of emergency. To proceed with operation choose "Continue". When calling up menu item "Goto...", the stage is positioned to the defined point. To define a free selectable position for the stage outside of the viewing field (to examine the sample in the cap). To switch to stage mode (the stage moves with mouse motion). With "Invert stage motion" activated, the stage moves opposite to mouse motion. When calling up menu item "Goto Reference-Position > Stage", the stage is positioned to the actual Reference-Position. To switch to freeze mode (the stage cannot be moved anymore and the video image is frozen). Only active in PALM CombiSystem. 1) "Define Reference-Position" must be used when saving elements for later re-using. The software needs a reference position to relocate saved elements with respect to a certain slide. When opening saved elements, you must move to the according Reference Position Mark and define it as new reference position. The software recalculates the position of all elements with respect to this reference position. To modify the actual speed of the movement ("Speed slower" and "Speed faster"). To center a certain element on the screen. With menu item "Cut Reference-Position" a Reference Position Mark is cut with its center at the defined reference position. The laser uses the actual settings for energy, focus and speed. If the Reference Position Mark is not sharp, you have to define a new reference position with modified settings. To determine speed settings for Stage, Arrow (Keys), Cut, LPC, Positioning and Scroll. 1) The PALM CombiSystem combines the cutting abilities of MicroBeam with the trapping abilities of Optical Tweezers. Contact PALM for extending your PALM MicroBeam System. Quick Software Guide MB (EN) 11

12 Menus Menu "Laser" Menu "Laser-Marker" To determine LPC-parameter for each selectable microscope lens. To determine energy and focus settings of the UV-Laser for each selectable microscope lens. To align the position of Laser-Marker with the actual position of laser focus (for UV- Laser). 1) Only active in PALM CombiSystem. 1) With "Layout Laser-Marker" the color, size, type and style of Laser-Marker can be defined (for UV-Laser). To start UV-Laser function after all settings are done. Only active in PALM CombiSystem. 1) With "View Laser-Marker" the Laser-Marker can be shown or hidden (for UV-Laser). 1) IR-Laser is only active in PALM CombiSystem. The PALM CombiSystem combines the cutting abilities of MicroBeam with the trapping abilities of Optical Tweezers. Contact PALM for extending your PALM MicroBeam System. To select the laser function for the next command "Start Laser function". To change the values for laser energy/ power and laser focus. The set values are valid for the actually in the Laser Tools selected laser function only (Cut or LPC; see page 14). During laser action this values can be changed via keyboard (see page 18) or Laser Tools (see page 14). 1) The PALM CombiSystem combines the cutting abilities of MicroBeam with the trapping abilities of Optical Tweezers. Contact PALM for extending your PALM MicroBeam System. Quick Software Guide MB (EN) 12

13 Menus Menu "Setup" General settings for saving images, saving elements and saving settings, for laser function and Reference Position Mark. To define significant names for the fluorescence filters instead of filter numbers (Ludl filter wheel) resp (Zeiss filter wheel)(rhodamin, DAPI, TITC etc.). To select a fluorescence filter. To get informations about the installed filter wheel. To calibrate the software with respect to the microscope setup. This is necessary for exact cuttings and measurements. The calibration is stored with respect to lens magnification. With "Factory Calibration" the default setting is called up. To open and close the shutter. To get informations about the shutter type. If your system is equipped with a Ludl filter wheel: Opens or closes the fluorescence shutter to activate or deactivate the fluorescence beam. If your system is equipped with a Zeiss filter wheel and a motorized microscope Zeiss Axiovert 200M: Opens or closes the microscope shutter to activate or deactivate the fluorescence beam. Menu "Help" To compensate the software to the offset of microscope objective (chromatic aberration). With "Factory Lens-Offset" the default setting is called up. To create a new or change an existing set of fluorescence settings. Defined fluorescence settings are activated via Fluorescence Tools (see page 6). The "PALM@Robo Instructor" gives help for various subjects. To list help topics. To save and load preset speed settings for: stage, arrow keys, cutting, LPC, positioning, scrolling, 3D and 3D Arrow and to save and load laser settings for: energy/ power, focus, lens value, LPC distances, and Auto-change. To show system informations. To get informations about the program (version number and manufacturer). To reset all settings to default settings. Quick Software Guide MB (EN) 13

14 Laser Tools 10 Laser Tools To select laser function Cut or LPC. With selecting a function the relevant controls and the actually set values are shown (see below). The preset values for energy and focus can be changed before each laser operation. In this way you optimize the parameters for each operation to obtain a precise cut and an effective catapulting. For fine adjustment the values can be changed even during cutting. Energy setting and Focus setting for laser function "Cut". Energy setting and Focus setting for laser function "LPC". Quick Software Guide MB (EN) 14

15 Speed Tools 11 Speed Tools To display and to change the preset speed for the following parameters: "Stage": "Arrow": "Cut": "LPC": "Position": "Scroll": Setting of the relation between mouse movement and movement of stage. Click with mouse into the checkbox for slow mode. Speed setting for the movement of stage with arrow keys. Speed setting for stage movement during Cut function. Speed setting for stage movement during LPC function. Speed setting for stage movement from element to element, to checkpoint or to reference position. Speed setting for scrolling in cursor mode. 12 Status Bar short descriptions for tools in Tool Bar or Graphic Tools, when moving the cursor over the buttons actually used setting file number of elements, or number of the centered element (x of...) and of all elements (... of y) (shown after using any function for centering an element) active mode: laser ON, stage mode, cursor mode, scrolling, position, continuous, calibration, Reference- Position. coordinates (x y) of actual stage position When the stage is moving the green ball shines yellow. Quick Software Guide MB (EN) 15

16 Element List 13 Element List The element list displays informations about all drawn elements and allows operations on them.. To show different types or all types of elements. Menus of Element list Menu "Stop" Click on menu item "Stop" to stop the UV-Laser function and the movement of the stage immediately in case of emergency. Menu "File" To prepare the software for drawing new elements. The columns in the upper table display the following informations about each element: first column: Nr: Type: cut,shot: Area: Comment: Counts: Position: color number type number of performed laser cuttings or catapultings area for elements of type "Line" and "AutoCircle" (in µm 2 ) a possibly added text the number of anchor points of the element the position (X,Y) relative to the reference position Table "Summary for Types and Colors" displays: Menu "Element" To open and save elements (see also page 9, Menu "File"). To import the element properties (Color, Nr, Type etc.) and the coordinates of the anchor points of the elements from a text file (*.txt). To export the element properties shown in the element list (Color, Nr, Type etc.) and the coordinates of the anchor points of the elements into a text file (*.txt).. To center the selected element on screen. To change the properties of the selected element. To renumber the remaining elements after deletion of elements. To delete selected resp. all elements. first column: Elements: Areas: reports the used colors for all types of elements total number of elements for each color and type total area of all elements of type "Line" and "AutoCircle" for each color (in µm 2 ) Quick Software Guide MB (EN) 16

17 Element List Menu "Laser" To select the laser function for the next command "Start Laser function". Menu "Close" Click on menu item "Close" to save and close the Element List. Tool Bar of Element List Stop To stop the UV-Laser function and the movement of the stage immediately in case of emergency. Export Elements To export the properties of the elements. Goto To center the selected element on screen. Change To change the properties of the selected element. Delete selected To delete the selected elements. Delete all To delete all elements. Renumber All To renumber the remaining elements after deletion of elements. Cut Tools of Element List/Start Laser To select the laser function for the next command "Start Laser function". For an overview of the Laser functions see page 19. Start Laser function. Quick Software Guide MB (EN) 17

18 Shortcuts 14 Shortcuts Shortcut Picto/Menu Shortcut Picto/Menu Help Move F1 Help File F2 Open Imaging... File > Open Imaging... F3 PALMPicture Ctrl+F Ctrl+S Ctrl+K Move > Speed faster Move > Speed slower Move > Goto Check-Point Ctrl+H Move > Goto Reference-Position > Stage F8 Move > Setup Speed... F7 Stage mode / Cursor mode Stage File > Open PALMPicture... Alt+F4 File > Exit PALM Robo Move > Stage mode Edit Esc Stop Ctrl+A Ctrl+C Ctrl+I Edit > Select / Deselect all Edit > Copy Edit > Select whole figure View Move > Stop Ctrl+V Edit > Paste Ctrl+L View > Menus > Laser Tools Backspace Delete last Ctrl+P Color Palette On Off Edit > Delete last View > Menus > Color Palette Alt+Backspace Edit > Undelete last F5 Element List Ctrl+Y Delete all View > Element List Edit > Delete all F6 Microscope Window Del Edit > Delete Alt+Enter Edit > Change View > Microscope Window Ctrl+U View > Menus > Cut Tools Ctrl+E View > Menus > Speed Tools Ctrl+G View > Menus > Graphic Tools Laser Page up Page Down Home End Laser > Energy/Power more Laser > Energy/Power less Laser > Focus up Laser > Focus down F9 Laser > Energy & Focus (UV-Laser)... F11 Start Laser Laser > Start Laser function Quick Software Guide MB (EN) 18

19 Laser functions - an overview 15 Laser functions - an overview Cut Cutting along the predefined line The laser cuts precisely along the predefined line yielding a clear-cut gap between the selected and non-selected material. Thus pure sample preparation is possible without danger of contamination. Close & Cut Close and cut glassmounted preparations, living cells, moist tissue samples The laser cuts precisely along the predefined line yielding a clear-cut gap between the selected and non-selected material. Thus pure sample preparation is possible without danger of contamination. LPC Laser Pressure Catapulting Only LPC dot-marked specimens are catapulted. This function is of special benefit for cytocentrifuged specimen, but is also used for isolated cells within a histological preparation or to individually catapult membrane-mounted specimen after cutting. AutoLPC Automatic catapulting of larger areas from glass-mounted preparations With glass-mounted preparations only a small amount of cellular material can be catapulted with each single shot. Therefore larger areas have to be catapulted with multiple shots. The distance between the shots depends on tissue characteristics and can be preselected as in the Setup menu. Close & Cut + AutoLPC Glass-mounted preparations:an open figure is closed and subsequently cut and catapulted Critical preparations have to be circumcised prior to AutoLPC to avoid contamination with neighboring tissue. Thus pure sample preparation can be warranted. Quick Software Guide MB (EN) 19

20 Laser functions - an overview RoboLPC Cutting and catapulting in a single step! Only possible with membrane-mounted specimen. The marked line is entirely closed leaving a small connecting piece from where the entire area is immediately catapulted with one single shot. The size of the connecting piece can be pre-selected within the laser Setup menu and displayed together with the Robo- LPC-dot. Auto Circle for glass-mounted preparations. Circular cutting and subsequent catapulting from the sample center. The circle radius can be predefined. Quick Software Guide MB (EN) 20