Manual for Deco1
Contents
1. PF The microscope can be controlled from the buttons and or Basic setting of microscope touchscreen alone if only eyepiece observations are q D needed Here we describe the main icons on the Contrast Method touchscreen and the functions of the buttons on either side Pa of the microscope Please refer to the Leica user manual for OY Magnification usage details m Stage and focus settings we LeicaScreen configuration buttons on left buttons on right PH phase contrast TL FLUO fluorescence last filter cube CHANGE TL switch through all TL processes CHANGE CUBE switch through filter cubes COMBI combination mode phase plus clockwise fluorescence SHUTTER FLUO open close fluorescence shutter 3 2 CIAN Deco1 short instructions version 2 July 2014 page 3of6 Image acquisition with Volocity Start Volocity by clicking the desktop icon Select 3DM acquisition and enter your user name and password Select Video Preview to start an acquisition session or Open Library to view existing images Select a light path using the colored icons To switch between eyepieces and camera use slider Fig 1 2 toggle between three positions 100 eyepiece 50 50 will reduce the signal strength by 50 for either one 100 camera Adjust camera sensitivity EM gain exposure time to optimize signal noise bleaching save settings for each light path by clicking little switch icon Open or create a library in order to a2. capture note that the frequency can be set in the device controls 7 Other tabs not used on this microscope Click OK to use immediately or save acquisition settings using a descriptive name save as export to Desktop for future use with Restore Scan direction Up Recommended z Order channels and Z by Z first then Channels z Manage shutters for Balanced Sample Protection ka Use Auto Exposure for each channel CIAN Deco1 short instructions version 2 July 2014 page 5 of 6 4 Handling and saving data Acquisition should always be to your folder After closing Volocity move data off the microscope computer onto a USB drive an optical disk or to the CIAN server To avoid data corruption DO NOT open Volocity files over a network Always make a copy on your local computer to view and analyze Volocity libraries Data is saved into a library A library contains individual images or image sequences i e images that belong to multidimensional image stacks The image sequences are also referred to as items The name of the Volocity library has no extension In your file structure you will see it as a folder directory It contains a data folder and a file that has the library name with the extension mvd2 and possibly two files named desktop ini and Folder ico All of these elements need to stay together to maintain the integrity of the data To open an existing library in Volo
3. SHORT INSTRUCTIONS FOR OPERATING Deco Leica DM6000B AT CIAN Version 2 July 2014 General reminders for Deco1 e Obey 10min 10min rule i e system has to be on for at least 10min and off for at least 10min e Avoid bumping into equipment leaning on table while acquiring e Move data off microscope computer after acquisition Useful abbreviations and terms BF bright field WFF wide field fluorescence PH Phase Contrast TL transmitted light IL incident fluorescent light FD field diaphragm AP aperture diaphragm also FA field aperture Light sources halogen lamp for TL Exfo mercury lamp for WFF More information The Leica DM6000B manual is found on the Deco1 computer and in the Shared folder of the CIAN server 1 Equipment Setup Fig 1 Basic setup of Deco1 1 EM CCD camera 2 slider eyepiece camera toggle 3 fluorescent filter turret 4 objective turret 5 stage with slide holder 6 condenser 7 condenser height adjustment 8 focus knob 9 fixed function buttons FD AP IL TL intensity 10 touchscreen Fig 2 control boxes 1 microscope 2 Exfo fluorescence lamp 3 camera CIAN Deco1 short instructions version 2 July 2014 page 2 of 6 2 Starting up Deco 1 Take the dust cover off the microscope put on hook table chair not the floor 2 Turn on Leica electronics box Fig 2 1 microscope control Exfo lamp Fig 2 2 and camera Fig 2 3 3 Turn on compu
4. city you open the library folder and select the mvd2 file within the folder To delete individual image sequences items within a library open the library in Volocity select the item right click on it and select Remove Item To import data also of other file types create or open a Volocity library then drag and drop the files or use File Import Export of individual image sequences within Volocity To extract individual image sequences items from a library with multiple items they can be exported as Volocity library clippings They can then be inserted into another or a new library Select the image sequence s item s in the library view on the left side of the screen File Export or right click item Export o Define name location o Format Item as Library Clipping The library clipping will show up as a file with the extension acff To view and manipulate the data open or create a library then import the clipping into the library by drag and drop or File Import Export into other file formats Image sequences items or the display of image sequences views can be exported into a number of scientific and presentation formats in the same way as described above by then selecting a different format Use of Volocity libraries in post acquisition software other than Volocity ImageJ Fiji Libraries can be opened if the LOCI Bio Formats plugin is present The plugin is part of Fiji If you don t have the plug
5. cquire images Take single images with camera icon or set up multi dimensional acquisition protocol section 3 2 2 To view images click on image icons in left screen panel BTA EE Fig 3 Volocity main screen device control elements 58 22 303 4095 22 6 dB lt Time absolute or time of acquisition ga i 24a lt Pixel intensity click to toggle for display options 2 a y TT 1 Ay seconds per Tmepont lt Acquisition rate frequency pull down to select E i a J J x KOO lt Acquisition setup green rectangle double click a9 a ecie lt Light path manager lt Button to save changes in current light path A O lt Fluorescence shutter 00 foo foo 020 lt j lt Exposure time ca lt Auto exposure use with caution ad pps lt Binning use with caution x D to Contrast lt Auto Contrast use recommended me lt Camera sensitivity EM gain lt Low high light setting lt contrast mode do not use to switch lt filter cube do not use to switch lt objective do not use to switch 33 09 um lt Leica focus control use to set up z stack 3 2 1 click button to open focus control window CIAN Deco1 short instructions version 2 July 2014 page 4 of 6 3 2 1 Z stack setup 1 focus on the sample Set Jon 2 click on the focus control button to open control window slider arrow on the SetBottom right can be used to control stage Z
6. er Set YFP BP 500 20 BP 535 30 YFP Filter Set CFP BP 436 20 455 BP 480 40 CFP
7. in in your ImageJ installation you can get information and download instructions here http loci wisc edu bio formats imagej Drag and drop the mvd2 file on the ImageJ Fiji icon or use File Open which will open a dialog window with display options Can open one or more items from a library with multiple items CIAN Deco1 short instructions version 2 July 2014 page 6 of 6 5 Shutting down Deco 1 or ye os Turn off Exfo lamp and the camera on their control boxes Fig 2 2 and 2 3 Exit the Volocity software log off or shut down computer Turn off power to Deco on the Leica electronics box Fig 2 1 Clean immersion objectives according to general instructions Carefully replace the dust cover on the microscope Appendix Technical data for Deco1 Microscope Base Leica DM6000B upright microscope motorized stage in z motorized optics Hamamatsu ORCA EM EM CCD camera acquisition software Volocity Improvision Perkin Elmer Objectives 10x 0 3 dry 40x 1 25 oil 63x 1 4 0 60 oil 100x 1 4 0 7 oil variable NA make sure to check that the aperture is all the way open before imaging Wide field fluorescence Light source Exfo metal halide lamp Lumen Dynamics Excitation Beam Emission f Filter set cube splitter Typical fluorophore Filter Set A4 BP 360 40 400 BP 470 40 DAPI Filter Set L5 BP 480 40 505 BP 527 30 GFP AF488 FITC Filter Set Y3 BP 545 40 565 BP 610 75 AF546 555 Cy5 Filt
8. position 19 00 M 3 choose lt Set Zero gt to set current position as Oum 4 find the top bottom of the sample click lt Set Top gt lt Set Bottom gt for gt 0 00 pm absolute stack definition 4 1 00 pm 5 alternatively enter relative distances from current position in the up down ge arrow fields for relative stack definition e g 5 and 5 for a 10um stack pa 18 00 pm Go To Zero T Set Zero _ 5 00 pm 3 2 2 Acquisition protocol setup 1 Open Acquisition Setup window by double clicking green rectangle in device control interface Fig 3 D stn i 2 Select the channel s for acquisition e g u Channels Z Time Points Stitch Autofocus Reference Rules Notes several fluorescent colors or BF and oe fluorescence 4 Change channels using light paths Change focus using ASI Stage Z 3 For Z stack acquisition select Leica stage Channel 1 GFP confocal GD capre with this 2 spacing Channel 2 RFP confocal sA A saa Capture this many slices control then choose one e Capture using Z spacing e Capture this many slices 4 Specify the order for channels and Z stacks 5 Shutter management balanced usually ae een ee ee eee ee ee ee um step size by moving ASI Stage Z upwards Capture multiple Z planes for each channel Shutters will be managed for balanced protection and works well 6 Time Setup the duration and frequency of a e
9. ter log in to your account launch Volocity program log into license server 4 Visually inspect objectives clean as needed see section 2 2 5 Adjust Kohler illumination see 2 1 2 1 Adjusting Kohler Illumination 1 Insert test slide adjust light intensity focus on specimen in bright field using 10x objective 2 Close Field Diaphragm Fig 1 9 FD to see edge focus it by adjusting the condenser height Fig 1 7 center if needed open FD just enough to illuminate field of view See images on the left 3 To adjust Field Aperture remove one eyepiece use buttons Fig 1 9 AP to adjust aperture to 80 of back of objective 4 Incase of poor images clean objective see section 2 2 repeat procedure if unsuccessful notify facility personel Repeat for every objective to be used or at least do it for objectives used in image acquisition en 2 2 Cleaning objectives Clean immersion objectives before and after use as follows e Take a lens paper and fold it in three into a long rectangle e Wipe the lens gently by holding both ends of the lens paper dragging it gently across the objective lens in a line Repeat with a fresh area of the tissue three times e Repeat as needed to remove excess of immersion oil If needed clean the objective more thoroughly e As above but with a drop or two of lens cleaner the blue fluid on the lens paper then water 3 Operating the microscope 3 1 Manual operation
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