Axioplan2 Metamorph user manual 041415
Contents
1. e Plan NEOFLUAR 100X NA 1 3 oil n 1 518 Brightfield DlC contrast III e ACHROPLAN 10X NA 0 3 W Ph 1 water n 1 33 Phase contrast DIC contrast II e ACHROPLAN 40X NA 0 8 W Ph 2 water n 1 33 Phase contrast DIC contrast III NA numerical aperture n refractive index Excitation Dichroic Fluorophore Filter Mirror Emission Filter DAPI Hoechst 340 360 380 400 420 Ip Ex 352 359 Em 455 461 Cyan FP CFP 426 436 446 455 460 480 500 Ex 430 Em 476 Cyan Topaz Energy 426 436 446 455 520 535 550 Transfer FRET FITC 450 470 490 500 515 535 555 Ex 493 Em 517 Narrow Band GFP 470 480 490 495 500 510 520 Ex 397 480 Em 515 YFP 490 500 510 515 520 535 550 Ex 520 Em 532 Y GFP Ip mutant 490 500 510 515 520 Ip Ex 498 Em 516 Cy 3 530 545 560 570 572 610 648 Ex 514 554 Em 566 615 Texas Red 540 560 580 595 600 630 660 Ex 555 595 Em 615 610 Ip Cy 5 590 620 650 660 662 700 738 Ex 649 Em 666 Wavelength numbers given in nanometers nm Excitation filter determines wavelength range low mid high limits Dichroic mirror transmits 50 of given wavelength Emission filter transmits maximum fluorescence with low mid high limits Ip transmits at and above given wavelength Using the Xenon Lamp Source This bulb can be used over the same spectra as the Mercury lamp having all the lines for epi fluorescence excitation Unless your project calls for Xenon use e g MetaFluor software please only us2. border color checked third box unchecked In the boxes next to the checked boxes to the right type in a number just below highest grayscale value for image can be obtained from the left side bar histogram using the mouse pointer and hit enter Note the higher the number the brighter the scale bar intensity will be the color will be that of the image by default for 16 bit tiff images The scale bar can be positioned quickly and easily by dragging the scale bar box with mouse cursor left click Click on Stamp and drag box away to see scale bar Undo will remove bar When finished Close dialog box Save image to save scale bar if scale bar is applied to an image already saved resave Combining Images as an alternative to Overlay Images Scale 16 bit DIC brightfield image to 8 bit tiff with Scale Image option can also scale 16 bit fluorescent images to 8 bit but not necessary Using Color Align assign any fluorescent images red green and apply Result combined is a new Untitled image file Using Arithmetic put merged combined Untitled image as Source 1 And put 8 bit DIC image as Source 2 Resulting image gt 24 bit Operation gt Add This produces an overlay image with 2 3 channels Converting 16 bit Tiff image files in MetaMorph to 8 bit color tif For example one can convert multiple images of planes in a stack file stk file to separate 16 bit tiff format files
3. using FILE menu under convert stack to TIFFs After creating individual 16 bit tiff files put files into a director folder e g images new folder_1 6to8 bit Then select DISPLAY gt gt Scale Image supply necessary information This allows one to convert a 16 bit file to an 8 bit color tif 0 256 grayscale range You can also process or convert the 16 bit file extension automatically in a batch fashion using the Journal file e Select JOURNAL menu go to LOOP and then lt all images in directory e Select the particular Journal written i e Copy to 8 Bit Batch JNL e Select particular path directory site where the files are e Click on open e Have checked all images and autosave Click on run starts operation e Dialog box will ask where to gt gt save in And also gt gt file name 8 hit copy of file name tif e Go through saving process manually by clicking on save box one can visualize the appearance of the new convened files 8 bit color tif in the folder box Note If the Journal 8bit copy and save to same location JNL is selected instead then after clicking on run all files will be processed automatically
4. 0X objective in viewing position Turn off microscope Dust cover placed over microscope if at end of day but do not cover the hot halogen light source box Note yes for shut down mode in log book 5 NOTE STOP TIME IN LOG BOOK Clearly indicate total lamp usage in log book MERCURY OR XENON LAMP USAGE MUST BE RECORDED 6 Turn off Mercury lamp ebq 100 light source YATTENTION IF THE METAMORPH PROGRAM SHUTS DOWN OR FREEZES DURING USE PLEASE DO THE FOLLOWING CAPTURE THE ACTIVE WINDOW S WHICH CONTAINS AND DESCRIBES THE ERROR ERROR REPORT USING ALT PRT SCRN COMMAND This function copies the window then open Word and paste the message into a new page The file can be named and placed in the reports folder on the desktop CAPTURING THE ENTIRE SCREEN CAN BE DONE USING CTRL PRT SCRN however this may not be necessary Exiting the program and completely turning the computer OFF Wait a few moments then back it ON again then logging back onto MetaMorph This can usually allow you to continue working COLOR CAMERA HOW TO ACQUIRE 24 BIT COLOR IMAGES Turn on microscope camera box monitor and computer Turn ON color camera CF with switch on left side back green LED light should go on next to switch position On microscope upper right front panel BP back port should be on selected for color camera site Log onto open MetaMorph with color camera ON for initialization N
5. Zeiss Axioplan2 and MetaMorph Imaging Software Microscope s User Manual Managed by Ja Shared Equipment mam iuthority For detail information about this instrument please contact AXIOPLAN2 MICROSCOPE SYSTEM START UP 1 DO NOT start up the system if the fluorescent lamp has not adequately cooled down after the previous user finished It will need 20 30 minutes to cool down To be sure check the log book to see the actual time the machine was turned off 2 MAKE SURE ALL COMPONENTS ARE OFF When the lamp is first turned on it sends off a magnetic pulse that can damage computers and electronic components This is why all other components must be off when the lamp is turned on This includes the computer monitor which has its own on off switch on the bottom right of the monitor face IT IS YOUR RESPONSIBILITY as a user to ensure that it is safe to turn the lamp on only then turn on arc lamp light source White power source ebq 100 with on off switch on upper left label no 1 NOTE YOUR LAMP START TIME IN THE LOG BOOK Allow the lamp about 5 minutes to stabilize before collecting imaging data 3 Turn on Axioplan2 microscope green switch on right back side of microscope label no 2 4 Turn on computer drive monitor label no 3 5 Turn on camera s The power source for the B W black and white camera Cool Snap HQ mounted on the top of the microscope is located on top of the computer tow
6. e this light source if the Mercury lamp is unusable Like igniting the Mercury lamp the monitor computer are OFF The settings on the power supply box are already set left knob 150w and right knob 0 250 watts ow oe eS Turn ON the Lambda 10 3 Sutter wheel control box Turn ON the RED power switch for Xenon lamp Trigger Manually push UP silver stick until click is heard Lamp light in left housing will be seen through grating at the top Switch lever between lamp housings toward the Xenon lamp turn to the left After computer and MetaMorph are ON have Lambda box in LOCAL mode push local button then have filter wheel in position 3 push number 3 and push S2 button to open filter wheel light shutter Note If using MetaFluor Lambda box will be automatically in ONLINE mode When finished push RED Xenon power supply switch to OFF and turn OFF Lambda box Scale Bar A scale bar can be applied to an Acquired image or to a single saved TIFF MetaMorph image or to all planes or single plane in a stack You can use click on Scale Bar box in the Taskbar or select Display Menu gt Graphics gt Calibration bar Have back up copy of image without scale bar if certain measurements related to grayscale intensity values are to be obtained for entire image Select type in bar thickness in pixels 5 15 is good Choose fonts in font section 24 or 26 is good Have upper 2 boxes under Bar
7. er black box labeled Cool Snap HQ label no 4 DO NOT alter the camera without permission The color camera Cool Snap CF is mounted on the back of the microscope Its power switch is located on the back left side of the camera and is the only part of the camera that should be touched Green LED indicates the camera is ON 6 Click Axioplan2 Icon and select the MetaMorph software program Logging in using the correct lab access and password The system is now ready to begin image acquisition 7 Verify during initial use that the imaging system is operational and was left in proper working condition e Left in a shut down mode see 4 of posted System Shut down procedure below e Objectives you use are clean and undamaged e No other visible alterations or damage are noticed on various system Components Please check yes or no in the log book to verify whether you began with the system in proper condition Make proper notes comments if there is a problem so that it can be addressed as soon as possible SYSTEM SHUT DOWN 1 Close software program only but do not shutdown computer yet 2 Turn off camera s 3 Shut down computer Be sure to turn off monitor 4 Microscope MUST BE returned to shut down mode before turning off the microscope Any objective used for oil immersion MUST BE cleaned using lens paper DO NOT use Kimwipes Stage should be left at lowest position Objective turret placed with 1
8. ote Acquire color dialog box for cool snap cf color camera use is different than the Acquire box for cool snap HQ B amp W camera use To select the Acquire Color dialog box for exposure settings capturing images etc go to the main menu under Acquire and choose Set Acquisition Channel there will be 2 lines 1 for Cool Snap HQ Monochrome 12 bit B amp W camera and 1 for Cool Snap CF Monochrome Mosaic 12 bit color camera Select Cool Snap CF to access the Acquire Color dialog box in the Acquire main menu For bright field color images choose Color Balancing menu and for Image type gt gt select brightfield and choose Acquire menu For Illumination gt gt DIC While for Image type amp Exposure gt gt gt color image Finally set time in ms 30 200ms Halogen light source levels can be 3 4 green dots Neutral density filters can be determined empirically e g 100 1 5 or 100 25 are functional settings i e looking in ocular attain appropriate color hue and this should be matched digitally on computer monitor DIC objective polarizer can be either removed or remains inserted Condenser diaphragm setting same as for DIC e g Il for 10x 20x or brightfield I H or 1 2 3 for respective objectives Alternatively 3200 K or level 9 can be used with appropriate filters subsequent white balancing and image acquisition might give a more true color To obtain an acti
9. ve image window gt gt gt click on start focusing box at bottom left If full field of view is not necessary define region of interest active region by selecting region and set box to desired areal dimensions This might work best for a specific area during white balancing White balancing on background area without specimen will set color scale for digital image acquisition to match extremely closely what is seen through ocular Choose Color Balancing menu and click on measure white ref box It might have to be done several times to set a true color tone for background There are controls for adjusting brightness and color RGB scale if 10 desired Color Balancing then Brightness and Adjustment However if microscope neutral density filters and camera white balance are well set this should not be necessary Images saved in default tiff format are 24 bit RGB 8 8 8 bit These are compatible with for example Windows image software If jog for example is chosen it will be 8 bit and image will lose information such as annotations Objectives for Zeiss Axioplan 2 e Plan APOCHROMAT 10X NA 0 45 air n 1 Brightfield DlC contrast II e Plan APOCHRON1AT 20X NA 0 75 air n 1 Brighttield DlC contrast II e Plan APOCHROMAT Korr 40X NA 0 95 air n 1 Brightfield DlC contrast III e Plan APOCHROMAT 63X NA 1 4 oil n 1 518 DIC objective DIC contrast III
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