Microscope Confocal LSM510 META

Transcription

1 Microscope Confocal LSM510 META Welcome to the Zeiss LSM 510 Meta Confocal tutorial. Before using the LSM 510 META, Log off any other computer that is open with your personal login. You will need to put down your name on the reservation system = on=com_login&task=loginform&current URL=%2Fres-loc%2Freservation.php%3F Version Thierry Laroche thierry.laroche@epfl.ch Int LSM 510 Meta tutorial

2 LSM510Meta Zeiss tutorial Waring; This tutorial version don t have any explication concerning the Meta PMT Reservation System. Startup Device. Start Up System. Create a new Data base. Igniting the Laser. Microscope Control. Microscope Parts. Microscopoe Manual Part. Viewing Specimen. Choosing the configuration. How to make a configuration for single Track. Image Acquisition Setup. Channel Setup. Scaning your sample. Adjusting for your Final Image. How to make a configuration for a Multi Tracking acquisition. Schutting Down the System. Resolution Rule, Objective/Format/Zoom. Various configurations channels. 2 LSM 510 Meta tutorial

3 System Start Up = 3 Step Turn on the remote control button, wait 15 second. Turn on the computer. Turn on the mercury lamp Input your user name and password. And click OK. 3 LSM 510 Meta tutorial

4 System Start Up Once you have entered your login, you will see the screen fill with several icons. Double click the LSM 510 META icon. The Zeiss software will startup and you will see the LSM 510 switchboard window. You will click Scan New Images and then Start Expert Mode. LSM 510 META 4 LSM 510 Meta tutorial

5 LSM Software When the LSM software opens you will want to create a new database or open your previous database. To create a new database: click on the new button and type in a name for your database. When you click create, a new database will open. Experiment name.mdb Experiment name.mdb 5 LSM 510 Meta tutorial

6 LSM Software To open an existing database click the Open button. Find your database and click on it and then click Open. Experiment name.mdb Experiment name.mdb Experiment name.mdb 6 LSM 510 Meta tutorial

7 Igniting the Lasers To acquire an image. Click the Acquire button on the control strip. Then click the Laser button. Click, if you need, each HeNe laser to the On position and Click the Argon laser to the Standby position ONLY if you need to use the 458, 477,488,and 514nm. 7 LSM 510 Meta tutorial

8 Igniting the Lasers Once the argon laser is warm, the status will read Ready and you can click it On. Output should not need to be over 30 % start at 25%. The current tubes should be around 5.3 to 5.7 A When all the lasers read On, close the laser control window. 8 LSM 510 Meta tutorial

9 Microscope Controls Change between direct observation and laser scanning Toggle between Vis to LSM Icon in Main menu, automatic switching between direct observation and laser scanning. 9 LSM 510 Meta tutorial

10 Microscope Controls Once you have the lasers on, you will need to find your sample with the microscope. Most microscope operations can be controlled manually or by the Zeiss software. To use the software controls, click the Acquire button on the top menu and then Micro on the bottom menu. 10 LSM 510 Meta tutorial

11 Microscope Controls From this menu, you can change the filter cube positions = Reflector, Objectives, and Transmitted light settings. Or you can also Click on the pull down arrow or the button, already assign, to install the option you want. Reflector = you can select the previous Filter as the Dapi, Fitc or Rohd. Objective = you can select the objective magnification you need. Reflected Light = Turn On or Off the mercury Lamp. Transmitted Light = Turn On or Off the halogen light. 11 LSM 510 Meta tutorial

12 Microscope Parts Becoming familiar with the different parts of the microscope.what they do will help you to collect a good image quickly. Transmission PMT Halogen Lamp LCD Display Condenser Meta Head The Eye pieces adjust your eyes by turning the dials of each lens 12 LSM 510 Meta tutorial

13 Manual Controls (Right side) You can focus (down or up) with the two buttons. (control the z stage) Turn On or Off the Mercury shutter. Turn On or Off the Halogen Lamp. The objective lens turret can be rotated by using the two buttons. You can rotate the filter cube turret with the two buttons below the focus knob. 13 LSM 510 Meta tutorial

14 Manual Controls The two buttons on the left side of the scope control the Z stage. Press the upper bottom for bring the objective near the focus position. Adjust the focus position with the focus knobs. Press the down bottom, the objective back to the previous and safety position. 14 LSM 510 Meta tutorial

15 Viewing Specimen With Brightfield Place your slide on the stage securing it with the slide clamp on the stage. Turn on the transmitted light by clicking the Transmitted Light box on the microscope window in the software. Be careful looking into the oculars. The light may be very bright. Start with the light around 3%. You will probably need to use the neutral density filters to protect your eyes. 15 LSM 510 Meta tutorial

16 Viewing Specimen With Fluorescence To view fluorescence you need select the proper filter cube for viewing Dapi, FITC\GFP or Rhod\Cy3\Texas Red using the Reflector turret menu in the Axioplan control box. Position 1 is an open position. Position 2 reads FSet10 wf, which is FITC. Position 3 reads FSet15 wf, which is Rhodamine. Position 4 reads FSet 49 wf which is Dapi. After you have selected the proper cube, push the Reflected Light button and you should see the reflected light come on. 16 LSM 510 Meta tutorial

17 Make your Configuration After you have a focused image on the microscope, you can acquire a scanned image. To acquire an image you should to toggle between Vis to LSM Icon in Main menu, 17 LSM 510 Meta tutorial

18 Choosing the configuration SINGLE TRACK. Use for single, double, triple labelling. MULTI TRACK. Use for double, triple, labelling and for the colocalisation. Simultaneous scanning only. Sequential scanning. ADVANTAGE ADVANTAGE Fast Acquisition. DISADVANTAGE crosstalk. Reduce dramatically the DISADVANTAGE Cross talk between channels. Slow Image Acquisition (One channel after one channel) 18 LSM 510 Meta tutorial

19 Make your Configuration for Single Track. Select Single Track. Click the Excitation button and select the laser line you need. Select the appropriate transmission value ( if you don't know, how much, used 10%) 20 LSM 510 Meta tutorial

20 Make your Configuration for Single Track. Select a appropriate DBS2 in the Dichroic Beam Splitter list 2 Select a appropriate DBS1 in the Dichroic Beam Splitter list 1 Select a appropriate HFT in the Main Dichroic Beam Splitter 21 LSM 510 Meta tutorial

21 Make your Configuration for Single Track. Select the appropriate Emission Filter in the list. Click on the Channel assignement panel for select the desired color bar. 22 LSM 510 Meta tutorial

22 Image Acquisition Set Up Click the Scan button under the Acquire menu and the scan control window will open. Make sure the window indicates Mode and that Frame scan is clicked. Click the Find button and the computer will approximate levels and generate a starting image. 23 LSM 510 Meta tutorial

23 Image Acquisition Set Up The mode window allows you to adjust digital resolution, scan speed, pixel depth, scan direction, scan averaging, zoom, rotation, and offset and it tells you what objective you are using. You can change objectives from here if you want to. When you first start learning on the confocal you should try out these settings to see how they effect your image. Initial settings should look as they appear in the picture. Start with a 512 x512 frame scan. Once you find the area you want to image you can increase your resolution. The same goes for scan speed and averaging. You will almost always want to use 8 bit single scans for your applications. 24 LSM 510 Meta tutorial

24 Image Acquisition Set Up Click on the Channels button of the Scan control window. This window gives you control over the pinhole diameter, brightness and contrast of your image during collection. It also lets you control the laser intensity. By clicking the Ch1 or Ch2 buttons, you can choose which laser line you wish to adjust. Always adjust both lines before making your final scan. Pinhole: the pinhole diameter determines the thickness of the optical section you will look at. By increasing the pinhole diameter/optical section, you are also increasing the out of focus regions. Try to use the 1 Airy Unit to start with. The ideal voxel size should be calculate before you acquire any image, this voxel size is dependant of the objective, the format and zoom factor. Check the Resolution Rule/ Format page; = 25 LSM 510 Meta tutorial

25 Channels Window Functions Detector gain: controls the sensitivity of the detector and will increase or decrease the brightness of your image. So if you have a dim sample you can increase the detector gain and boost the intensity of the light detected in your sample better than opening the pin hole so always try this first. This is a gain setting and will amplify your signal including noise if set too high. Ampl. Offset: will adjust the black level, or the background so it affects the image contrast of your sample. If the black level is too high, you may be blocking out some of your signal so use sparingly in most cases. Ampl. Gain: this will boost your signal by increasing the overall intensity of the collection. Because of this you also will increase the noise in your collected image very bad. Always try to keep the gain as low as possible. Excitation: controls the intensity of the excitation lasers. The HeNe lasers can be boosted to 100% with out any problems. The argon laser should not need to be run over 30% when possible. If your signal is too dim, try adjusting the detector gain or try using a broad band dichroic during collection before boosting the laser. 26 LSM 510 Meta tutorial

26 Scanning Your Sample Your goal is to produce an image which represents the way your sample looks not the way you want it to look. This takes practice. First click Fast XY and using the fine focus on the microscope, focus up and down through your sample. Stop at the brightest area of the sample. Now you will use the Palette to help you produce a pseudo-colored image. 27 LSM 510 Meta tutorial

27 Warning The following instructions are meant to be used as a guide in collecting a standard sample that being said, there are no standard samples. If you are having trouble obtaining an image of your sample, do not waste time trying to figure it out on your own Call me (Thierry int.39603) and I will get you going. I can help you on your first couple of sessions with your sample, to make sure you are getting what you want. Let me know when you sign up so I can get you on your way. If you have a double labeled sample it is best to scan it using the Multi- Tracking option to prevent bleed through. 28 LSM 510 Meta tutorial

28 Scanning Your Sample First click Palette on the image window tool bar and the Color Palette window will open. Click Range Indicator. Now you will have an image where the red represents the white saturated pixels and the blue represents the black saturated pixels. Click fast XY on the scan window and change the Detector gain and Ampl. Offset to adjust your image to have just a few red and a few blue pixels. Make sure you do this for each channel. Click stop, palette and then no palette to see your image. 29 LSM 510 Meta tutorial

29 Adjusting for Your Final Image Once you are satisfied with the range of saturation on all channels, you can create your final image. Go back to the Scan Control window by clicking the Mode button. First perform a Single scan. At this point you can need increase the resolution or frame size by clicking on You can clean up details of the image by averaging your scan. Usually you will do a Line average with a Mean calculation and a average number of 4 8 or 16 (best). 30 LSM 510 Meta tutorial

30 Adjusting for Your Final Image Here is the difference in the initial image, the final image and view of the individual channels. 31 LSM 510 Meta tutorial

31 Saving Your Image When you are satisfied with your image you need to save it to your database. Press save as in the image window and the save window will open. Choose your database or create a new database by clicking the New MDB button. Name the file and include any other information in the description of notes sections. 32 LSM 510 Meta tutorial

32 Make your Configuration for MultiTrack. Select Multi Track for sequential mode. Select a stored SINGLE TRACK (from one channel) and pull down menu, click on Apply. Add a stored second (and third) SINGLE TRACK channel and pull down in the menu, click Apply 33 LSM 510 Meta tutorial

33 Shutting Down the System Click the Laser icon and turn Off the lasers. The HeNe lasers will turn Off and the Argon laser will turn off but you will need to wait about 5 minutes for the fan to cool the laser. When the laser is cooled you will hear the fan go off. 34 LSM 510 Meta tutorial

34 Shutting Down the System While you are waiting for the Argon laser to cool, you can exit the Zeiss LSM software by clicking the X in the right corner of the LSM 510 menu bar. A message will come up reminding you not to power down the system until the laser is cool. Click OK. Somebody would like use the microscope after you.choose the LOGOUT option. This will log you out of the system. If you do not logout, the system will continue to charge time to your account. Nobody use the microscope after you.go to Start and shutdown to shutdown the computer once the computer is safe to be shut down and the fan has clicked off, turn off the remote control. Leave the objective in place. Make sure you have cleaned off any oil with lens paper and glass cleaner. You are finished thank you for your careful cooperation. 35 LSM 510 Meta tutorial

35 Resolution Rule The sampling frequency is an important parameter which governs the resolution of the acquired image. Regarding the Nyquist theorem the smallest resolvable structure (in this case defined by the optical resolution limit) must be sampled (at least) twice in order to record all necessary information. The maximum optical resolution resel* can be defined as the radius of the first dark fringe in the diffraction pattern, or half the diameter of the Airy disc. *Robert H.Webb, Confocal optical microscopy, Rep.Prog.Phys. 59 (1996) ) Resel, confocal xy-plane = 0.44 NA Resel, confocal axial = 1.5 n NA² Example 500nm, n =1.518, NA = 1.4 XY- plane resel = 157 nm Axial resolution = 580nm With Nyquist criterion the ideal voxel is 78.5 nm in XY and 290nm in Z 37 LSM 510 Meta tutorial

36 Resolution Rule The following table shows two different voxel sizes. One is the optimal voxel size following the Nyquist theorem the other voxel size is proposed by SVI for doing deconvolution (Huygens). For doing Deconvolution it is recommended to use the voxel size proposed by SVI if you sample allows it. But be aware of the fact that a smaller voxel size leads to more photobleaching. If bleaching is an issue, you can also use a voxel size following the Nyquist theorem. For standard imaging a voxel size following the Nyquist theorem is totally sufficient. If you don t have to optimize for maximum resolution even undersampling (larger voxel size) can be an option. LSM 510 VOXEL SIZE TABLE ideal voxel size for confocal microscope ( in 488nm SVI Resel lsm510 xy z xy z 20x 0,4 air x 0,6 air x 0,8 imm x 1,0 oil x 1,4 oil SVI Formula XY = lex/(8 n sin a) Z = lex/(4 n (1- cos a) Resel* /2 XY = (0.44 lex/na)/2 Z = (1.5 n l/na2) / 2 Nyquist Calulator from SVI = Resel* = the sesolution can be defined as the radius of the first dark fringe in the the diffraction pattern, or half the diameter of the Airy disc. *(Rober H. weeb, Confocal optical microscopy, Rep. Prog. Phys. 509 (1996) ) 36 LSM 510 Meta tutorial

37 Various configurations Channels Rule about: laser/dichroïde filter/emission filter/channel, setting for 1 Channel Dapi with a LP Filter Fitc/Gfp with LP Filter Dapi with a BP Filter Laser Line Labeling DAPI DAPI Main Dichroide 405/488/543/ 405/488/543/ Second Dichroide Mirror Mirror Third Dichoide Mirror Mirror Emission Filter LP 420 BP Channel 2 2 Pinhole PH2 PH2 Fitc/Gfp with BP Filter Fitc/Gfp with BP Filter Laser Line Labeling FITC-GFP FITC-GFP FITC-GFP Main Dichroide HFT 488 HFT 488 HFT 488 Second Dichroide Mirror Mirror Mirror Third Dichoide HFT 490 HFT 490 HFT 490 Emission Filter LP 505 BP BP Channel Pinhole PH3 PH3 PH3 Cfp with a LP Filter Yfp with a BP Filter Laser Line 514 Labeling YFP Main Dichroide HFT 458/514 Second Dichroide Mirror Third Dichoide HFT 490 Emission Filter BP Channel 3 Pinhole Cfp with a BP Filter Cfp with a BP Filter Laser Line Labeling CFP CFP CFP Main Dichroide NFT 488 NFT 488 NFT 488 Second Dichroide Mirror Mirror Mirror Third Dichoide Mirror Mirror Mirror Emission Filter LP 475 BP BP Channel Pinhole PH2 PH2 PH2 Please look the next page setting for 1 Channel PH3 38 LSM 510 Meta tutorial

38 Various configurations Channels Rule about: laser/dichroïde filter/emission filter/channel, setting for 1 Channel Rohd TexRed LPFilter Rohd TexRed BP Filter CY5 a LP Filter Laser Line Laser Line 633 Labeling ROHD CY3 TexasR ROHD CY3 TexasR Main Dichroide HFT 488/543 HFT 488/543 Second Dichroide Mirror Mirror Labeling Main Dichroide Second Dichroide CY5 UV/488/545/633 Mirror Third Dichoide HFT 490 HFT 490 Emission Filter LP 560 BP Channel 3 3 Third Dichoide HFT 490 Emission Filter LP 650 Channel 3 Pinhole PH3 PH3 Pinhole PH3 Please look the next page setting for 2 Channels 39 LSM 510 Meta tutorial

39 Various configurations Channels Rule about: laser/dichroïde filter/emission filter/channel, setting for 2 Channels Dapi and Fitc/Gfp Laser Line labeling DAPI FITC Main Dichroide 405/488/543/ 405/488/543 Second Dichroide Mirror Mirror Third Dichoide NFT 490 NFT 490 Dapi and Rohd/Cy3/TexasR Laser Line labeling DAPI ROHD CY3 TexasR Main Dichroide 405/488/ /488/543 Second Dichroide Mirror Mirror Third Dichoide NFT 545 NFT 545 Emission Filter BP LP 505 Emission Filter BP LP 560 Channel 2 3 Channel 2 3 Pinhole PH2 PH3 Pinhole PH2 PH3 Dapi and Cy5 Laser Line labeling DAPI CY5 Main Dichroide UV/488/543/633 UV/488/543/633 Second Dichroide Mirror Mirror Third Dichoide NFT 490 NFT 490 Emission Filter BP LP 650 Channel 2 3 Pinhole PH2 PH3 Please look the next page setting for 2 Channels 40 LSM 510 Meta tutorial

40 Various configurations Channels Rule about: laser/dichroïde filter/emission filter/channel, setting for 2 Channels Fitc/Gfp and Rohd/Cy3/TexasR Laser Line labeling FITC-GFP ROHD CY3 TexasR Main Dichroide HFT 488/543 HFT 488/543 Second Dichroide Mirror Mirror Third Dichoide NFT 545 NFT 545 Emission Filter BP LP 560 Channel 2 3 Pinhole PH2 PH3 Fitc/Gfp and Cy5 Laser Line labeling FITC-GFP CY5 Main Dichroide UV/488/543/633 UV/488/543/633 Second Dichroide Mirror Mirror Third Dichoide NFT 545 NFT 545 Emission Filter BP LP 650 Channel 2 3 Pinhole PH2 PH3 Cfp and Yfp/Gfp Rohd /Cy3/TexasRed and Cy5 Laser Line Laser Line labeling CFP YFP Main Dichroide 458/ /514 Second Dichroide Mirror Mirror Third Dichoide NFT 515 NFT 515 labeling ROHD CY3 TexasR CY5 Main Dichroide UV/488/543/633 UV/488/543/633 Second Dichroide NTF 635 VIS NTF 635 VIS Third Dichoide NTF 490 none Emission Filter BP BP Emission Filter BP Meta Channel 2 3 Channel 3 Meta Pinhole PH2 PH3 Pinhole PH3 PH1 Please look the next page setting for 3 Channels 41 LSM 510 Meta tutorial

41 Various configurations Channels Rule about: laser/dichroïde filter/emission filter/channel, setting for 3 Channels Dapi/Fitc or Gfp/Rohd TexR Dapi/Fitc or Gfp/Cy5 Laser Line Laser Line Color Dapi Fitc Rohd Color Dapi Fitc Cy5 Main Dichroide 405/488/ /488/ /488/543 Main Dichroide UV/488/543/633 UV/488/543/633 UV/488/543/633 Second Dichroide NFT 545 NFT 545 NFT 545 Third Dichoide NFT 490 NFT 490 NFT 490 Second Dichroide NFT 635 VIS NFT 635 VIS NFT 635 VIS Third Dichoide NFT 490 NFT 490 NFT 490 Emission Filter BP BP Meta Channel 2 3 Meta Pinhole Ph2 Ph3 Ph1 Dapi/Rohd TexRed/Cy5 Laser Line Color Dapi Rohd Cy5 Main Dichroide UV/488/543/633 UV/488/543/633 UV/488/543/633 Second Dichroide NFT 635 VIS NFT 635 VIS NFT 635 VIS Third Dichoide NFT 545 NFT 545 NFT 545 Emission Filter BP BP META Chanel 2 3 Meta Emission Filter BP BP META Channel 2 3 Meta Pinhole PH2 PH3 PH1 Fitc or Gfp/Rohd TexRed/Cy5 Laser Line Color Fitc Rohd Cy5 Main Dichroide UV/488/543/633 UV/488/543/633 UV/488/543/633 Second Dichroide NFT 635 VIS NFT 635 VIS NFT 635 VIS Third Dichoide NFT 545 NFT 545 NFT 545 Emission Filter BP BP META Chanel 2 3 Meta Pinhole PH2 PH3 PH1 Pinhole PH2 PH3 PH1 Please look the next page setting for 4 Channels 42 LSM 510 Meta tutorial

42 Various configurations Channels Rule about: laser/dichroïde filter/emission filter/channel, setting for 4 Channels Dapi/Fitc/Rohd TexRed/Cy5 Laser Line Color Dapi Fitc Rohd Cy5 Main Dichroide UV/488/54 3/633 UV/488/54 3/633 UV/488/5 43/633 UV/488/54 3/633 Second Dichroide NFT 545 NFT 545 NFT 545 NFT 545 Third Dichoide NFT 490 NFT 490 NFT 490 NFT 490 Emission Filter BP BP Meta META Channel 2 3 Meta Meta Pinhole Ph2 Ph3 Ph1 Ph1 43 LSM 510 Meta tutorial