Zeiss Laser Scanning Microscope 510. Zuidhorst 178. For more information contact. Tom Groothuis. ZH153,

Transcription

1 Zeiss Laser Scanning Microscope 510 Zuidhorst 178 For more information contact Tom Groothuis ZH153,

2 Start Hardware Remove the blue dust cap from the microscope. If needed, turn on the mercury short arc lamp light switch (will not start before next step) Note: Whenever the mercury lamp is turned ON, it should be left on for at least 30 minutes. Once the lamp has been turned OFF, it should not be turned on again for 30 minutes. Turn on the whole microscope system with the remote control Turn on the PC power (Note: if the computer was already on before the rest of the system, the computer might not recognize the system and you cannot make images)

3 Start Software Log on to windows XP with the account: user and password Confocal Start the Zen software by clicking the Zen icon The Zen program will start and show a selection menu were you can choose between Start System and Image Processing. Choose the first one, after which the hardware will be initialized. This may take up to 1 minute. Tool groups with tools: I.Setup Manager I.i.Laser I.ii.Imaging Setup I.iii.Light Path II.Online Acquisition II.i.Acquisition Mode II.ii.Channels II.iii.Focus II.iv.Regions III.Multidimensional Acquisition III.i.Z-Stack III.ii.Bleaching III.iii.Time Series III.iv.Information On Experiment

4 Choosing and Changing Objectives Corrections Magnifi cation N.A. Spec. Optical Immer sion Cov.Gl. (mm) Work dist. (mm) Plan Neofluar 10x 0.3 DIC Plan Neofluar 20x 0.5 DIC/Ph LD-Achroplan 40x 0.6 DIC C-Apochromat 63x 1.2 DIC Water Select a proper objective. The objective should suite your desired MAGNIFICATION and THICKNESS of the material between the specimen and the objective. LD-Achroplan 63x 0.75 Ph Plan-Apochromat 63x 1.4 DIC Oil Change objectives: I. In the Light Path tool - Ocular tab II. With the Objective buttons on the microscope Select a proper sample holder from the 4 holders available. (e.g. cell culture plate holder, object glass holder, heated holder). Note: make sure the red dot of the holder (blue arrows) is in the same corner as the red dot on the microscope stage (white arrow) and clamp it.

5 Mount and Find the Specimen Change the microscope-light path to direct the light (white or fluorescence) to the eyepieces by clicking on the Ocular button in the Zen toolbar. FL HAL Reflector The mercury-lamp-light can be shuttered ON and OFF by the FL button on the body of the microscope on the right hand-side. Bright-field, halogen light can be switched on and off HAL button on the body of the microscope. The fluorescence filter block (a.k.a. reflector) carousel can be switched back and forward using the two buttons marked Reflector on the body of the microscope, behind the right hand focus knob. The reflector contains 3 filter blocks: Name Ex. D.M. Em. DAPI (01) BP 365/12 FT 395 LP 397 FITC (09) BP FT 510 LP 515 Rhod. (15) BP 546/12 FT 580 LP 590 Once you have found your specimen, redirect the light-path to in the confocal by clicking the LSM button. The microscope will automatically change the filterposition and shutter the light sources

6 Starting the lasers Open the Laser Tool and activate the lasers you need for your experiment. Remember, the argon multi-line laser has to first be put to standby for a 5 minute warm-up before it changes to on. The activated lasers are indicated with a color icon of a laser warning sign. When all the power statuses of the desired lasers read On, close the Laser Tool.

7 Configure the Lasers and the Light-Path Open the Light Path tool - Acquisition tab. The Light Path tool displays the selected track configuration which is used for the scan procedure. You can change the settings of this panel using the following function elements: Activation / deactivation of the excitation wavelengths (check box) and setting of excitation intensities (slider). If necessary open the Laser Control tool (see above). Selection of the main dichroic beam splitter (HFT) or secondary dichroic beam splitter (NFT) position through selection from the relevant list box. Selection of an emission filter through selection from the relevant list box. Activation / deactivation of the selected channel (check box in front to icon) for the scanning procedure and assigning a color (Look Up Table, LUT) to the channel. Open the Imaging Setup tool. The Detection Bands & Laser Lines assigned in the previous step are displayed in a spectral panel to visualize the activated laser lines for excitation (vertical lines) and activated detection channels (colored horizontal bars). You can save the settings of a Track at the bottom of the tool in pro mode (click ʻproʼ to activate/deactivate).

8 Setting the Parameters for Scanning Open the Acquisition Mode tool (pro-mode) Set the image size to the size you want your images to have (512 x 512 is a good start setting). Set the scan speed (faster scanning gives smaller time interval between images, but lower signal-to-noise ratio; slower scanning gives longer time intervals, but higher signal-to-noise ratio). Averaging will increase the signal-to-noise ratio, but longer time intervals. You can select to average frameby-frame or line-by-line in pro-mode. For quantification of your images, select a bit depth (dynamic range) of either 8-bit (256 intensity-values) or 12-bit (4096 intensity-values). Set the Scan Area by changing the zoom and the xyrange with the sliders or by dragging the square to the site of interest; you can rotate your image with the rotation slider. To reset a setting, push the respective button on the right hand side of the setting: C, 0 or 1.

9 Acquire Preliminary Confocal Image Open the Channels tool Set your preferred pinhole size for each channel: click 1 AU for high z-resolution (confocal) images, but lower fluorescence signal; click Max for low z-resolution, but maximum fluorescence signal. Click the Find button of the action buttons in the upper left corner. This will start the laser scanning for automatic preadjustment of detector gain and offset in the Channels tool. If applicable, Zen will also generate a new tab for the image. NOTE: Zen will only open a new tab if there is no image open or if the previous image has been saved manually. The preview image will look something like the image shown on the left. For multi-label experiments, it is best to have all single channels displayed separately. To do this: click the Split tab next to the image.

10 Optimizing the Settings Expose (if not already visible) the View Dimensions section of the screen. To toggle between the ascribed LUT and the range indicator LUT simply click on the color field in the button under the channel button (blue circle) Note: To get an optimal image that you can use for quantification it is necessary to use the full dynamic range you selected above (8- or 12-bit). The dynamic range can be shown with the Range Indicator LUT (Look Up Table; the microscopeʼs photomultipliers (PMTs) catch photons, which donʼt have a color, therefore you gave them a false color representation (a LUT) in the Light Path configuration above). The split view of a Green, Red and Merged image (check box in lower left corner of the image) with range indicator LUTs will appear as shown below. Toggle a channel On - Off with the respective button (blue arrow) and select a different LUT with the pulldown menu (white arrow).

11 Optimizing the Settings (2) Expose (if not already visible) the Channels tool. For continuos scanning during optimization click either the Fast button or the Continuous button of the action buttons Note: Continuous differs from Fast in that it is already using the Parameters for Scanning you assigned above, and Fast does not. So continuous will give a more representative image of the end result, while Fast allows fast optimization. Adjust the Gain and Amplifier Offset for each channel such that you get an optimal image. You may also decrease the scanning speed or increase the laserpower to get more signal, but beware of photobleaching and sample heating in the latter case. After you optimized the channels, stop scanning by pressing the Stop button. Acquire the final image by pressing the Start button. Your image will look like shown below.

12 Saving and exporting the image(s) To Save an image click the respective icon in the Open Images section. Save an image as an LSM 5 file to preserve all metadata (objective, zoom, light path etc. used). This metadata is important for calibration (x,y,z dimensions of an image in µm) and Reuse of the settings. Saving an image can also be done via the File menu. Store your images on the D:\ partition of the hard-drive. The folders are being shared, you can find the computer in the TNW directory: BFT0151 You can Reuse the settings of an image by opening the image and then click the Reuse button in the bottom of the View Dimensions section (Note: this will NOT turn the lasers to the ON state, do this manually). Use the View Overlay section to overlay the image with text / scalebar / arrows etc. Once your image is complete, use Export in the File menu to export the image as you like. There are several options, please check them to see if the result is the same as you intended.

13 Acquiring a Z-series Open and activate the Z-Stack tool in the Multidimensional Acquisition. Note: To activate the tool, click the check-mark of the tool (blue arrow), this will activate z-stack imaging as shown by the little icon (blue circle) below the action buttons. I. Make sure you have a proper pinhole setting for z- stacks. II. Activate scanning by using the Fast or Continuous action button. III. Change the z-level manually at the microscope to the top most position you want to image and click the Set First button. IV.Change the z-level manually at the microscope to the bottom most position you want to image and click Set Last button. V. Click the Optimal Interval button so the software can determine the optimal number of slices for the chosen Range (First and Last) and Pinhole thickness (indicated on the Optimal interval button). VI.Activate the scanning procedure with the Start button. The result can be displayed in Gallery view (below) and some other views as indicated by the view-tabs.

14 Multitrack imaging (= sequential imaging) When you are using multiple dyes, fluorescence of one fluorophore may also be detected in a ʻwrongʼ channel. This phenomenon is named cross-talk, leak-through or bleed-through. Fluorescence from one channel detected in another is shown below (blue circles). You can test for leak-through by setting the transmission of a laser to 0% in the Channels tool during Fast or Continuous scanning. This will be so if there is still fluorescence detected in e.g. the red channel when the 543 nm laser is set to 0%. To overcome the problem of leak-through you can make use of multitrack scanning, this will make a scan of each label one after the other. Open the Imaging Setup tool Use the Track + and - buttons to add or remove a track. Configure a Light Path for each track as above. Optimize the settings (as in Optimizing Settings) for each channel separately by un-checking the check-box in front of the other track. Push either the Fast or Continuous action button to start the scanning. You can Save the Configuration in the top of the Imaging Setup tool.

15 Shutting the System Down Turn Off the lasers in the Laser tool In the Laser control window turn each laser Off or to Standby if somebody is using the system within the next hour (check if the person shows up!!!). Remove Specimen and clean microscope Wipe-off water or oil from objective and specimen. Move to a low power objective (10 or 20x). Lower the objective nosepiece using the lower focus button on the right hand side of the microscope base. Turn off the epifluorescence lamp if nobody is using the system within the next hour (check if the person shows up!!!). Cover the microscope avoiding the hot mercurylamp-housing. Exit the software Exit the Zen software. A message will come up reminding you not to power down the system until the laser is cool (about 5 minutes). Click OK. If you have left the lasers on for the next user, you will also be asked whether you want the lasers switched off. Click No. Burn your data to CD or copy across network. Once you have finished with the computer, LOGOUT of SHUT-DOWN. Power down the system If nobody has booked for the next hour, please shutdown the system If the next person is the last booking of the day, please call them and confirm that they will be using it. Shutting down requires that the remote control be switched off.

16 Offline Image Analysis and Manipulation Opening your images offline Zeiss Zen A downloaded file of Zen is ready for installation in D:\Software\ and can be used for Off-line use, no license needed. You can also download the newest version yourself at After the animation you can find a link in the lower left corner. This does require to fill in a registration form. Requires Microsoft Windows XP ImageJ An open source Java application for multiple platforms: Has a lot of plugins available, including Zeiss plugin:

17 Troubleshooting: Error messages The Error Log pops-up with a COM2 Error The 2-photon, Chameleon laser can also be in use on another microscope next-door, therefor you have to book the Chameleon separately from the LSM. If the Chameleon is in use on the other system the LSM computer is not able to communicate with the laser, which will generate COM2 Errors. This is NO problem for using the rest of the features of the microscope. Leave the Error menu in the background or minimize it with the _ button in the upper right corner. If you encounter other COM errors, please contact the administrator. When you are loading a configuration (Track or Reused) you might get the warning displayed on the left. Loading a configuration can change objectives, filters, dichroic mirrors etc. but CANNOT turn a laser On, so do this manually.

18 Troubleshooting: Strange circles My images have strange fluorescent rings If you have multiple colors, please make sure you are not detecting one of the laser-lines, which are partially reflected by the coverglass. The reflected laser-light may end up in one of the detectors, but will be relatively strong compared to the fluorescence signal. A detected laser-line (543 nm) will appear as shown in the green channel below. Solutions: Use the BP nm filter for channel 2, so the 543 nm line will not be detected. Use a multitrack procedure.

19 Light Path Hardware Main dichroic beam splitter (HFT) The main dichroic mirror deflects the indicated laser lines onto the specimen and allows the emitted fluorescent light to pass through. A HFT KP XXX/YYY is a combination of a HFT YYY and HFT KP XXX used for simultaneous IR multi photon and single photon excitation. Example: HFT KP 700/488. A HFT KP XXX (KP = Short Pass) is a special type of a main dichroic used for IR multi photon excitation (Chameleon). The HFT KP 650, deflects laser light with a wavelength longer as 650 nm onto the specimen and allows fluorescent emission light in the visible range below 650 nm to pass through. NT XX/YY = Neutral Density Filter, works as an attenuation filter. NT 80/20 passes 80% of the light, cuts 20% of light. Secondary dichroic beam splitters (NFTs) A secondary beam splitter is used to split the emitted light which will be guided into separate channels. A cascade of NFTs (NFT 1,2,3) allows distribution of the emission light to more than two channels/ detectors. A None or Plate will let all light pass. A Mirror will reflect all light. A NFT XXX will deflect light with shorter wavelength than XXX nm, while light with longer wavelength passes the NFT. A BG 39 (Blue Green glass) blocks infrared excitation light by absorption.

20 Light Path Hardware Channel 1: KP 685 LP 475 Remark: BP = Band Pass LP = Long Pass KP = Short Pass BP IR = a band pass filter suitable for detection of IR excited dyes, which blocks the IR light. LP 505 LP 530 LP 560 BP IR LP 650 Channel 2: KP 685 BP IR BP IR BP IR BP BP IR LP 505 Channel 3: LP 560 BP IR BP IR BP IR BP IR Laser Lines: Chameleon 680 to 1080 nm