Zeiss LSM880 Operating Instructions. UTMB Optical Microscopy Core Jan. 16, 2018

Transcription

1 Zeiss LSM880 Operating Instructions UTMB Optical Microscopy Core Jan. 16,

2 1. Power up the microscope Sing the LOGBOOK Steps below will provide power to the computer and all of the microscope components. Turn the MAIN SWITCH on Wait 2 s Turn the SYSTEMS/PC on (STOP HERE FOR COMPUTER USE ONLY) Wait 2 s Turn the COMPONENTS on 2

3 2. Open the front and top doors of the plexiglas incubator Unless live cell imaging is planned, open 4 doors (2 front and 2 top) to allow easy access to the microscope stage. 3

4 3. Start ZEN software After the windows has been loaded, login by clicking on the LSM User icon. No password is required. On the desktop, click on the ZEN Confocal (black) icon to start confocal acquisition software. (ZEN blue software can be used for controlling the Axiocam and image analysis.) Click on the Start System button. The software will start recognizing the motorized components of the microscope. 4

5 4. Set the objective lens to be used IN ZEN software: In the Locate tab navigate to the Microscope Control window. In that window click on the objective icon to bring up a list of the available objectives (5X, 10X, 20X : AIR; 40X: WATER immersion; 63X: OIL immersion). Select one. ALTERNATIVELY using the touchscreen: Click: Control > Objectives > 20x. When changing from air to immersion objective, or in reverse, a warning to change the immersion media is shown. Click OK, when done. 5

6 5. Apply immersion media IF required Only 63X and 40X objectives require immersion media! 63X Oil immersion objective (has a rubber band around it) 40X Water immersion objective Use Immersol 518 F only (n=1.518). Open the bottle and apply a drop of the immersion oil with the applicator cap to the top of the lens. Use distilled water only (n=1.330). Open the tube and apply a drop of water with a plastic pipette to the top of the lens*. REMEMBER TO WIPE CLEAN THESE OBJECTIVES WITH LENS PAPER ONLY AFTER EACH USE AND ALSO BEFORE USE. *For live-cell experiments at 37C, Immersol W, a viscous water based media must be used to prevent evaporation. Ask the OMC personnel for guidance. 6

7 6. Put objective into the load position First, make sure the objective is in its lowest position (load position). To put the objective into the load position: press on the load button on the right side of the microscope or on the load button on the touchscreen. OR 7

8 7. Place your sample onto the stage Illum. head Adaptor frame Make sure that the stage adaptor frame is not loose/shaky. Tilt the illumination head, if needed. Place your sample coverslip down onto the stage. Do not clamp your slide tight. Pull the illumination head back forward. Using the joystick that controls the motorized sample stage center your specimen above the objective lens. 8

9 8. Bring the immersion objective up IF USING AN AIR OBJECTIVE READ, BUT SKIP DOING THIS STEP! Using the coarse/fine focus knobs on the microscope body bring the objective up (clockwise direction) until the immersion media just touches the coverslip. 9

10 9. Bring your sample into focus In the Locate tab, depending on the fluorophores in your sample, click on any of the four buttons to turn the excitation light on: TL/DIC : white light for brightfield Blue: for near-uv excitation (DAPI, EBFP) Green: for ~ 488 nm excitation (Alexa 488/ EGFP) Red: for ~ 540 nm excitation (Rodamine, Alexa 568/594) The UV source illumination intensity may be changed in the Microscope Control window by clicking on the UV lamp icon and dragging the slider. While looking into the oculars, bring the objective slowly up until you can clearly see your specimen. (If you are using the 40X objective, and the image appears fuzzy. You may then need to adjust the objective s collar ring to the appropriate thickness of your coverslip (default is 0.17mm)). Using the stage joystick to move the sample around find a suitable imaging area. (Minimize sample exposure time to reduce fluorophore bleaching!). Click on Set Work position button on the touchscreen, if present. After finding the spot click on the ALL OFF button to turn the light off. 10

11 10. Load confocal acquisition settings A) If you used this microscope before and saved your images in the.czi format, you may reload your previous settings: At the top of ZEN, click open file. Locate your.czi image and open it. In the Dimensions tab, locate the Reuse button. Click it to load settings. Beware: if the objective in the light path does not match to the one used before, the pinhole setting will not be set properly. See the Info tab and adjust the pinhole manually. B) If you are imaging for the first time or have a new set of dyes, you may then choose from one of the pre-set imaging protocols. In the Acquisition tab, navigate to the Experiment Manager field, click on the open file icon to bring up a list of available pre-set imaging protocols. 11

12 The list has protocols for confocal and airyscan imaging modes. Airyscan mode is not part of this learning module. Confocal protocols start with the word confocal followed by a set of numbers that correspond to the excitation wavelengths and image scan mode (Line/Frame). Line vs. Frame mode: in each of these protocols, images of fluorophores are acquired sequentially by switching on and off lasers in different acquisition tracks. In the line mode, lasers are switched on/off after each image line. In this mode, no hardware movements are allowed (pinhole, emission and excitations settings must remain static for all channels, only lasers can be switched on and off). It is the fastest mode. In the frame mode, lasers are switched on/off after each full image scan. It is the fastest mode if hardware movements are needed (setting pinhole for each track/channel, overlapping detection ranges, etc.). Example: For a sample stained with DAPI (ex. 360 nm), Alexa Fluor 488 (ex. 488 nm) and MitoTracker Red (ex. 569 nm) one may choose either Confocal-561_488_405 Line Zeiss or Confocal-633_561_488_405 Line Zeiss protocol. If the last protocol is chosen, the 633 channel/track can be deactivated or removed after protocol loading. 12

13 After selecting and loading of a protocol, required lasers will be automatically powered on. The status of each laser can be seen in the Laser window. Argon laser needs 5 mins of warmup after start before a stable emission is achieved. THIS IS FOR EDUCATION ONLY, DO NOT CHANGE: In the Imaging Setup window, one can switch between imaging tracks by clicking on their respective buttons (561 is selected in the image) Track switching mode selector line/frame. The bottom of the Imaging Setup window shows the light path configuration for each selected track. 13

14 LIGTH PATH CONFIGURATION (THIS IS FOR EDUCATION ONLY): Currently selected imaging track (561) Delete current track Graphical representation of the activated laser lines (vertical line(s)), theoretical emission spectra of fluorophore(s), and emission wavelengths being collected (shaded area(s)). One can change emission collection by dragging/resizing the bottom slider. Shows activated detectors in the current track (checked), Dye (its selection updates emission graphics above), Color (selects palette to display the image), Detector s name (total of 3, but 1 is used in this track), Range (shows emission collection range shaded area in the above graphics). IMPORTANT: Ranges must not overlap between tracks if track line switching to be uses! Clicking on either Visible or Invisible Light icon on the right one can checkmark laser lines to be turned on during this track activation. Correspondingly, the correct MBS or main dichroic beam splitter on the left must be chosen that would reflect the selected laser light onto the sample. Optionally, one can checkmark the transmission PMT (T- PMT) to also collect brightfield images in this track. 14

15 DETECTORS (THIS IS FOR EDUCATION ONLY): Ch3 Ch2 GaAsP Ch1 The emission light from fluorophore(s) is directed onto the diffraction grating that linearly disperses the light according to its wavelengths (similar to a prism creating a ray of rainbow from sunlight). The resultant rainbow can be cut and bend with sliders and prisms, respectively, to direct its portions to separate photomultipliers (Ch1, Ch2 GaAsP, Ch3) for intensity/photon flux detection. Ch2 GaAsP is a special detector with enhanced sensitivity to the visible light. 15

16 11. Optimize your detection channels In the Channels window, uncheck all but one channel (start with the shortest excitation wavelength channel), highlight the checked channel (by clicking on the line) to update the detector settings below. Important: The goal of channel optimization is to ensure that most of the pixels in your final image are not black or saturated (e.g. for an 8-bit image [0 to 255 intensity gray values; 0 black and 255 white(saturated)]), to accurately quantify differences in fluorescence intensities. At the very beginning of the Acquisition tab click on the Live button to star image scanning from the selected channel. At the bottom of the imaging window, in the Dimensions tab, check the Range Indicator box. This will color all 0 intensity pixels (below detection threshold) in blue and 255 intensity pixels in red (saturated) in the live image. 16

17 In the Display tab click on the Reset button to ensure that you are adjusting your image to the full histogram [0-255]. If you are using the line mode of switching between tracks, you must adjust the pinhole for one track and keep it the same for others. Adjust the pinhole by clicking on the 1AU button in the Channels tab*. *Note: 1AU sets the pinhole diameter to 1 Airy Unit (AU) (diameter ~ ex. wavelength). 1AU is the best compromise between light efficiency collection and optical section thickness. For dim samples pinhole diameter can be set wider (e.g. 2xAU) to increase light collection with a compromise in the axial/lateral resolution). Adjusting channels using Laser Power, Master Gain and Digital Offset sliders: Laser power adjustment slider to brighten pixels (too much laser >10% bleaches sample quicker) Master gain adjustment slider to brighten pixels (keep it >500 but <950 to avoid detector noise) Digital offset adjustment slider (controls background/blue pixels) Too many blue pixels & low signal. Solution: 1. Focus better 2. Increase digital offset to eliminate blue pixels 3. Increase laser power or master gain (to brighten pixels up). Too many blue pixels & high signal. Solution: 1. Focus better 2. Increase digital offset to eliminate blue pixels 3. Decrease laser power or master gain (to desaturate pixels up). Well-adjusted channel: No blue, no red pixels. Pixels are well stretched in the histogram. 17

18 After a successful channel adjustment click on the Stop button at the top of the tab to stop the live scan. In Acquisition mode window: After adjusting all channels one by one, check all of the channels. Uncheck the adjusted channel, check and highlight the next channel to be adjusted. Repeat the adjustments as before, but remember not to change the pinhole! Set image size in pixel by pressing on the X*Y button, typically 1024 by 1024 is sufficient. (Using the Optimal button sets the image size automatically for best possible resolution.) Speed slider adjusts pixel exposure time : the lower the speed the better the signal to noise ratio. Keep it around 7 or 8. Set the number of lines to average for a better signal to noise performance (scans # of lines, calculates and shows the average). To start, keep at 2 lines. Select bit depth (the precision with which fluorescent intensity is determent), typically 8 or 12 bit will suffice. Select bidirectional scan for faster scanning. Using these sliders, one can optically zoom into the sample, shift and rotate the scan area. (Leave it) 18

19 12. Taking a snap Click on the Snap button to take a picture of every channel, which will be displayed as an overlay in the image window. If you do not like the current focus. Click on the Live button, refocus or move the stage, and then click on Snap again. Pixel distribution will appear well stretched between 0 and 255 gray intensity values in the histogram for channels (shown in different colors) that have been optimally adjusted. 13. Saving your images In the upper right corner, locate the Images and Documents window. The current image will be highlighted, click on the Diskette icon to save the image. When it has been saved, the new file name will appear and the exclamation sign will be gone. Save images in the.czi format to be able to reuse your setting later on. 19

20 14. Finding new imaging area Click on the Live button. While scanning your can use the Stage window to move the motorized stage position (in place of the joystick) using inner slices by small steps, outer slices by large steps. Find another area, re-focus and take another snap. Save your Image. Small step Large step 15. Taking a Z-stack of images To start acquiring a z-stack or images: at the top of the Acquisition tab check the Z-stack checkbox. This will activate the Z-Stack window at the bottom of the Acquisition tab. Scroll to the bottom, find the Z- Stack window, click and pull it out to any convenient place on the screen 20

21 In the Z-Stack window click on the First/Last tab. Click on the Live button to start scanning. While scanning, use the fine focus knob to focus on where you would like the image stack to start (such as bottom of your sample) and then, click on the Set Last bottom. Then using the same knob focus on where you would like the image stack to end (such as the top of your sample), click on the Set First button. Stop the live scan. OR After setting your scan range, now you have to set the number of slices to be acquired. For a 3D reconstruction later on: Keep Interval selected, then click on the optimal interval button (number). This sets the interval between the slices at the Nyquist s sampling frequency (50% overlap between adjacent slices) for all channels to be scanned. If no 3D reconstruction is planned, or the number of slices is too many to scan with the optimal interval, you can select Keep Slice and then type the number of slices you would like to acquire into the Slices field. 21

22 After finishing with the Z-stack setting, click on the Start Experiment button. The microscope will begin taking pictures of your image slices. After the scan is done Save your Z-stack the same way as image snaps, see page Microscope shut down 1. Exit ZEN by clicking on the X icon. The software will prompt you to turn the lasers off, select OFF next to every laser. 2. Copy your images to your USB storage device. 3. Clean the objectives and close the incubator doors. 4. Safely unplug your storage device from the computer. 5. Shut down the computer. 6. If 5 mins have not yet passed since turning off the lasers in the software, WAIT for 5 mins. Then, turn all the switches off Components > Systems/PC >Main Switch. 7. Sign the logbook. 22

23 Confocal Imaging Suggestions Channel adjustment must be performed for every series of samples stained under identical conditions. It should be done at the onset of imaging and then the settings to be reused during later imaging sessions for similar/same samples, provided that you do not change staining conditions. Channels need ideally to be adjusted to the brightest sample/area in the series. Alternatively, channels may be adjusted to 50-80% of the maximum gray value to leave room for a higher intensity signal to prevent saturation. Example: Sample set: BHK 21 cells treated with increasing concentrations of Bengamide B and untreated controls. Cells are fixed with 4% PFA, permeabilized, blocked and stained with a primary antibody against NF-kB followed by a conjugated secondary antibody. It is advisable, in addition to treated and untreated BHK 21 cells to have these controls for imaging: unstained BHK 21 cells, BHK 21 cells incubated with only secondary antibody, and if more than one target/fluorophore is used, BHK 21 cells stained only for one target/fluorophore. IMPORTANT: Any change in the staining protocol (fixation method or duration, non-specific blocker change, change in antibody dilutions or incubation times) may require a readjustment of imaging channels, which may prohibit pooling with the previous results. 23