Zeiss 880 Training Notes
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Zeiss 880 Training Notes

Zeiss 880 Training Notes

Zen 2.3
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Zeiss 880
Training Notes
Zen 2.3

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  • Page 1 Zeiss 880 Training Notes Zen 2.3...
  • Page 2 Switch. Switch Switch Do you need the Argon Go to the Acquisition Tab and Start Zeiss Zen via Elan Laser initiate the warm up Tracking Software. 458, 488, or 514 nm lines? procedure for the laser. Set up sample on microscope.

  • Page 3: Logging Into The 780

    CAM credentials; be sure you are logging into the proper domain. Double click on the Zen icon, the one • labeled with “AUT-Zeiss LSM 880” or similar text. The idea elan tracking and billing software will initialize after clicking on the Zen icon.
  • Page 4 Starting Zen with idea elan Select your scheduled appointment • and/or click the login button as directed by the software. Confirm your appointment and the Zen • software will be initialized.

  • Page 5: Starting Zen

    Starting Zen 2.3 Select Start System from the Zen 2012 login window. This will start up the hardware and software. Image Processing is for using the system in an offline mode and this will ignore all the hardware components.

  • Page 6: Introducing The Zen Workspace

    Introducing the Zen Workspace The Zen workspace is designed in a tabular fashion allowing you to step you through initially looking at your specimen via the Oculars tab, in the control panel, to acquiring images via the Acquire tab. The remaining tabs add off- line Processing and Maintenance options.
  • Page 7 Introducing the Zen workspace Your imaging configuration and your work space configuration can be stored separately. In addition, your imaging configuration and acquisition parameters can easily be retrieved from the Reuse function when you have a previously saved image loaded in the workspace.

  • Page 8: Getting Light To The Oculars

    Getting Light to the Oculars Locate Tab 1. Click on the objective icon to access the list of possible objectives. Be sure to note the immersion type; air, oil or water. Please do not change from oil to air/dry or from water to oil and oil to water without cleaning your specimen first.

  • Page 9: Getting All The Tools

    Getting all the tools Acquisition Tab In order to view all the options available via the software, be sure to go to: View>Show All (global) Select >Show all Tools. Make sure to do this each time you use the software otherwise the required features, like the lasers, will not be accessible to you.

  • Page 10: Acquisition Tab

    Acquisition Tab 1. Experiment Manager The manager allows you to save your scan configuration settings for your experiment and reload the settings for a quick start. This method does not record the acquisition parameters. 2. Scan Control Tool Bar AF (Find Focus) – computer determines focus based •...

  • Page 11: Using Smart Setup

    Using Smart Setup Smart Setup The best and easiest way to get started is to use the Smart Setup tool. Click on the down arrow to open the dye selection menu. Select your dye from the drop down menu. Repeat this procedure to select additional dyes.

  • Page 12: Smart Setup - Fastest

    Smart Setup - Fastest Acquires the image with the simultaneous excitation and detection of one or multiple probes. This option produces a single image, with a single scan however it may result in bleed through between the different channels.

  • Page 13: Smart Setup - Best Signal

    Smart Setup – Best Signal Acquires the image with the sequential excitation and detection of the different probes. Each probe is scanned separately and the final image is the result of an overlay of the individual channels. This option avoids bleed through between the different channels however multiple scans are require to produce the image which can lead to bleaching or other laser...

  • Page 14: Smart Setup - Smartest (Line)

    Smart Setup – Smartest (Line) Smartest acquires multiple channels at the same time while quickly toggling laser lines to acquire a second track. There are limitations to this option, especially if you are acquiring 4 different probes. a) You must you use the same dichroics between the two tracks.

  • Page 15: Smart Setup - Linear Unmixing

    Smart Setup – Linear Unmixing This option creates a confirmation for computationally subtracting bleed through between channels. This can be performed after you acquire individual, pure spectral profiles of the probes you are using.

  • Page 16: Using The Advanced Scanning Modules

    Using the Advanced Scanning Modules Advanced scanning modules are available as a single option or they can be used in combination with multiple modules. The modules are initiated via the Start Experiment button. Time Series - collect a series of images based on a specific time interval and duration.
  • Page 17 Using the Advanced Scanning Modules (cont.) Regions – drawing tools used Bleaching – module for Tile Scan – create larger to customize and restrict the FRAP (fluorescent images by tiling/stitching scan area. recovery after smaller images together. photobleaching.)

  • Page 18: Using The Lasers

    Using the lasers The lasers that are available for use are: Diode 405 nm Argon 458,488,514 nm DPSS (Diode-pumped solid state) 561 nm HeNe (Helium-Neon) 633 nm The Argon, has a start-up and shut-down procedure. This can be monitored by opening the Laser Properties panel. red highlighted color indicates the laser is not available for use.

  • Page 19: Looking At The Light Path

    Looking at the Light Path 1. Tracks Create and/or delete tracks which contain the • scanning configuration for each probe or combination of probes that are scanned. The configuration consists of the laser line, excitation dichroic, detector and emission range that is captured.

  • Page 20: Imaging Setup And Light Path

    Imaging Setup and Light Path Track – defines the fluorophore(s) that is(are) being detected. A maximum of four tracks can be created which specify the dye, laser excitation line and the emission detection range. Visual representation of excitation line(s) and the emission profile(s) which you can set by adjusting the slider bars or alternatively you can enter the range at the drop down arrow.
  • Page 21 Imaging Setup and Light Path Tracks are defined by dye, pseudo color, detector, and emission range. The check mark indicates the track is enabled and will scan upon activation. Detectors – select from standard PMTs, Ch1 and Ch2 or the QUASAR detector ChS 1-8, and the transmitted light detector ChD (not confocal).

  • Page 22: Acquisition Mode

    Acquisition Mode Objective – change objectives, this is an alternative to going to the locate tab. Scan Mode, Frame Size and Line Step – Scan Mode - Frame, Line, Spot • Frame Size – defaults to 512 x 512. Select •...
  • Page 23 Acquisition Mode (cont.) Averaging – improves image by increasing the signal-to-noise ratio. Select from Line or Frame averaging and select the number to average by. Averaging helps to reduce photobleaching. Bit Depth – select from 8 bit (256 gray levels), 12 bit (4096 gray levels), or 16 bit (65536 gray levels).

  • Page 24: Working With Channels

    Working with Channels The channels dialog allows you to select an individual track and adjust the corresponding settings to improve the image quality. Tracks – as defined from the light path. Be sure to highlight the channel you wish to modify. If a track has a “check mark”, it is active and it will be scanned.
  • Page 25 Working with Channels(cont.) Gain, Digital Offset, Digital Gain Gain (Master) – amplifies the voltage on • the photomultiplier tube; regulates brightness of the image. Digital Offset – sets background/threshold • level or dark current of the image. Digital Gain – post acquisition •...

  • Page 26: Using The Range Indicator

    Using the Range Indicator Remember, when acquiring your images, you want to be within the dynamic range of the detector. This is especially critical when collecting images that will be used for quantitative analysis. Use the Range Indicator to adjust the gain •...

  • Page 27: Using The Focus Tool

    Using the Focus Tool The 780 offers the ability to manually control the focus or to use the software for more precise control of the focus. Step size – set a fixed step size, in microns. Move up or down in the focal plane based on that step size.

  • Page 28: Using The Stage Tool

    Using the Stage Tool Click in the direction you wish to move your Moves according to sample. set step size. The 780 allows you to manually control the stage via the joy stick or to use the software to control the stage.

  • Page 29: Acquiring A Z-Stack

    Acquiring a Z-stack Used to collect multiple images, of different focal planes, within a single file for 3-D analysis/display. You can enable the z-stack dialog from advanced features menu. Scroll down to access and then open the Z-Stack dialog.

  • Page 30: Z-Stack Dialog

    Z-stack Dialog First/Last or Center Establish the stack acquisition parameters. • Optimize Sectioning and Step Ensure the pinholes are set to the same section • thickness for multiple wavelengths. Correction Options for adjusting refractive index and/or • auto brightness to ensure even illumination through the stack.

  • Page 31: Defining First/Last

    Defining First/Last Set the first and last starting positions for the stack and acquire the slices within that range. Set Last – marks z position furthest away from the specimen, as mounted on the cover glass. Range – total thickness of stack, in microns. •...

  • Page 32: Optimize Sectioning And Step

    Optimize Sectioning and Step Match Pinhole – for multiple wavelengths, ensures that the pinholes are yielding the same section thickness. Optimal - calculates each slice to be ½ the z-resolution for proper Nyquist sampling. Undo – clears selection X:Y:Z=1 – sets the z interval so the voxel is the same for x, y, and z dimensions.

  • Page 33: Z-Stack Display Options

    Z-Stack Display Options Ortho – orthogonal display, xy, yz and xz options Cut – display a particular slice of stack and have options for manipulating that slice 2.5D – layered depth display 3D – reconstruction of z-stack...

  • Page 34: Image Display Options

    Image Display Options 1. 2D – x-y view of image 2. Gallery – display of z-stack or time series images. 3. Split – view multi-channel images as individual and overlay displays.

  • Page 35: Advanced Image Display Options

    Advanced Image Display Options Profile – display pixel intensities along defined regions or lines. Co-localization –display spatial overlap of fluorescent regions in different channels. Histogram – display of image intensities and their frequencies within the image. Information – summary of image file.

  • Page 36: Image Display Features

    Image Display Features Dimensions Tab Z position – step through optical z-stack slices Zoom – digital zoom feature Channels – range indicator option, pseudo-color selection, turn on/off channels that are displayed Ruse – load image acquisition parameters for image being displayed. Crop –...
  • Page 37 Image Display Features Display Tab Channel selection– modify display settings for all or individual channels. Min/Max, Best Fit – methods for adjusting contrast settings. Black/White, Gamma - set max and minimum levels and non-linear histogram adjustment.
  • Page 38 Image Display Features Player - controller for reviewing z-stack or time series data. Graphics – image overlays include scale bars, text and other various drawing elements.
  • Page 39 Exit Zen and log off the computer. Are you using the argon laser? Within the software, put the Exit Zen and log off the Argon laser in Stand-by. computer. Shut Down Sequence Verified Next User Coming...
  • Page 40 Within the software, Select Are you using the argon LET THE LASER COOL DOWN Stand-by and then select laser? Shut-Down. Has the laser fan turned Exit Zen and shut down the WAIT off? computer. Turn off Turn off the Systems PC Turn off the Components Main Power Switch.

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