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Z-stack Dialog

1. First/Last or Center

•

Establish the stack acquisition parameters.

2. Optimize Sectioning and Step

•

Ensure the pinholes are set to the same section

thickness for multiple wavelengths.

3. Correction

•

Options for adjusting refractive index and/or

auto brightness to ensure even illumination

through the stack.

1

2

3

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Summary of Contents for 880

Page 1: ...Zeiss 880 Training Notes Zen 2 3...

Page 2: ...h Turn on the HXP 120V Lamp Turn on the Components Switch Turn on the PC and log into your account Start Zeiss Zen via Elan Tracking Software No Create scanning configuration 880 Start Up Sequence Set...

Page 3: ...omain 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 Enter your UCHC ema...

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: ...n 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...

Page 6: ...shion 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 an...

Page 7: ...figuration 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...

Page 8: ...e 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 2 Select your light source from Transmitted ON Tra...

Page 9: ...he software be sure to go to 1 View Show All global 2 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 t...

Page 10: ...ntrol Tool Bar AF Find Focus computer determines focus based on contrast settings Set Exposure the computer automatically calculates the gain and black level settings Live initiates a fast scan regard...

Page 11: ...ye from the drop down menu Repeat this procedure to select additional dyes Depending on your imaging needs the Smart Setup will offer you up to four different configurations for imaging your sample Fa...

Page 12: ...t 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...

Page 13: ...erent 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 multipl...

Page 14: ...ile 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...

Page 15: ...near 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...

Page 16: ...or they can be used in combination with multiple modules The modules are initiated via the Start Experiment button 1 Time Series collect a series of images based on a specific time interval and durat...

Page 17: ...Modules cont Bleaching module for FRAP fluorescent recovery after photobleaching Regions drawing tools used to customize and restrict the scan area Tile Scan create larger images by tiling stitching s...

Page 18: ...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 The red highlighted color indicates the laser i...

Page 19: ...Setup application your tracks will automatically be configured for you 2 Switch track every Line tracks are switched line by line Laser line laser intensity and channels can be changed between tracks...

Page 20: ...um of four tracks can be created which specify the dye laser excitation line and the emission detection range 2 Visual representation of excitation line s and the emission profile s which you can set...

Page 21: ...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 2 Select laser wav...

Page 22: ...ize defaults to 512 x 512 Select Optimal for appropriate number of pixels depending on numerical aperture and excitation wavelength Line Step scan every line or less than every line 3 Speed adjust sca...

Page 23: ...to reduce photobleaching 5 Bit Depth select from 8 bit 256 gray levels 12 bit 4096 gray levels or 16 bit 65536 gray levels Be sure your software can handle the information 12 and 16 bit images are rec...

Page 24: ...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 2 Lasers enable or disable and modify laser power 3 Pinhole set pinhole to 1 AU...

Page 25: ...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 p...

Page 26: ...alysis Use the Range Indicator to adjust the gain and offset Blue minimum Red maximum 0 and 255 for 8 bit image 0 and 4095 for 12 image 0 and 65536 for 16 bit image Adjust Detector Gain and Amplifier...

Page 27: ...fixed step size in microns Move up or down in the focal plane based on that step size 2 Z position specifies location of focal plane with regard to a user set zero position 3 Home zeros the focal pos...

Page 28: ...dinates indicates stage position 2 Step move specified step size in microns 3 Set zero establish a zero point 0 0 on the stage 4 Move to zero move to the zero point on the stage 5 Marks generate a lis...

Page 29: ...to collect multiple images of different focal planes within a single file for 3 D analysis display 1 You can enable the z stack dialog from advanced features menu 2 Scroll down to access and then ope...

Page 30: ...uisition parameters 2 Optimize Sectioning and Step Ensure the pinholes are set to the same section thickness for multiple wavelengths 3 Correction Options for adjusting refractive index and or auto br...

Page 31: ...of stack in microns Slices number of slices making up the stack Interval distance between each slice in microns Smallest calculates the slice to be the z resolution for proper Nyquist sampling Keep In...

Page 32: ...gths ensures that the pinholes are yielding the same section thickness 2 Optimal calculates each slice to be the z resolution for proper Nyquist sampling 3 Undo clears selection 4 X Y Z 1 sets the z i...

Page 33: ...ay Options 1 Ortho orthogonal display xy yz and xz options 2 Cut display a particular slice of stack and have options for manipulating that slice 3 2 5D layered depth display 4 3D reconstruction of z...

Page 34: ...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 3 2 1...

Page 35: ...intensities along defined regions or lines 2 Co localization display spatial overlap of fluorescent regions in different channels 3 Histogram display of image intensities and their frequencies within...

Page 36: ...parameters for image being displayed 5 Crop isolates a smaller region of the image and optically zooms the image 6 Positions when you click on the image a crosshair will be displayed this will corres...

Page 37: ...ab 1 Channel selection modify display settings for all or individual channels 2 Min Max Best Fit methods for adjusting contrast settings 3 Black White Gamma set max and minimum levels and non linear h...

Page 38: ...Image Display Features 1 Player controller for reviewing z stack or time series data 2 Graphics image overlays include scale bars text and other various drawing elements v 1 v 2...

Page 39: ...re you using the argon laser Within the software put the Argon laser in Stand by Yes No Exit Zen and log off the computer Shut Down Sequence Verified Next User Coming Exit Zen and log off the computer...

Page 40: ...lect Shut Down Yes No Turn off the HXP 120V lamp Exit Zen and shut down the computer Has the laser fan turned off LET THE LASER COOL DOWN Yes No WAIT Complete Shut Down Sequence 1 Turn off the Compone...

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