zeiss 710 confocal microscope user guide - cornell
TRANSCRIPT
Cornell University - Biotechnology Resource Center – Imaging Facility
Zeiss 710 Confocal Microscope User Guide B46 Weill Hall
BRC Imaging Facility 8-6-2014
1
Table of Contents
Startup – General Procedure 2
Microscope General Info 2
To see your sample thru the microscope 3
Zen software – Locate (Eyepieces) 4
Zen software – Acquisition 5
Configurations 5
Smart Setup 5
Initial Settings for Live Scan 7
Confocal Scanning 8
Saving Image Files 9
Z-Stack 10
Saturated Pixels 11
Scale Bar 11
Optimizing Image Quality Chart 12
Multi-tracking (Sequential Imaging) 13
Time Series 14
Tile Scan 14
Reflected Light Confocal 15
Transmitted Light (Bright Field) 15
Positions or Multi X-Y-Z 16
PhotoBleaching (FRAP) or Photoactivation 16
Definite Focus 17
Zen Light 2012 Browser 18
Adjusting the Brightness and Contrast 19
DIC 20
Specifications 21
Common Errors and Problems 22
2
Startup – General Procedure
See instructions posted Login to computer with net id and netid password
Start Zen software
Start System
Choose objective, add immersion if needed
Place sample on stage
Focus on sample with bright field or fluorescence
Switch to Confocal Acquisition
Load configuration
Start Live Scan
Microscope General Info
Objectives Right focus knob, far buttons will change objectives.
Objectives are covered with spill protectors but are in
this order:
10x is dry –has large top lens and yellow ring
25x is multi-immersion, has red and green top rings
o Remove protective cover
o Set black line on knurled ring for immersion
Oil use red tape setting
Water use blue tape setting
40x is water immersion, has blue ring, set knurled ring
for cover slip thickness (black line to 0.17 = #1.5)
63x is oil immersion, has black ring and paper collar Note: See Stats at end for more info about objectives
40x H2O cover slip setting 63x Oil 40x Dipping
10x Dry 25xOil/H20 H2O setting Oil setting
Ending your Session:
Check schedule
Save your images
Exit Zen software
Transfer your files to
Imaging Lab FileShare
Logoff Windows
Lower objective, remove sample
Wipe oil or water off objective
STOP HERE
Total Shutdown:
Start as above
See instructions posted
Also:
Shut down Windows
Cover microscope
NOTE: after hours, if the next
person is not there,
Do Total Shutdown.
When in doubt, do total shutdown.
Change Objectives
with buttons on
Right focus knob
3
Stage control
X-Y joystick - Top button toggles between very slow and
normal speed (beeps)
Focus
o Manual coarse and fine knobs on each side
o Away is up (see arrows above left knob)
o You must manual focus the first time
Quick Focus
o Auto up and down with buttons on right focus knob,
see arrows on scope
o Always lower objective with down button before
placing or removing samples.
o Up button returns to previous position
Do not use up button until you have found the focus manually.
Sample loading onto microscope Put oil or water on objective (not 10x)
Put sample on stage, cover slip down, center with joy stick
Manually focus up (away) until immersion liquid contacts sample, lower slightly but don’t
lose contact with immersion liquid
Find focus through eyepieces.
To see your sample thru the microscope Without using Zen
Brightfield Touchscreen
Press ‘Set Work Position’ to get rid of this screen
Home-Make it Visible for viewing bright field
Look thru eyepieces and focus up
Wiggle the stage as you go, look for movement
Fluorescence
You must Start Zen software first, see next page
Once you set up for fluorescence, you can change the filters on the
microscope:
Fluorescence Filter Cubes: Use 2 back buttons on left focus knob to change filters
Last button
o Blue->green->red (towards red)
Second last button:
o Red->green->blue (towards blue)
If spinning, hit same button twice
Note: Fluorescence at the microscope uses the Mercury-Halide lamp, not the lasers. The Blue and Green filters are Long Pass (LP) meaning you will see all colors through them. These filters are not used for confocal imaging.
Make it Visible
for bright field at the eyepieces
Right Focus Knob
Quick focus
Left Focus Knob
blue>green>red
red>green>blue
4
Zen software – Locate (Eyepieces)
Choose Bright Field or Fluorescence
If no buttons or buttons don’t work: Turn on desired lamp
o (12% for fluorescence)
Open shutter
Close other shutter
Choose filter (for fluorescence)
Find sample with eyepieces, focus
When ready for confocal:
o Hit Light Off avoid photobleaching
o Acquisition—to do confocal
Bright Field Fluorescence
5
Zen software – Acquisition Configurations To use an existing configuration you must
open the folder and choose the config
Or, open a previous image and Reuse
To make a new configuration or change an
existing one, use Smart Setup
Show all tools should always be checked
Note: You have to load a configuration each time you start Zen. It does not load automatically. If the software asks about the 561 laser and you have a red dye, say yes to turn it on.
Smart Setup Use this rather than making your own or adding to an existing configuration. Adding a dye to
an existing configuration often will not work.
Choose dyes and colors, starting with lowest wavelength
Choose Current (default)
Choose Smartest (Line)
Apply
6
Smart Setup Options
Name Autoscan Frame size Scan Speed Bit Depth Scan direction Pinhole
Current No 512 x 512 9 8 single 1 AU
Quality Yes 1700 X 1700 6 12 single 1 AU
Speed Yes 400 x 400 10 12 bi-directional 1 AU
Standard Yes 512 x 512 9 8 single 1 AU
Widefield Like Yes 512 x 512 9 8 single Max
Note: numbers may vary. Recommended to use Current (no autoscan) and set these parameters yourself.
See Optimizing Image Quality for guidance.
Lasers For Green dyes the 488nm laser is turned on with the key on the laser control box
For Red dyes (rhodamine, dsRed) use 561nm laser.
405nm (DAPI) and 633nm (AF647) lasers automatically go on as needed
Note: 488, 514 and 458 nm lines are all from the same laser
Light Path Adjust emission (spectral) regions if desired
MBS (Main Beam Splitter) Filters
o must match laser lines in use
For a Bright field/DIC image click on T-PMT
(transmitted light detector)
Note: bright field uses whatever lasers are on as the light source, not a separate lamp
Detector ChS1 This channel can be divided into many colors,
designated S1, S2, etc. ChS only has one master
Gain. To adjust the brightness of the individual S
channels, use the digital gain. See next page.
Notes on layout and windows with blue bars: Can be open or closed – click on blue bar Can be expanded (Show all on blue bar) or not Can be moved, drag blue bar (arrow in upper right corner
returns to home) To make a new column, click in grey area and drag over You can save your arrangement of windows using
Workspace config in the upper right corner of the software
o Zen2012 is the default o Once loaded or saved, it will load automatically
every time
.
7
Initial Settings for Live Scan
Note: see Optimizing Image Quality for more information.
Acquisition Mode window Show All
Check that correct objective is listed
Pixel format: Click on X*Y to change
Use 512 x 512 or 1024 x 1024
Bit Depth: use 12 bits for measuring
fluorescence, else 8 bits
Use Bi-directional scanning unless using
very high resolution
Channels window Laser Power (2% is a good starting point)
Pinhole: 1 AU for optimal section thickness
Master Gain (~500-750)
Digital Offset (keep near 0)
Digital Gain leave at 1.0
For 4+ colors, use the Digital Gain to
adjust the S1, S2 channels separately
8
Confocal Scanning
Live for a fast continuous scan
Continuous for a high quality continuous scan
(slow)
Snap to take an image
Save Immediately or open a new window
See Saving Images (p8).
Split to see individual color channels
Zoom-Crop Box You must stop scanning to use
Position box on object of interest
Resize, move, rotate
Live to see zoomed image
Live Zoom Active while live scanning
Adjust zoom value
Move, resize, rotate with
o Graphic
o Values
o Sliders
o Little arrows (most accurate)
Reset All to return to zoom 1, center
You can zoom out to 0.6 but the corners and
edges may be less bright
Notes on Zoom: Real Optical zoom Increases Resolution Decreases Image Area
Resolution Collect 2-3 pixels over the smallest object you
want to resolve
Do not collect smaller than the minimum pixel
size
Use higher mag/NA lens if needed
Minimum pixel size 10x pixel size ~ 0.30 um (300 nm)
25x pixel size ~ 0.10 um (100 nm)
40x pixel size ~ 0.08 um (80 nm)
63x pixel size ~ 0.07 um (70 nm)
Note: To get small pixels but a larger field you can collect 1024 x 1024 pixels or use tiling.
Can be adjusted while Live Imaging
Stop First, then Crop
9
Saving Image Files
Single images - Save Immediately
or open a new document window.
Z-stack, Time series, Tile, etc are not
overwritten. You can save later.
Save files to
D:\ALL USER IMAGES HERE
Save files as .czi.
stores all the hardware settings
Reuse the settings from any
image
Information to see settings
Copy Images to Z:Imaging Lab
FileShare
Retrieve from CU campus only
Retrieve and backup in a timely
manner
Opening .czi files later Zen Lite 2012 Blue or Black
Free (see below)
Find in the Imaging Fileshare
Zeiss 710 folder
Zen 2012 Full version
o On our workstation
FIJI (is Image J)
Free software for PC or Mac
Autosave Saves your file automatically
Breaks up your .czi files
Separate files for each channel, z-plane, time point or position (not tiles)
Save as .czi
o If you save as .tif, the colors will be wrong
Name is a base name and will be followed by date, time and other identifiers (ch, z, t)
Saving .tif images
Batch Export
There is a macro that will export a list of files to .tif all at once.
File- Export to .tif
See Zen Browser
10
Z-Stack
Check Z-Stack box and open window
Focus (down) to desired starting point
Set First
Focus (up) to desired ending point
Set Last
Stop scanning
Choose optimal step size
Start Experiment
Note: always focus down to set First.
Zen only collects against gravity.
Set Zero
Only if you want, not required
Go to Focus window
Show all
Manually set zero position
Optimize Sectioning and Step
Optimal: 50% section overlap
o Best for 3D imaging
Match Pinhole: no overlap, no gaps
X:Y:Z=1 gives cubic pixels
Imaging your Z-Series
Gallery to see all slices
Ortho- gives you a cross section at
any location, cover slip (start point)
is to the outside
3D – to do a live rotation.
o Use Maximum Projection
o House icon to return
o Create image to save
Cut—slices in another direction
Note: Volocity is another software option for doing 3D
visualization and analysis
11
Saturated pixels
Range Indicator
Check Range Indicator
Saturated pixels are red,
Black (zero value) pixels are blue.
Use Split view and adjust each channel
Aim for a few red and a few blue pixels,
max
For quantification, avoid all red and blue
pixels
collect 12 bit images
See Optimizing Image Quality
Note: You can always make your images brighter and the background blacker, with image processing, but you cannot unsaturate pixels after acquisition
Merged Image-Fewer channels To remove a channel from the Merged Image,
Click on the name of the channel above the color
bar, eg Ch1 or T-PMT. This only changes what
you view, the image is still acquired and saved.
Scale Bar Overlay
Click on ruler
Click on image and drag to set length.
Change color, etc
To save scale bar on image:
Change to single image (2D vs Split)
Use 100% for screen zoom
File-Export
Choose High resolution contents of image
window
Choose channels
Save as tif
Note: scale bar is saved as a screen shot. Saving contents of image window will save exactly what you see in the Image Window. You can also save a series.
12
Optimizing Image Quality
Option Optimal Range What it Does Pros Cons
Increasing Brightness
Master gain - increase 450-750+
Amplifies signal, makes
image brighter
No effect on
photobleaching High gains amplify noise
Laser Power - increase 2% - 20% More excitation light No increase in noise Can increase photobleaching
Pinhole - open wider 1AU - >
Collects thicker slice, more
emission light
No effect on
photobleaching Decreases Z resolution
Adjust Display
Best Fit or
min/max Only affects view, not data See low signal better Use with 12 bit images
Increasing Signal to Noise Ratio
Master Gain - decrease < 700 Lower brightness of image Image less noisy
Need to increase laser power
Line Average - increase 2-8
Repeats scans to collect
more excitation light Image less noisy Slow, increases photobleaching
Scan Speed - decrease 7-9
Slower speed collects more
excitation light Image less noisy Slow, increases photobleaching
Increasing Speed
Scan Speed >9 Scans faster Fast, less damage Collects less light
Bidirectional Scanning On or Off Scans in both directions
Cuts time in half, no
loss of light
May produce artifacts at high
resolution
Increasing Resolution (decreased pixel size)
Zoom
up to minimum
pixel size Scans smaller area Pixel size is reduced
Image area reduced,
increases photobleaching
# pixels 1024 x 1024
Divides image into more
pixels
Image area not
reduced Less light per pixel
Zoom + tiling
up to minimim
pixel size Scans a larger area Pixel size is reduced
Takes time, increases
photobleaching, poor stitching
High NA objective 1.2-1.4 Can resolve smaller
objects Thinner slices Also higher magnification
13
Multi-tracking (Sequential Imaging)
Use Smartest (Line) in Smart Setup
Check
Channels
Laser Lines
Colors
Pinhole
Main Beam Splitter Note: Set Pinhole to 1 AU on track with longest laser wavelength Start Live scanning
Adjust
Gain (use Digital Gain for ChS1 and ChS2)
Laser Powers, etc
Save Configuration in upper left corner of
Zen
Note: With line method, only the channels (on/off) and lasers (on/off) can change between tracks Line method is preferred unless you absolutely need to change something else between tracks, like pinhole, or beam splitter. Then you need the frame method. This is often the case with reflected light.
14
Time Series
Click on time series at top
Set interval and total number of scans
Start Experiment
o If you also have Z-Stack checked,
you will get an xyzt series
Definite Focus will keep sample in focus
over time.
See more notes below
Tile Scan Use to get large field at high resolution
Set number of tiles (eg)
3 x 3 (odd numbers) puts current
image in the center
2 x 2 (even numbers) cuts up the
current image
2 x 8- can be non-square
Rotate image to fit tile grid
Scan overview image for quick view
Set# tiles
Set objective
Start Experiment
o You can do a z-stack at each tile, set
up as usual
Note: Tile Scan creates one large image and the
stitching is not perfect. To get a perfect, stitched
image, or to get separate tiles, you can use the
Multitime Macro. It tends to be much slower and
does not rotate. We will be getting a better stitching
algorithm.
15
Reflected Light Confocal
Light Path
Set Main Beam Splitter to T80/R20
Check box for Reflection
Create small region under desired laser line
Can do fluorescence and reflected light
Note: For brighter fluorescence, use frame sequential
Will not work with 405 line.
Transmitted Light (Bright Field) In Light Path window,
Click on T-PMT.
Adjust gain and offset
Gain = brightness
Offset = blackness or contrast
o Can be negative to increase contrast
Uses whatever lasers are active
o Changing any laser power affects this
image
Note: This image is not an optical section For a high quality image see DIC
Reflected Light
Transmitted Light T-PMT
16
Positions or Multi X-Y-Z
Click on positions in upper left
Got to desired X-Y-Z location
Add position, repeat
Return to first position
Start Experiment
For Z-stack at several positions
Use the center option to define the z-stack at the first position
Then just add each location when in the center of the object.
o You must do the same range and step size
for each position
o X-Y-Z coordinate is recorded
o Software sets an offset to relate to center
of the first position
Center Option for Z-series
Choose Center vs First/Last
Manually focus to the center of sample
Hit Center
Choose Interval, then calculate the # of steps
needed to get the distance you want and set
steps
o Use the focus window to set the center to zero, if desired
Range Select
Will do x-z scan, you can change center or top/bottom on this image
PhotoBleaching (FRAP) or Photoactivation
Choose Bleach at the top
o Time series and Regions should also activate
Choose # of before scans
Iterations is the number of scans during the bleaching
o More iterations will bleach longer
Repeat bleach-don’t use this, it repeats the before scans, too.
Set laser and laser power
o 405nm at high percent will give fastest bleaching
o Slower scan speed will give better bleaching, use fewer iterations
o Zoom bleach can be faster but is less spatially accurate
Define region(s)
o Define control regions, too
o Choose which to bleach and analyze
Set up time series
o Time series does not include the bleach step, just pauses
Start Experiment
Note that a larger region than defined may be bleached, esp above and below the plane
17
Definite Focus
Uses a very far red laser (750nm) to find the coverslip and maintain the same distance from
the objective to the coverslip.
It does not follow your moving cells
Works with a time series +/- Z series, positions, tiling.
Helps to have >10 sec between acquisitions (end to begin)
o Other things are possible with the Multitime Macro
Touch Pad:
Home, Settings
o Set how often to check focus (this is disabled during the time course and Zen uses
its own interval so you don’t really need to do this)
Home, Microscope, XYZ, Definite Focus
o ON or Once – watch on Touch Pad or on Power supply
o Will be ‘Waiting’ between focus checks on control box
Watch it on the Touchpad. Let it check once or twice.
Hit OFF
Focus Devices and Strategy (Zen Blue Bar)
Need to have Time checked
o Choose Definite focus
o Choose how often to check
Start Experiment
You will see it ‘Setting Focus’ on the control box
Or watch the Touchpad to see it check focus.
This is usually fast but sometimes can take up to 10 seconds
Batch Export There is a macro that will export a list of files to .tif all at once.
To Load the macro the first time
Macro-macro
Assign macro
Menu entry - choose 1 or whatever
….Browse, find C:\zen\macros\Batch export
Text, type in Batch Export (this will appear in the dropdown menu)
Apply, wait
Close window
To Use, Macro – Batch Export
Find directory (folder) – Open
Highlight files – choose only single image or series files, do not mix
Single image with raw data or Series with raw data
For individual channel tifs, check the box for Monochrome
For merged tifs, leave unchecked and choose channels
You cannot mix these, you have to do them separately
You can only use Red, Green, Blue, no other colors
No greyscale for brightfield images
Click box for Long file names (?)
Do not click box for overwrite files
Name will be the original file name plus ch1, etc
Choose .tif
Start Batch Export
18
Zen Light 2012 Browser
Get this free browser from the fileshare where you retrieve your image files. Look under
Zeiss710. Install on any PC anywhere. They now have a Blue and a Black edition. Both have
3D capabilities, but the Black has more confocal features and the Blue has better export and
image processing features. If you have a 32bit computer, you can only use the Blue edition.
Zen Installation Instructions for a 64-bit PC: (Note: this takes a while.)
1) Find the folder Zeiss 710 on the fileshare where you download your files from
2) copy this file to your computer: ZEN_2012SP1_black_SP2_blue.exe
3) double click to extract
4) follow instructions to install but X-out AxioCam, CanCheck, System Maintenance Tool and
MTB (see pic below)
5) Install drivers (there are many)
6) This will install both the black and the blue versions
a. The black has confocal features-recommended
b. The blue has more image processing/analysis features
c. Both will do 3D rotations
Zen Installation Instructions for a 32-bit PC:
1) Download and install the 32bit.exe version. This is only the blue version but you do
get the 3D rotation feature. (I think)
Sorry -- there are no Zen options for a Mac
Mac and PC users can open .czi files with the FIJI version of ImageJ available at
http://fiji.sc/Fiji.
19
Using the Zen 2012 Browser
This looks just like the acquisition software, so you should be able to do basic things.
Here are some extras:
Export to .tif
Use File-Export- Export Raw Data
Choose single plane or series, RGB image or pallet for single color
Chose tif, 12 bit tif, 16 bit tif
Choose Full resolution image window makes the pixel size of the image match the pixel
size of the monitor. This is the best way to capture a screen shot.
Use this to:
Save a scale bar or other overlay items (numbers, arrows, regions, etc)
Save a greyscale image or an overlay including greyscale (use single, not tile)
Save any colors other than Red, Green Blue.
Contents of image window gives a screen shot of whatever is in the image window. If you zoom
up (make image pixelated) you will get only the area visible on the screen.
Processing-Copy-Subset: to remove frames from a Z-stack or Time series or X-Y crop
Select current image, Adjust, Apply
3D
You can do lots of 3D stuff with ZenLight 2012.
Rotations
Surface shadowing
Make AVI movies
Etc. You should just play around.
For better 3D try Volocity (see Carol or Johanna)
Adjusting the Brightness and Contrast of your image
Photoshop (is the BEST)
Image--Adjustments--Levels
Choose color(s)
Adjust histogram with little triangles on right or left of histogram
Center triangle is gamma, this effects linearity of brightness, use with care
Gamma can brighten dim areas without saturating already bright areas.
Save with new name as this is permanent and changes the image data.
Adobe Elements does the same thing, just look here:
Enhance—Adjust Lighting—Levels (Ctrl-L)
ImageJ (Fiji)
Image-Adjust-Brightness/Contrast or Color Balance
This gives you both the histogram adjustment and, brightness and contrast adjustment.
No gamma Note: Save with a new name. Always save your raw image or .czi file. These procedures change the pixel values of your image!
20
DIC (Differential Interference Contrast or Nomarski) Note: Get help with this the first time, at least
At microscope (Ocular)
Set Kohler Illumination First:
Close Field Stop near top of microscope
Adjust condenser height to see and focus on small spot of light (polygon)
o Should have sharply focused edges
Center with centering screws
Open field stop to fill field Note: you should set Kohler every time you collect a bright field image
Condenser Settings
On left is aperture -- Open fully for DIC
o Close partly for Bright Field (to increase contrast)
Closing too far lowers resolution, better to increase contrast by lowering
offset in software
On right are settings—must look on Touch Screen to see current setting
o Options are PH2, PH3, BF, DICII, DICIII
o Use DICII for 25x, DICIII for 40x or 63x Note: you must have this setting on the Touch Screen even when in confocal acquisition mode. You may have to resave your config so it stays.
Filter Cube Setting (right focus knob)
Use POL for DIC. See setting on Touchscreen.
o This is only for eyepieces, it will change when acquiring an image.
Upper Polarizer
This must be rotated to the left when using the eyepieces
When switching to confocal, rotate to the right
Clicks into correct position
Wollaston Prism
This is a small filter with an adjustable screw just below the objective. Each objective
has one (except 10x) but they are not kept on the microscope.
Must be inserted (and removed) each time and this is best to do before moving objective
into position.
Adjust the screw as desired (try to get your hand in there while looking thru the
eyepieces)
o The center of the adjustment should be darkest, as you move away in either
direction the DIC effect maximizes and then goes away.
o Light / dark shadows flip from one side to the other as you cross the center. You
may get nice colors in the eyepieces.
Confocal Acquisition of DIC
Rotate upper polarizer to right
Make sure condenser setting is still on DICII or DICIII
Click on T-PMT
Feel free to lower offset to a very negative value, increase gain as needed. This
increases contrast.
Adjust Wollaston prism while scanning if desired
21
Specifications
Lasers 405nm – 30mW
488nm (also 458nm and 514nm) - 25mW
561nm -15mW
633nm - 5mW
Objectives Mag/ NA Imm Name Coverslip WD(mm) Ring Color
10x/ 0.3 dry EC Plan-NEOFLUAR - 5.2 Yellow
25x/ 0.8 Oil/W* LCI Plan-APOCHROMAT +/- 0.55 Red/Green
40x/ 1.2 Water C-APOCHROMAT 0.14-0.19** 0.28 Blue
63x/ 1.4 Oil Plan-APOCHROMAT +0.17 0.19 Black
*25x: Set to Red mark for oil, Green mark for water
**set for cover slip thickness. #1 cover slip is 0.13 μm, #1.5 cover slip is 0.17 μm.
#1.5 cover slip is recommended, esp for oil immersion
WD = Working distance: how far the objective can focus past the coverslip
Focus Coarse 1 revolution = 2mm Fine 1 revolution = 0.2 mm (200μm)
Filters in Microscope – Axio Observer .Z1
Position Set# Ex BS Em
1 empty
2 02 G365 FT 395 LP 420 (Long Pass)
3 09 BP 470/40 FT 510 LP 515
4 15 BP 546/12 FT 580 LP 590
5 POL Polarizer for DIC
6 Mirror for LSM
Stage Speed Focus Speed
10x 7 4
25x 5 3
40x 5 2
63x 3 1
Resolution
Collect 2-3 pixels over the smallest object you want to resolve
Maximum zoom (at 512x512) and minimum pixel size
Objective Max Zoom Minimum pixel size
10x 5.0 0.30 um (300 nm)
25x 6. 0.10 um (100 nm)
40x 6.3 0.08 um (80 nm)
63x 4.0 0.07 um (70 nm)
To get smaller pixels but a larger field you can collect 1024 x 1024 pixels or use tiling.
22
Common Errors and Problems Inkubator
When starting, small box in lower right will say “Error…Inkubator…”
Ignore this. Unless you are trying to use the incubator.
No light at microscope in Ocular mode Did you hit Online?
Is the lamp on in the screen graphic?
For fluorescence is the power >0% ?
Is the shutter open?
If yes to all of these, hit Offline, then Online again. This usually works.
Zen not loading
Blue bar is stops moving after ~1/4 inch:
Stop and exit
Find large black box on the table in the
corner, it looks like a computer and is
called the Real Time Controller (RTC).
Open front (like a door—far side opens).
Hit reset button (yellow triangle).
Watch for red flashes, takes a minute
Restart Zen.
Blue bar stops moving near the end:
You will also get more error messages in
the lower left box.
o You will need to restart everything.
Stop Zen and restart Windows
Turn off all buttons on white control box.
Then quickly turn Main back on
Wait 5 sec and turn on 2 small buttons
Turn the laser key off and on again.
o You may have to actually shut down the computer, not just restart.