Cornell University - Biotechnology Resource Center – Imaging Facility
Zeiss 710
Confocal Microscope
User Guide
B46 Weill Hall
BRC Imaging Facility
8-6-2014
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
1
Startup – General Procedure
Ending your Session:
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See instructions posted
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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
Total Shutdown:
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
10x Dry
40x H2O
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
25xOil/H20
cover slip setting
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.
H2O setting
63x Oil
Oil setting
40x Dipping
Change Objectives
with buttons on
Right focus knob
2
Stage control
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X-Y joystick - Top button toggles between very slow and
Right Focus
normal speed (beeps)
Focus
Quick 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.
Knob
Sample loading onto microscope
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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:
Make it Visible
for bright field at
the eyepieces
Left Focus Knob
Fluorescence Filter Cubes:
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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
blue>green>red
red>green>blue
Note: Fluorescence at the microscope uses the MercuryHalide 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.
3
Zen software – Locate (Eyepieces)
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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
Fluorescence
Bright Field
4
Zen software – Acquisition
Configurations
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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
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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
5
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
Widefield Like
Yes
Yes
512 x 512
512 x 512
9
9
8
8
single
single
1 AU
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
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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
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6
Initial Settings for Live Scan
Note: see Optimizing Image Quality for more information.
Acquisition Mode window
Channels window
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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
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
7
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
Stop First, then Crop
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
Can be adjusted while Live Imaging
Notes on Zoom:
 Real Optical zoom
 Increases Resolution
 Decreases Image Area
Resolution
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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.
8
Saving Image Files
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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
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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
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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
9
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
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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
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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
10
Saturated pixels
Range Indicator
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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
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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
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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.
11
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
Laser Power - increase
2% - 20%
More excitation light
Can increase photobleaching
Pinhole - open wider
1AU - >
Best Fit or
min/max
Collects thicker slice, more
emission light
No increase in noise
No effect on
photobleaching
Only affects view, not data
See low signal better
Use with 12 bit images
Image less noisy
Need to increase laser power
Image less noisy
Slow, increases photobleaching
Image less noisy
Slow, increases photobleaching
Adjust Display
High gains amplify noise
Decreases Z resolution
Increasing Signal to Noise Ratio
Master Gain - decrease
< 700
Line Average - increase
2-8
Scan Speed - decrease
7-9
Lower brightness of image
Repeats scans to collect
more excitation light
Slower speed collects more
excitation light
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 Speed
Increasing Resolution (decreased pixel size)
Zoom
# pixels
up to minimum
pixel size
Zoom + tiling
1024 x 1024
up to minimim
pixel size
High NA objective
1.2-1.4
Image area reduced,
increases photobleaching
Scans smaller area
Divides image into more
pixels
Pixel size is reduced
Image area not
reduced
Scans a larger area
Can resolve smaller
objects
Pixel size is reduced
Less light per pixel
Takes time, increases
photobleaching, poor stitching
Thinner slices
Also higher magnification
12
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.
13
Time Series
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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.
14
Reflected Light Confocal
Reflected Light
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
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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
Transmitted Light T-PMT
15
Positions or Multi X-Y-Z
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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
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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
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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
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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
16
Definite Focus
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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
17
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.
18
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!
19
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
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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
20
Specifications
Lasers
405nm
488nm
561nm
633nm
– 30mW
(also 458nm and 514nm) - 25mW
-15mW
- 5mW
Objectives
Mag/ NA
10x/ 0.3
25x/ 0.8
40x/ 1.2
63x/ 1.4
Imm Name
dry
EC Plan-NEOFLUAR
Oil/W* LCI Plan-APOCHROMAT
Water C-APOCHROMAT
Oil
Plan-APOCHROMAT
Coverslip
+/0.14-0.19**
+0.17
WD(mm)
5.2
0.55
0.28
0.19
Ring Color
Yellow
Red/Green
Blue
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#
1
2
02
3
09
4
15
5
POL
6
10x
25x
40x
63x
Stage Speed
7
5
5
3
Ex
BS
empty
G365
FT 395
BP 470/40
FT 510
BP 546/12
FT 580
Polarizer for DIC
Mirror for LSM
Em
LP 420 (Long Pass)
LP 515
LP 590
Focus Speed
4
3
2
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.
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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.
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