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.