User – Manual

Zeiss - Confocal
(10 – 31 - 2018)
From: Karl Ferdinand Ziegler

1
Contents
1. Start/Stop the System ........................................................................................................................... 3
1.1. Hardware ...................................................................................................................................... 3
1.2. Software ........................................................................................................................................ 3
1.3. Turn the system OFF ..................................................................................................................... 4
2. Find your sample ................................................................................................................................... 5
2.1. Place the sample and setup the objective ......................................................................................... 5
2.2. Locate your sample ............................................................................................................................ 5
2.2.1. Transmitted light (Bright field/DIC) ............................................................................................ 5
2.2.2. Epi-fluorescence.......................................................................................................................... 7
3. Pre-setup an experiment ...................................................................................................................... 8
4. Acquire an 2D image ............................................................................................................................. 9
5. Acquire a Z-Stack image ...................................................................................................................... 11

2
 1. Start/Stop the System
1.1. Hardware

1. Turn ON the Hardware in the order shown in the image above 2. If the PC is not already ON, start
by flipping the switches the PC after the Hardware is run-
ning

1.2. Software
1. Prior to starting ZEN, move the objective turret to position #1

2. After the Hardware is running and the objective turret is in position #1 you can start the Software

3. Double click on the icon “ZEN Black confo- 4. Click on “Start System”
cal”

3
 1.3. Turn the system OFF
1. Save/transfer your data

2. Click on the “X” on the top right corner

3. Click on “OK” in the window shown below to turn off the Lasers

4. Now you can turn off the hardware in the opposite way as shown in Point 1.1 by flipping the switches.
DON’T TURN OFF THE PC.

4
 2. Find your sample
2.1. Place the sample and setup the objective
1. After the System is running, place your sample on the stage

2. Place the sample on the sample stage and 3. Move the Objective in working distance by
choose the objective for your experiment by rotat- rotating the knob as shown above. If necessary,
ing the Objective turret until the number are over- put Oil/Water on top of the sample before you
lapping with the white lines (as shown above). move the Objective down.

4. If you need a different objective on the microscope than contact Andy Schaber

2.2. Locate your sample

2.2.1. Transmitted light (Bright field/DIC)
1. After the sample and the Objective is in the right place you can use the software to find your sample.

2. Click on the Tab 3. Choose either DIC or Bright field

“Locate” on the top
left corner

5
5. If you want to use DIC please make sure you follow the next steps, otherwise skip to step 10.

6. Make sure the DIC prism is in the Objective 7. Check if the prism matches the Objective with
the list close to the microscope.

8. Rotate the condenser turret to match the ro- 9. Contrast can be adjusted by rotating the wheel
man numbers between the 2 prisms (objective on the polarizer under the condenser
and condenser)

6
10. Use the Eye piece to locate the sample, either with or without DIC

11. Change the focal posi- 12. Rotate the wheel to change the 13. Move the joystick to change
tion by rotating the knob. light intensity the X,Y Position of the sample

2.2.2. Epi-fluorescence
1. After the sample and the Objective is in the right place you can use the software to find your sample.

2. Click on the Tab 3. Select a Filter set for your experiment 4. Press the wheel to turn ON/OFF
“Locate” on the top the LED light source
left corner

5. Change the focal position 6. Rotate the wheel to change the 7. Move the joystick to change
by rotating the knob. light intensity. the X,Y Position of the sample

7
 3. Pre-setup an experiment
1. After you found the sample through the eye piece you are ready to pre-setup the system with the right
laser and detection combination
2. Click on “Acquisition” on the top left corner. Then pre-configure the system by one of the next meth-
ods.

Method a:

3a. Pre-configure the system by

loading an old data set and press
the “Recycle” button.

Method b:

3b. Click in the field “Experiment 4b. Click through each “Track” and make sure:
Manager” on the button shown - the right channel is active
above and choose one of the pre - detection spectrum is at the right position and has the
setups from the drop down menu. right size

8
Method c:

3c. Select “Smart Setup” in 4c. Select the dyes you will use in 5c. Then choose between three dif-
the “Experiment Manager”. your experiment from the drop ferent modes: fastest, best signal or
down menu after clicking on the smartest and press apply.
arrow. Choose as much as you
need.

4. Acquire a 2D image
1. After you found the sample through the eye pieces and you have setup the system you can acquire an
image.

2. In the window “Acquisition Mode” you can setup your scan parameter.

Frame Size: This option allows you to change the

pixel density for your scanning area. Higher
frame size led to longer acquisition times and
more photo bleaching.

Speed: Changes the Pixel Dwell time or the

frame per second (fps). Faster scanning reduces
photo bleaching but decreases the signal to
noise ratio (SNR).

Averaging: Allows you to increase the SNR by

scanning the Frame/Lines multiple times. In-
crease photo bleaching as well.

Scan Area: Changes the size of your scanning

area. The optical zoom increases the magnifica-
tion and decreases the field of view.

Scan Time: Indicate the time which is necessary

to acquire an image.

Recommended settings to start with:

- Frame size 512x512 or 1024x1024

- Speed max
- Averaging 1

9
 3. If using mul- 4. Activate the tab “Dis- 5a. Click in the “Experi- 5b. Click in the “Experi-
tichannel, play” and check the box ment Manager” on “Live” ment Manager” on “Con-
change the “Auto”. That enable the to get an impression of tinuous” to get impression
displayed view auto-contrast function of your sample regardless of of your sample in regards
to Split the Look Up Table. your acquisition mode. to your acquisition mode.

6. Press “Expand All” to visualize all track settings

7. Set the Pinhole size by clicking one of the button (1) “AU”. The active Pinhole size counts for all
tracks/channels. You cannot set a different Pinhole size for each track.

8. Change the Laser Power (2) and the Master gain (3) for each tracks/channels that your signal reaches
2/3 of the scale shown in the “Display” tab.
1

1
2

3
2
1

2 3

10
9. When everything is setup up and you are satisfied with the image then press the button “Snap” to
acquire a single XY image.

5. Acquire a Z-Stack image

1. After you configure the system and are able to acquire a 2D image you can also collect a Z-stack (3D)
image

2. Activate the Z-Stack experiment

manger by checking the box

3. Choose between two different

modes: “First/Last” and “Center”.
First/Last: Define the bottom and top
boundary by moving the focus position
up/down and click “Set Last”, “Set
First” respectively. Change the slice
thickness to the optimum (Indicated by
the button “Optimal”). After that you
can press “Start Experiment”.
Center: Move the focal position to the
center of your sample. Set the top and
bottom boundary by tipping the dis-
tance to the center position. Change
the slice thickness to the optimum (In-
dicated by the button “Optimal”). After
that you can press “Start Experiment”.

11
 6. Troubleshooting
If something doesn’t work well, you need help or the microscope have malfunction, please contact Andy
Schaber (Facility manager) by phone: (765) 496-3148 and/or email: schaber@purdue.edu

12