Turku Bioscience Zeiss LSM780 User Manual

Zeiss LSM780

User manual

Cell Imaging & Cytometry

CONTACT:

[e-mail]

[phone](office)

050 369 7532 (mobile)

Turku Bioscience 5th floor, room 5077

Contents

1. Information ..................................................................................................... 1

2. Before Imaging ................................................................................................ 2

2.1 Check your sample with an ordinary fluorescence microscope ............................. 2

2.2 Clean your slides ................................................................................................. 2

2.3 Check the environment ....................................................................................... 2

2.4 Start the heating earlier if possible ...................................................................... 2

2.5 Cancellation ....................................................................................................... 2

2.6 Unsure? .............................................................................................................. 2

3. Working with immersion objectives ................................................................. 3

4. Starting up and shutting down the instrument ................................................. 4

4.1 Start up .............................................................................................................. 4

4.1.1 Definite focus ........................................................................................................ 4

4.1.2 Main switches ....................................................................................................... 4

4.1.3 Argon laser ............................................................................................................ 5

4.2 Shut down .......................................................................................................... 5

5. Starting the Zen software ................................................................................ 6

5.1 Start the ZEN software ........................................................................................ 6

6. Using the fluorescence microscope .................................................................. 7

6.1 Visualizing the sample in ocular mode ................................................................. 7

6.2 Using the confocal microscope settings ............................................................... 8

6.2.1 Reusing settings from previous experiments ....................................................... 8

6.2.2 Setting up new experiment .................................................................................. 8

6.2.3 Using Smart Setup ................................................................................................ 8

6.2.4 Manually create light paths .................................................................................. 9

6.2.5 Visualise sample on screen ................................................................................... 9

6.2.6 Adjust the Channels ............................................................................................ 10

6.2.7 Acquire images ................................................................................................... 10

6.3 Experiments ..................................................................................................... 11

6.4 Z-stacks ............................................................................................................ 11

7. Live Cell experiments ..................................................................................... 12

7.1 Live imaging equipment set up .......................................................................... 12

7.2 Setting up time-lapse ........................................................................................ 13

8. Troubleshooting ............................................................................................ 14

8.1 Do not see fluorescence in the Locate mode ...................................................... 14

8.2 Weak signal with the Argon laser ...................................................................... 14

8.3 Argon does not start ......................................................................................... 14

1

1. Information

The Cell Imaging and Cytometry Core is responsible for maintenance of this microscope.
Each user must follow the CIC user policy.
More information: https://bioscience.fi/services/cell-imaging

 Only authorized users may use the CIC instruments.

 Users must report any malfunction or problem to the CIC

personnel.

 The user is responsible for removing their data from the hard

drive and should do so immediately.

 Files older than 30 days are automatically wiped from the

system without prior notice

2

2. Before Imaging

2.1 Check your sample with an ordinary fluorescence microscope

It is a good practice to check the quality of your sample before making use of a
more expensive instrument.

2.2 Clean your slides

Clean the remaining salt and mounting medium off the coverslip. Dirty coverslip
compromises the image quality. You can use ethanol to aid the cleaning. Do not
use the microscope lens tissue. Clean your slides beforehand in your own lab, as
it is impractical to use microscopy time for cleaning.

2.3 Check the environment

Switch on the lights and check if the microscope environment is tidy.
If there are oil spills or other issues, please inform CIC personnel.
([e-mail])

2.4 Start the heating earlier if possible

When doing live cell imaging, it is good to switch on the heating at least two hours
in advance. If someone is using the instrument, ask whether it is possible to
switch it on. If no one is using the instrument, start the instrument and switch on
the heating.

2.5 Cancellation

Cancellation must be done 24 hours before the reservation starts. However, if
you suddenly cannot use your time, inform the next user and cancel your
reservation.
If you are the last user of the day and cannot come, you are responsible for the
instrument shutdown.

2.6 Unsure?

If you feel that you need support, please contact CIC personnel.

3

3. Working with immersion objectives

 The immersion medium should match the objective.

 The 40x and 63x objectives need water or oil with a refraction index of 1.334. For

fixed samples and short time experiments, MilliQ water is appropriate

 For long experiments water immersion oil is more suitable. The water immersion

bottle is marked with a W; the water-oil is very fluid and must be applied very
carefully.

 The 100x objective needs oil with a refraction index of 1.518. This oil is more

viscous than the water immersion oil.

 The image will be suboptimal when incorrect oil is applied. Do not mix immersion

oils.

 A small drop of oil is enough, as too much will make a mess, damaging the

instrument.

 Start imaging with the objective in the lowest position. Then, focus the objective

upwards until the oil touches the coverslip. Next, focus the sample visually through
the eyepieces.

 After imaging, wipe the oil off from the objective softly with a dry lens tissue. Then,

finish the cleaning by wiping softly with a new lens tissue moistened with
isopropanol. Only lens tissue may touch the objective lens.

 When changing the sample with the immersion objective, the objective must be

lowered (inverted microscope) in between.

 Press Load position in the remote unit, in the software (Focus window) or on the

top of the right focusing knob (closest, front most button). Objective moves down.

 Remove the sample and add oil if needed
 Place the new sample. If you are changing the objective in between, wipe the

objective clean with a dry lens tissue. Otherwise, oil might spill into the microscope.

 Press Work position in the remote unit, software or on the top of the right focusing

knob (second button). The objective will move up into the position before Load was
pressed. It should be very close to the correct focus position.