Zeiss LSM 710 SIM User manual

ZEISS LSM 710 SIM
Superresolution microscope
Manual/Quick guide
confocal image SIM image
Matyas Molnar, Biovis 2016

Starting the microscope
Condenser, tiltable
Laser safety box;
make sure all
openings are closed
before acquisition is
started
No need to touch…
Microscope stand
Computer (left side)
Real time PC, open
front and reset if
ZEN cannot start
Argon confocal laser
Main switch
Fluorescent lamp
Confocal and SIM laser boxes

1. Check the microscope if everything looks clean and normal. If not, report
it in the logbook.
2. Start the system with the Main switch (on the microscope panel, located
on the laser box)
3. Switch the Systems/PC ON (on the microscope panel, located on the
laser box)
4. Switch the Components ON (on the microscope panel, located on the
laser box)
5. Switch on the left computer
6. Login to the computer with your account
7. Wait 1 min so the system is fully booted
8. Start the ZEN software
9. If there is a connection problem with the Real time PC (PC on the right
side), switch off everything and reset the real time PC with its reset
button on the front.
10. If you want to use the followings, turn on:
a.Argon lasers: small black box located on the laser box, turn on with
the key on the box and put the switch on the LASOS control panel
to “RUN”

b.Fluorescent lamp: switch on with the button on its control box
(white box located on the laser box, marked with X-cite sign)

Using the ZEN software
Locate tab
Use the locate tab to focus onto the sample and to get a first overview with the eyepiece.
Turning the fluorescence lamp ON/OFF:
1. Put into ON position
2. Tune it to 12% at least
3. Open the shutter
Switching between fluorescence
channels (OBS! The only
available filter is the FSet77 HE,
which is a double filter for green
and red channels)
Changing objectives
Turning ON/OFF the
transmitted (brightfield)
lamp
Changing the intensity of
the transmitted
(brightfield) lamp
1-2
3

Acquisition tab, confocal imaging
This is the tab where image acquisition can be started and change the properties of the
imaging. Always check “show all tools” here and for all the blue menus in the view menu
(show all global).
Laser menu:turning the
confocal and SIM (Elyra)
lasers ON/OFF
Acquisition mode:
changing the quality of the
imaging (resolution, image
speed, averaging, dynamic
range setup)
Imaging setup: selecting
the excitation and
emission for the different
channels. Choosing
between simultaneous
and sequential imaging
Channels:controlling the
excitation laser power and
the gain on the PMT
detector
Starting/Stopping
acquisition
Experiments:activating
advanced acquisitions
Smart setup:quick setup
for channels

Smart setup (only for the confocal mode)
Smart setup is a quick and easy way to create the settings for fluorophores and channels.
Unexperienced users should start with the smart setup. Please note that smart setup might not
working with the multiphoton laser so multiphoton channels should be setup manually.
•Select your fluorophores
•Select between simultaneous or sequential imaging. Fastest is simultaneous,
best signal is sequential imaging (imaging all fluorophores in different tracks).
Start/stop acquisition
Find the focus
in the sample
Auto-adjust
exposure
Real time, fast, low
quality live mode to
find places in the
sample, adjust the
correct sample
intensity and to set
up Z-stacks
Slow live mode to find
places in the sample and
adjust the correct sample
intensity. The quality of
the continuous mode is
taken up from the
settings of “Acquisition
mode” menu
Making an image
(acquisition)

Experiments
A possibility to select advanced imaging methods such as:
•3D imaging (Z-Stack)
•Time lapse imaging
•Bleaching experiments
•Tile scan (no stitching possibility exist for this instrument)
•Imaging with specific regions
Laser menu
SIM lasers (Elyra) can be turned on here. All the other lasers are either always on (HeNe
lasers, 405 diode) or turned on manually on a laser box (like the Argon laser).

Imaging setup
A tool to manually setup channels for fluorophores either is for simultaneous (two detector is
activated for one or more track) or for sequential imaging (all fluorophores are in different
tracks).
Changing between WF
(Widefield), SIM, LASER WF,
LSM (confocal), or lambda
mode (using the instrument
as a spectrometer)
Emission profile for
fluorophores
Scanning windows for each
PMT detectors
Adding/removing tracks.
Selecting excitation lasers.
Visible lasers (Argon or
HeNe) or 405 invisible
Selecting the laser guide
filters for the selected
excitation lasers.
Activate the brightfield (T-
PMT) detector for imaging
brightfield in a separate
simultaneous channel.
Activating or deactivating PMT detectors. If
more than one detector is active, then
simultaneous imaging is ongoing.

Channels
Here the intensity of the signal for the different channels can be adjusted. When adjusting the
intensity for a channel be sure that the desired channel is highlighted and activated (checked)
otherwise settings cannot be modified, or settings are modified for a different channel.
Adjusting the correct intensity with the channels menu
The best way is to adjust the correct intensity of a channel is to use the range indicator in the
bottom of the image window. If range indicator is activated we see the lowest intensity pixels
(pixel value: 0) in blue; and the overexposed pixels in red. The overexposed pixels are out of
the detector range, their intensity information is lost. We must avoid seeing overexposed (red)
areas. By changing (decreasing) the detector gain or the laser AOTF in the channels menu,
we can move the overexposed areas into the range of the detector.
Which one should we change, the laser or the detector? There are no rules here, both have
advantages and disadvantages. If we use high laser power with low gain we see a good quality
image with low noise, but we can bleach the sample, so as always COMPROMISE between
Existing tracks and
channels.
Active lasers
Digital gain:forget it…
Existing channels
AOTF control
(“power”) for a laser
Gain:applied voltage to the
detector of this channel
Digital offset: change the
background (darkest level) to
the detector of this channel
Pinhole: changing the thickness of
the optical section. Click 1 AU to get
confocal imaging. There is no pinhole
adjustment for the NDD-multiphoton
setup.