Zeiss LSM 710 SIM Manual de usuario

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

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.

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