Leica Stellaris 5 Manuel utilisateur

LEICA Stellaris 5

Confocal microscope

Manual/Quick guide

Matyas Molnar, Biovis 2024

Starting the microscope

1. Turn ON the computer, wait till Windows is booted

2. Log in with yourUU AKKA account

3. Turn ON the scanner box switches below the table:

i. Power buttonON

ii. Laser button ON

iii. Turnthe laser interlockkey to vertical position (emission light ON)

4. Waitapprox. 3 minutes

5. Start the LAS Xsoftware andstart“machine” configuration and “DMI8” microscope:

The microscope stand

Visualizing the sample through the eyepiece using fluorescent light (IL)

If you don’t see any signal through the eyepiece, make sure that you are using the following

settings on the TFT screen:

Visualizing the sample through the eyepiece using brightfield light (TL)

If you don’t see any signal through the eyepiece, make sure that you are using the following

settings on the TFT screen:

Follow these steps to have light through the eyepiece:

- Select the desired objective and use the correct immersion mediumfor it. On the TFT

screen the objective buttons blink when there’s a change inthe immersion medium.

Press them againafterchanged the immersion medium.

- Port: Eyepieces 100%

- Magn.-Changer: 1

To change the intensity for the TL or IL:

Using the LAS X software

In LAS X, hover the mouse on any button to get info about it.

Configuration tab

Under the configuration tab, several optionscan be changed and lasers canbe turned ON andOFF.

-In the Laser config menu: laser can be turned ON orOFF. We have twolasers, a

fixed 405nm anda tunable White light laser (WLL) 485-790nm.

-In the Hardware menu: dynamic range canbe set to 16-bit (thedefault is 8-bit)

-In the USB Panel, the sensitivity of theknobs can be changed

Acquire tab,confocal imaging

This is the mostused tab, here the imaging setup can be configured andimaging canbe started.

Acquisition mode:

Typeof imaging,

2D,3D, time-lapse,

spectral scanning

Navigator. Quick spiral

scanning, tile scanning.

Info and help menu.

Use it when you need!

ROI Bleaching experiment

; : Expand orcollapse amenu.

Auto mode. If you are unsure what

you are doing,start setting up your

imaging here.

Image compass

Lasers, detection windows, etc.

Acquisition mode:

Type ofimaging, 2D,3D, time-lapse,

spectral scanning, simultaneous or

sequential imaging

Acquisition menus

Detectionconfiguration.

Three HyD detectors can be

turned ON-OFF, and their

parameterssuch as detection

range, gain and operating

mode can be modified.

Capture an image

Objectives canonly

Start to acquire 3D;

time-lapse;

or spectral scanning,

etc.

Fast live mode to check the

sample and find locations

with quick scanning (speed

and resolution settings are

NOT utilized here).

Objectives

Live mode. Same as

“Capture Image” but on

continuous mode (speed

and resolution settings are

utilized here).

Auto mode (Dye Assistant)

Auto mode is aquick and easyway to create the settings for fluorophores andchannels.

Unexperienced userscan start with the auto mode. Please note that auto mode might not work

perfectly in special cases orhas to be fine-tunedmanually. Users shouldn’trely on automodes, the

safest way is toset up theimaging as manually as possible.

-Select your fluorophores

- Selectbetween simultaneous or sequential imaging, line or frame

Manual mode, semi-manual mode (Image Compass)

Setting up the channels with the image compass is a love andhate thing. It was made user friendly

and super-overcomplicated at the same time where you needtoalternate between clicking buttons

and dragging-dropping items. Watchout for high numbers of buttons,check-in marks, padlocks,

menus and small signs. They all have a feature.

Sometimes you need to delete settings by dragging and dropping it to and emptyspace, sometimes

to click on it and then click on the delete button.

Semi-manual mode: 1. Drag and drop your

fluorophore to a

detector

2. Theexcitation laser

and detection

window for the

detectoris selected

automatically

3. Continue doing this

with all your

fluorophores starting

from the blue range

(HyD S 1 detector) till

thered-infrared

range (HyD S 3

detector)

4. Toimage

sequentially,add a

new “Setting” with

the “+” sign and drag

and drop a

fluorophore to a

detectorin the new

setting. Select

“Stack, frame or line”

mode.

5. Change the laser

intensity by clicking

on thelaser line and

changing the

“intensity”(knob can

be used)

6. Change the detector

gain by clicking on

the detector and

changing the “gain”

(knob can be used)

7. Todelete a fluorophorefrom the settings, put your

cursor on its emission curve and drag anddrop it to

an empty space. OBS ! If you wantto only delete the

laser line, click onit and push the thrash button (top

right corner)

8. Navigation between Setting 1 and2. You can:

- Select a setting by clicking on it

- Uncheck (deactivate) a setting with the

check-in boxon the top left corner

- Remove/delete a setting by the“X” button

on the topright corner.

Manual mode:

Start withthe semi-manual mode (by dragging and dropping a fluorophore to a detector) and modify

the automatically generated channel (detection range, tuning theWLL onto a different laser

wavelength,etc.).

“Add Laser” (drag and drop this button to a desired laser wavelength) and turn ON a desired detector

and set up the detection window, laser intensity anddetector gain.

It’s encouraged to try out what all the different buttons and features do. Hover themouse over a

buttonto see the info about it.

If lost, don’t forget to use thebuilt-in HELP function of thesoftware by clicking on the closest“i”

button. This is one of the best features of LAS X.

Get HELP and

tutorial. Use it

often.

Adjusting the correct intensity with the Detection configuration menu orthe knobs

The bestway to adjust the correctintensity of a channel is to use the Over-/Underexposure tool on

the left side ofthe image window (first button). If Over-/Underexposure tool is activated we see the

overexposed pixels in blue.

The overexposed pixels areout ofthe detector range, their intensity information is lost. Wemust

avoid seeing overexposed (blue) areas. By changing (decreasing) thedetectorgain (voltage) or the

laser intensity, we can move the overexposed areas intothe range of thedetector. For multichannel

simultaneous imaging, besure to click onthe image of the channel you want to change, the other

channel’s detector is not changing meanwhile. Whichoneshould we change, the laser or the

Over-/Underexposure tool

These channels are notselected

(not clicked), we change the

detectorgain for the other channel

Overexposure (blue color); the

histogram is clipped on the right

side. To avoid clipping and to

remove the blue, lower the “gain”

or “laser power”

This channel is selected(clicked),

we change the detector gain for

this channel

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