Leica Stellaris 5 User manual

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)
1.
4.
4.
4.
5.
7.
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.).
OR
“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

detector? There are no rules here,both have advantages anddisadvantages. If we use high laser
power with low gain we see a good quality image with low noise,but we can bleach the sample, soas
always, COMPROMISE between quality and time/bleaching! Don’t use a default laser orgain settings,
always change them freelyto get the best image without ruining thesample.
Note thatif you change thepinhole or detection range, thesignal is collected in adifferent Z/spectral
range (intensity is changed),therefore newintensity adjustment is needed.
Acquisition menu
Optimize resolution (pixel size)
Set resolution (pixel size) manually.
If you are unsure, use the optimize
button. For thick 3D imaging use
small, like 512x512to save
time/avoid bleaching.
Scanning speed (Hz): go up with the
speed to decrease, go down to increase
the quality. No golden rule, itis sample
dependent,if unsure try the sample with
different speeds, and use the best for the
real acquisition. Again COMPROMISE
between time andquality, decide whatis
more important.
Bidirecional scanning: Before
starting ascan always testif X
phase is correct. If unsure, don’t
use it, itcan ruin you imaging.
Averaging: noise (random pixels)
are removed with averaging
process. Selectthe number of
image to be used for averaging
(again time vs. quality!) and choose
between line and frame averaging.
Use the check-in box to apply the
settings to all sequences.
Pinhole: changes the thickness of
the optical section. Click 1 AU
button to getconfocal imaging. If
the signal is weak, open the pinhole
more to collect photons from a
thicker optical plane.

Z-stacking (3D imaging)
Deconvolution (Lightning)
1. Go to Live or Fast Live mode, use
the focus knob to find the end of
the sample and push Begin to
select the starting positionof the
sample where the first 2D stack
should be made.
2. Focus to the other end of
the sample and hitEnd to
select the position where
the last stack should be
made.
3. Select thenumber of 2D
stacks. Twoways of doing it,
onecan selectZ-step Size
and typein the interval
betweentwo stacks or
select the Number of steps
and typein the desired slice
number. If unsure,go for
the System Optimized
buttonto not lose any
information between two
stacks.
4. Advanced optimization setup
5. Setup forlinear laser or
detectorgain compensation
when signal is getting lostin
deep samples.
With the Lightning module, on-the-fly deconvolution can
be usedto increase resolution and signal to noise ratio.
Use the slider to decide a balance between speed and
resolution.
Strategy: Adaptive is always better,use this (numbers are
based on and calculated for parts of the image with
different signal tonoise ratio).
Select the used mounting medium or select“custom” and
type in manually the correctrefractive index.

The image window
Over
-
/Underexposure
LUT
Auto contrast
View histogram
Hide/view channels
View all channels
separately
3D viewer
(opens upin new module)
Add
scalebar
and
annotations
Zoom
Right click on the image
to Snapshot a view (for
MIP or orthogonal view
forexample)

Saving, exporting
In LAS X projects can be createdand saved. In a project, many images,datasets,snapshots can be
saved and later re-used.Saved data will end upin .lif files thatcan be opened inLAS X, ImageJ, and
Imaris.
Always save yourimage in RAW format (.lif) as itcontains all the settings and information. If image
file is needed, right click ona dataset andselect Export.
Right click onan Image and
selectProperties to see all the
imaging settings for the
dataset. Tore-use the settings
for a newscanning, hitApply
settings in the pop-up
window.
New project Open project Save all
Show/Hide gallery
Save project
Right click onan Image and
selectExport if image files or
video files are needed of the
dataset

Shutting down the microscope
1. Clean after yourself, put in the smallest objective andcover the microscope stand with its
dust cover.
2. Use the logbook and type in yourimaging session’sdetails.
3. In LAS X softwaresave everything,and turn OFF all lasers
Toturn OFF the lasers, use ONLY the ON-OFF sliders in the Laser Overview window (in the
Acquire tab) or in the Laser Config window (in the Configuration tab).
4. Close down the LASX software, wait till fully OFF
5. Shut down the computer, wait till fully OFF
6. Turn OFF the scanner box switches below the table:
i. Turn the laser interlock key to horizontal position (emission light OFF)
ii. Laser button OFF
iii. Power button OFF
6. Wait 10-15 sec so the instrument turns itself off. Don’t turnoff anything else that is not
mentioned here.