Leica TCS SP5 X User manual

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Cell Imaging Unit – UIC
LEICA TCS SP5 MICROSCOPE MANUAL

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TABLE OF CONTENTS
ANATOMY OF THE LEICA SP5 CONFOCAL MICROSCOPE 3
INITIALISATION PROCEDURE 4
SP5 MICROSCOPE 5
1. RIGHT SIDE OF MICROSCOPE (DETAILED VIEW) 5
2. FRONT SIDE OF MICROSCOPE (DETAILED VIEW) 6
3. LEFT SIDE OF MICROSCOPE (DETAILED VIEW) 6
4. CONTROL PANEL BUTTONS 7
STARTING THE LAS SOFTWARE 8
1. CONFIGURE FOR ACQUISITION 8
1.1
THE LEFT PANEL ALLOWS YOU TO CONFIGURE THE IMAGE
ACQUISITION PARAMETERS
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1.2
THE RIGHT PANEL ALLOWS YOU TO LOAD A PRE-MADE
CONFIGURATION OR TO CREATE YOUR OWN ONE
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1.3
CREATE A SEQUENTIAL ACQUISITION 10
2. ACQUIRE IMAGES 11
3. ACQUIRING A Z-STACk 12
4. SAVE AND EXPORT YOUR IMAGES 13
SHUTDOWN PROCEDURE 14

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ANATOMY OF THE LEICA SP5 CONFOCAL MICROSCOPE
1. Research inverted microscope 1. Transmitted light source
2. Scanhead 2. Stage
3. TCS Workstation 3. CLSM port
4. Microscope control unit 4. z -Drive
5. Laser and power supply 5. Display
6. Computer table 6. Controller
7. Microscope table 7. x-Drive
10. Control panel 8. y-Drive
11. Control monitor 9. z-Drive
12. Image Monitor 10. Eyepieces
13. Supply control 11. Condenser head
12. field diaphragm
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INITIALISATION PROCEDURE
1. Turn on the “PC”, “SCANNER” and “FAN” switch.
1. Turn the LASER key on
2. For fluorescence samples, turn on the Fluorescence light power source (check
first if the lamp is not hot), otherwise leave it off and proceed
3. Turn on the microscope
4. Log on into your personal/group account
5. Start the “Leica Confocal Software -LAS” available both as a Desktop
shortcut and the Windows start menu.

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SP5 MICROSCOPE
The microscope is fully automated which means that you have now button in stead of
knobs. It you know what you do in a conventional microscope, it takes 5 minutes to
understand what the buttons are for.
1. RIGHT SIDE OF MICROSCOPE (DETAILED VIEW)
Displays the following information:
1. Microscope stage up movement (DO NOT USE).
2. When used with 7 is sets the maximum and minimum threshold (DO NOT
USE).
3. Microscope stage down movement (DO NOT USE).
4. Speed of knob 7
5. Change objective to high magnification.
6. Change objective to low magnification.
7. Focus knob.

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2. FRONT SIDE OF MICROSCOPE (DETAILED VIEW)
Displays the following information:
1. Change cube for blue excitation filter cube – FITC, GFP
2. Change cube for green excitation filter cube – RFP, CY3
3. Use if for cutting some “umpolarized light” from the laser (might be
interesting for good DIC)
4. Empty position
5. Empty position
6. Empty position
7. Fluorescence shutter
3. LEFT SIDE OF MICROSCOPE (DETAILED VIEW)

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Displays the following information:
1. Change from transmitted light to transmitted light detector (not confocal
detector)
2. Enables switching from oil to dry objectives and vice-versa, minimizing the
risk of having oil in dry objectives
3. Move one filter cube position
4. Change from confocal mode to visualization mode
5. Change aperture diaphragm just for florescence
6. Change field diaphragm just for transmitted light
7. Change light intensity in both fluorescence and transmitted light, depending
on which mode you are
8. Toggles between transmitted light and fluorescence
4.CONTROL PANEL BUTTONS
Displays the following information:
1. Smart gain
2. Smart offset
3. Rotation
4. Pinhole
5. Zoom
6. Z position

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THE LAS SOFTWARE
1. Double-click on the LAS AF icon to start the program.
2. In the window coming up check that MACHINE is selected under
CONFIGURATION and START.
3. In the software window, go the CONFIGURATION tab, LASER and turn on
the lasers you need
4. Set the percent power on the Argon laser (25-30% is recommended).
1. CONFIGURE FOR ACQUISITION
It’s time now to configure the software for a confocal acquisition:

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1.1THE LEFT PANEL ALLOWS YOU TO CONFIGURE THE
IMAGE ACQUISITION PARAMETERS:
•Acquisition type. Standard mode is XYZ.
•Image size in pixel. Standard size is 512x512 and acceptable for
publication, although in some instances 1024x1024 may be desirable
for publication. Lower and higher resolutions are available in the drop-
down menu, but beware, the higher the resolution the larger the file.
The pixel size and image size are shown.
•Acquisition speed in Hz de 10-1000 Hz. Standard speed is 400Hz.
Faster scanning speeds may be required for imaging live cells or
moving specimens. For even faster scanning, you can activate
bidirectional scanning (“Bidirectional X”). This doubles the scanning
speed, but requires “Phase” adjustment.
•Pinhole size. The default setting of 1 Airy Unit (AU) is the
recommended setting. (The pinhole setting of 1 Airy Unit will display
a different physical pinhole aperture size on the control panel for each
objective). For faint specimens, increasing the pinhole size may help,
but the images you are capturing may no longer be truly “confocal”
images.
•Zoom level. Refer to notes on “Zooming and selecting a Region of
Interest”. By zooming in the pixel size will decrease. For high
resolution imaging optimal pixel size according to the Nyquist
criterion should be ensured. Note that the pixel size can also be
decreased by enhancing the format.
•Level of averaging used to reduce the background noise. It’s
unnecessary to use an average of more than 8x. To determine whether
you should average your image more times, try capturing with more
averages and see whether the image is improved. Line averaging is
faster and therefore recommended for live cells. For fixed specimens, it
doesn’t really matter whether you use line averaging or frame
averaging or a combination, although frame averaging may be better
suited for some applications to allow for a brief recovery from photo-
bleaching.

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1.2 THE RIGHT PANEL ALLOWS YOU TO LOAD A PRE-MADE
CONFIGURATION OR TO CREATE YOUR OWN ONE
1.2.1 If you want to use a pre-made configuration, just use the drop
down menu « Load/Save single settings »:
1.2.2 If you want to create your own configuration, follow this step-by-
step:
•Activate the laser lines you need (« fluo » and/or « Visible »).
•Tune their output power (usually between 15 and 50 % depending on the line
and the sample that is used).
•Activate the PMTs (detectors) that you need (check « Active »). Use first the
PMTs located just under the spectral band you want to detect.
•Choose the bandwidth according to your staining for each PMT.
1.3 CREATE A SEQUENTIAL ACQUISITION:
This mode allows you to avoid « crosstalk » problems by acquiring each channel one
after the other.
Click on the « sequential » button then create a configuration only one
channel with only one excitation wavelength.
Add a new sequence by clicking on the « + » button, the preceding sequence being
used as a template for the new one. Create the configuration for the next channel. You
can add as many sequences you want. To delete a sequence, click on the « - » button,
and the last one created will be deleted.
Click here to
select a pre-made
configuration

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2 ACQUIRE IMAGES
Once the acquisition configuration is done you can start to acquire images.
The intensity of the image can be controlled by the laser intensity and the gain
(amplification of photons) and offset (threshold) adjustment of the detector.
6.1 Start preview scanning by clicking on LIVE.
6.2 Enhance the gain until you see an image. By default the gain is on
0V (black screen) when you start. Adjust the focal plane with the
z-knob until you see the brightest plane of interest.
6.3 Adjust gain and offset: to aid adjustment of gain and
offset choose the «glow-over-under» display by clicking
into the QLut icon (left border on right screen).
6.4 In the display mode the saturated pixels are represented by blue
dots and the black signals (grey level 0) are shown in green.
While scanning adjust the offset and gain so that there are a few green pixel in the
background and some blue speckles in the object. The aim is to have the whole range
of grey levels in the image.

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3ACQUIRING A Z-STACK
Activate the Z-stack window. The cube represents the specimen. The yellow
plane marks the current z-position. This position is specified in the field below
the cube (z position µm).
7. 1 Set the z-stack range: Select the top and the bottom of the sample
and define them by clicking into the Begin and End arrow which will than
turn red.
7.2 Set z-step size: By default the software calculates the optimal z-
step size and number or steps (accept this frequency for subsequent
deconvolution or 3-D reconstruction). However, often such a high z-imaging

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frequency is not necessary. You can also define the number of images of a z-
stack and the distance between them manually.
4SAVE AND EXPORT YOUR IMAGES
The captured images displayed in the “Experiment” menu are not safely saved until
you save them. It is therefore strongly recommended that you start saving your images
from the very first capture you make, and that you update your save after each image
capture.
4.2 You can open the properties pages of a picture and restore the acquisition
parameters that were used:

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SHUTDOWN PROCEDURE
1. Log out and shut down the computer in windows.
2. Turn off the microscope.
3. Turn off the camera.
4. If you used the HBO light source and there is no one using the microscope for
the next hour or you’re sure that the next user won’t use this light source, then turn the
lasers off (in the key) and the fluorescence light power source off. Also turn off the
“PC” and “SCANNER” switch. Turn off “FAN” switch after 10 min (for laser
cooldown)
5. If you used any immersion lenses be sure to clean them.