Leica TCS SP8 User manual

Leica TCS SP8
Quick Start Guide

Leica TCS SP8 System Overview

Start-Up Procedure
1. Turn on the CTR Control Box, EL6000 fluorescent light source for the
microscope stand.
2. Turn on the Scanner Power (1) on the front of the Compact Supply Unit (CSU)
3. Turn on the Laser Power (2)
4. Enable the lasers by turning the key
from the Off position to the On
position (3).
5. Turn on Workstation and Monitor
6. Log into user profile
7. Double click on the LAS X icon to start the software.
8. Select Machine for the Configuration and DMi8 for the Microscope. (the
simulator is used to view and analyze images and not acquistition)
10. Click OK to start the initialization of
the LAS AF (Leica Application Suite
for Advanced Fluorescence)
11. A message will appear asking whether you want to initialize the stage. Initializing
the stage is required to activate the Tiling and Mark & Find features in the software.
If you select yes, the stage is going to
move to all 4 corners to calibrate the
position of the stage.
Make sure there is nothing on the
stage that will hit the condenser
before you select yes!

Shut-Down Procedure
1. Turn off the lasers in the software
2. Lower the objective to the lowest position, remove specimen and clean all of the
objectives
3. Save all unsaved data and exit LAS X.
NOTE: Do not turn off scanner and/or CTR control box for the microscope before the
software is closed
4. Turn off the switches on the front of the laser supply unit in the reverse order (#3-#1)
of start-up (ie. turn key off, then turn off Laser Power and then turn off Scanner
Power)
5. Turn off microscope control box

The LAS X software will open in the Acquire tab. There are 3 portions to this window:
Scan Parameters Light Path Image

Turning on the Lasers:
1. Click on the “+” in the Laser Lines Box in the Beam Path Settings Window to directly
open the main Laser Control Window
2. Activate the lasers you require
3. Alternatively, click on the Configuration tab and the Laser icon to open the main
Laser Control Window
NOTE: Only turn on the laser(s) with the appropriate laser line(s) that will excite
the fluorophores you are using

Additional Tools in the Configuration Tab…
1. Customize the USB Control Panel -assign various parameter and sensitivities to
the various knobs of the control panel
2. Specifications of the Objectives equipped on the microscope
Save and Load the customized settings
Alternatively , click on the Control
Panel Icon in the Beam Path Settings
Window as a short cut to the USB
Control Panel Window

Additional Tools in the Configuration Tab Continued…
3. Dye Database with the excitation and emission spectra of common fluorochromes
Emission spectrum from a lambda scan can be added to the dye database.
Many manufacturer’s will also provide the data for fluorophores that can added to
the database (see LAS AF Help)
Another great resource is the Leica FluoScout
interactive tool
http://www.leica-microsystems.com/fluoscout/

Beam Path Setting
Option #1: Manual setting of the beam path configuration
1. Click On to activate
the lasers 2. Adjust the laser intensity
of the appropriate laser
line(s) by moving the
slider up or by directly
entering the level (start
low as a suggestion).
4. Use Autoselect to select the beam splitter
(inactivate and manually select if BSC is red)
3. Select the appropriate objective
6. Select the appropriate emission
spectrum (if available) from the
dye data base to use as a guide
8. Define the of emission to be collected with the sliders
5. Click On to activate the
appropriate detector(s)
Keep in mind that the emission spectra
displayed are to be used as a guide and are not
defining the wavelengths collected. The
specific wavelengths collected are determined
by the position of the gates.
Alternatively, double click on
the slider to open a window
that allows you to directly
enter the start and end
position of the gates.
7. Select the Pseudocolour

Beam Path Setting
Option #2: Dye Assistant to set beam path configuration
The Dye Assistant offers suggestion on system configuration based on the spectral
characteristics of the fluorescent dyes being used. The user can select the appropriate
option for his or her application
1. Activate the Dye Assistant.
Select the dye from the database Select the type of detector (if applicable)
Use the “+” or “-” to
add or delete a dye
The different configuration options for acquisition are suggested
here and the overlap is shown graphically:
•Yield: Intensity yield of the individual dyes
•Crosstalk: Intensity of the crosstalk in other channels
Edit and Apply
the settings.
(see next page)

The following settings are mode for image acquisition when clicking Apply:
•Selection of the laser lines
•Selection of the detectors
•Setting for the detection range
•Assignment of the fluorescent dyes to the respective detectors
•Assignment of the appropriate colour look-up table (LUT) for the
respective fluorescent dyes\
All other settings for image acquisition are made as usual

Beam Path Setting
Option #3: Load/Save specific settings
The settings can be saved for subsequent experiments with the same or similar
specimens.
SUGGESTION: Include your name or initials when saving the settings to identify who
created the configuration.
Alternatively, save an image as a sample “configuration” image and
apply those settings from the Experiment tab (ie. Save an image and
open it to apply the settings)
To Load the instrument parameter settings, click on the arrow keys beside “Load/Save
single settings” and select the appropriate setting
Click on the diskette
icon to save a setting

Beam Path Setting - Sequential Acquisition
1. Click SEQ to open the Sequential Scan
control window
2. Set the light path configuration for the first
sequence (ie laser, beam splitter, detector,
emission window, etc,)
3. Select switching mode and click +
to add a sequential scan
4. Set the light path configuration for
the new scan
5. Repeat if necessary
NOTE: Sequential scan settings
can be saved and loaded as an
alternative.
Turning on or off lasers and detectors
is very fast whereas changing the beam
splitter and/or position of the
detection gates is much slower.
Therefore, in order to switch image
acquisition after each line, these
componentsmustbethesameforeach
of the scans

Acquisition Parameters
Format or # of pixels in the image
(start with 512 x 512)
Scan Speed (start with 400-600 Hz)
Option for bidirectional for ~2x faster
acquisition (may need to adjust phase)
Adjust zoom factor various ways by:
•Adjusting slider
•Entering specific zoom factor
•Activating the Zoom In and drawing
a ROI in image
•Zoom knob on Control Panel
The Image Size and Pixel Size will
change accordingly as the Format
and/or Zoom Factor is adjusted
Select Optimize xy Format to set the
optimal # of pixels correctly over-
sample (resolution depends on NA of
objective)
Set Average and/or Accumulation
NOTE: Averaging removes noise and in
general, less averaging is required with
a lower gain setting for the detectors.
The HyDs have very little, if any, noise
and therefore requires less averaging
than the PMTs.
Rotate image if necessary
(Optical rotation)
Panning to position
specimen in image
window
The pinhole will automatically default to
1 Airy unit (optimal) and will adjust
accordingly with the different objectives.
The diameter can be be adjusted
manually with the slider (or control
panel)

1. Select xyz as the Acquisition Mode
2. Use the Control Panel to move the focus
position to beginning position of the stack
3. Click Begin to set the start position
4. Use the Control Panel or the
SmartMoveto move the focus position to
the end position of the stack and click End
5. Select Z-Wide to indicate the focus drive of
the microscope stage will be used to control z
position during the stack.
Note: Z-Galvo refers to Z-control through a
Super Z Galvo Focusing stage, which in not
configured with the particular system.
6. Zoom in to help visualize the schematic
representation of the z stack
Acquisition Modes:
ZStack(xyz)
Select the appropriate Acquisition Mode
•xyz – single image or z-stack
•xzy – xz image
•xyt – time series
•xyzt -- z-stack and time series
•xy– lambda scan
7. Manually define the number of images
of a z-stack (Nr. of steps) and the
distance between the images (z-step
size), or have them optimized
automatically (System Optimized).

Z Stack (xyz) continued…
Use arrows to move to the
set Begin or End position
Delete Begin and
End Positions
Use Set Focus to define
current position as the
focal plane
Move to set focal
position
Move to centre
position of stack
Use mouse to move
position of objective Alternative to setting the Begin and
End position, a stack can be define
around current position.
Activate Z Around Current and
indicate the size of the stack
Change direction of
acquisition of the
stack
Time Series (xyt, xyzt, etc.)
1. Select the appropriate acquisition
mod that includes time (t)
2. Set the Time Interval (for no delay
between images, select Minimize)
3. Define the parameter for acquisition
to stop

Additional Acquisition Mode – Tile Scan
1. Activate Tile Scan acquisition mode
NOTE: the stage must be initialized
during the start-up
Adjust Zoom slide to
visualize better the
current stage posistion
Option 2 (easiest):
1. Move the specimen to the position
that will be the centre of the tile
scan using the SmartMove
2. Enter the dimensions of the tile
scan (ie. 2 x 2 for a 2 image by 2
image tile scan)
3. Click on Start to begin the
acquisition
Option 1
1. Move the specimen to the position that will a
corner of the tile scan using the SmartMove
and Mark the Position
2. Move the specimen to the opposite
corner of the tile scan and mark the
new position.
The # of tiles will be calculated to
accommodate the marked
positions.
A
dditional
p
ositions can be marked
if necessary
3. Click on Start to begin the
acquisition
Activate Merge Images for automatic stitching and
smoothing of seams after acquisition is complete.
Both the single images and merged images are added to
the data container. The single images can be re-tiled
using the Merge tool in the Process Tab.
The % overlap used for the automated stitching can be
set under the Configuration Tab in the Stage Control
window

Additional Acquisition Mode – Mark & Find
1. Activate Mark & Find in acquisition mode
NOTE: the stage must be initialized
during the start-up
Move the stage using the SmartMove and
mark the position of interest. Repeat
as many times as necessary
Adjust Zoom slide to
visualize better the
current stage position
Click to delete current saved
position or all saved
positions with the trash can
Move the stage to a specific
saved position
Z-position set with Z-Galvo
stage is also save with the xy
coordinates