Zeiss LSM 510 META User manual

Zeiss LSM 510 META User manual
Zeiss LSM 510 META - Guided Tour
Confocal microscopy
Zeiss LSM 510 and Zeiss LSM 510 META
Visualisation of biological structures in 3D
Carl Zeiss - Training Application and Support Center
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Zeiss LSM 510 META - Guided Tour
Confocal Principle
Signals from above
and below the plane
of focus fall outside
the pinhole and are
blocked
Carl Zeiss - Training Application and Support Center
Only signals from
plane of focus pass
the pinhole and are
detected - producing
a “optical section”
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Zeiss LSM 510 META - Guided Tour
Upright Zeiss LSM 510 confocal microscope
LSM 510 scan head
Axioplan 2 imaging
Carl Zeiss - Training Application and Support Center
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Zeiss LSM 510 META - Guided Tour
Inverted Zeiss LSM 510 confocal microscope
Axiovert 100M
LSM 510 scan head
in base port position
Carl Zeiss - Training Application and Support Center
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Zeiss LSM 510 META - Guided Tour
Upright Zeiss LSM 510 META confocal microscope
LSM 510 META
scan head
Axioplan 2 imaging
Carl Zeiss - Training Application and Support Center
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Zeiss LSM 510 META - Guided Tour
Inverted Zeiss LSM 510 META confocal microscope
LSM 510 META
scan head
Carl Zeiss - Training Application and Support Center
Axiovert 200M
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Zeiss LSM 510 META - Guided Tour
Contents
• Starting the Zeiss LSM 510 microscope, software and laser
Selecting an objective and focusing the microscope
• Selecting an objective and focusing the microscope
• Configuring the laser scanning and detection for confocal image
acquisition
• Acquiring a Z- and Time - Series
• Data storage
Descriptions also include the LSM 510 META
Carl Zeiss - Training Application and Support Center
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Zeiss LSM 510 META - Guided Tour
Contents
• Starting the Zeiss LSM 510 microscope, software and laser
Selecting an objective and focusing the microscope
• Selecting an objective and focusing the microscope
• Configuring the laser scanning and detection for confocal image
acquisition
• Acquiring a Z- and Time - Series
• Data storage
Descriptions also include the LSM 510 META
Carl Zeiss - Training Application and Support Center
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Zeiss LSM 510 META - Guided Tour
Start the Zeiss LSM 510 Confocal Microscope
1) First switch on the mercury lamp
2) Turn on the remote control switch
3) Wait for the computer to boot up
and Login by simultaneously
pressing the Ctrl, Alt and Delete
keys
Carl Zeiss - Training Application and Support Center
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Zeiss LSM 510 META - Guided Tour
Starting the LSM 510 software
1) Double click the
LSM 510 icon
2) Select “Scan New
Images”
3) Select “Start
Expert Mode”
Carl Zeiss - Training Application and Support Center
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Zeiss LSM 510 META - Guided Tour
Creating a database for acquired images
1) In the main menu File select New database
2) Select drive C or D: from pull down menu
3) Create a new directory if needed
Carl Zeiss - Training Application and Support Center
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Zeiss LSM 510 META - Guided Tour
1) Select Acquire
Turning on the lasers
2) Select Laser
3) Switch required
laser/s to Standby
4) When status is
Ready, click On
5) Set Output [%] so that
the tube current is
between 5.5 and 6.5 A
Carl Zeiss - Training Application and Support Center
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Zeiss LSM 510 META - Guided Tour
Change between direct observation and laser scanning
Upright Microscopes: Axioplan
2 imaging and Axioskop 2 FS
For direct observation of
transmitted light and
fluorescence:
Set slider to “VIS” (push it in)
For laser scanning image
acquisition:
Set slider to “LSM”
(pull slider out)
Carl Zeiss - Training Application and Support Center
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Zeiss LSM 510 META - Guided Tour
Change between direct observation and laser scanning
Inverted Microscope: Axiovert 200 M
Toggle between Vis and LSM button in main menu, automatic switching
between direct observation and laser scanning (no slider)
Carl Zeiss - Training Application and Support Center
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Zeiss LSM 510 META - Guided Tour
Contents
• Starting the Zeiss LSM 510 microscope, software and laser
Selecting an objective and focusing the microscope
• Selecting an objective and focusing the microscope
• Configuring the laser scanning and detection for confocal image
acquisition
• Acquiring a Z- and Time - Series
• Data storage
Descriptions also include the LSM 510 META
Carl Zeiss - Training Application and Support Center
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Zeiss LSM 510 META - Guided Tour
Selecting an objective and focusing the microscope
1) Select Micro (Main menu: Acquire)
(For Axioskop 2 FS these settings have to
be adjusted manually)
2) Microscope settings can be stored
and up to 8 buttons assigned for fast
retrieval and adjustment
3) Objective lens can be selected from
a pull down menu by clicking onto the
Objective button
Carl Zeiss - Training Application and Support Center
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Zeiss LSM 510 META - Guided Tour
Focusing the microscope in fluorescence mode
Click onto Reflected Light to open
the shutter of the HBO lamp
Fluorescence is observed by
selecting the appropriate filter set
in the pull down menu of Reflector
Carl Zeiss - Training Application and Support Center
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Zeiss LSM 510 META - Guided Tour
Focusing the microscope in transmitted mode
Click onto Transmitted Light
and move the slider to set
the intensity of the HAL
illumination
Use no reflector cube in the
reflector turret, chose None
Carl Zeiss - Training Application and Support Center
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Zeiss LSM 510 META - Guided Tour
Contents
• Starting the Zeiss LSM 510 microscope, software and laser
Selecting an objective and focusing the microscope
• Selecting an objective and focusing the microscope
• Configuring the laser scanning and detection for confocal image
acquisition
• Acquiring a Z- and Time - Series
• Data storage
Descriptions also include the LSM 510 META
Carl Zeiss - Training Application and Support Center
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Zeiss LSM 510 META - Guided Tour
Choosing the configuration
SINGLE TRACK
MULTI TRACK
Use for single, double and
triple labelling
Use for double or
triple labelling
Simultaneous scanning
only
Sequential scanning, line by
line or frame by frame
ADVANTAGES
ADVANTAGES
Faster image
acquisition
When one track is active, only
one detector and one laser is
switched on. This dramatically
reduces crosstalk.
DISADVANTAGES
Cross talk between
channels
DISADVANTAGES
Slower image acquisition
Carl Zeiss - Training Application and Support Center
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Zeiss LSM 510 META - Guided Tour
Configuration of the filters and storage of the track configurations
SINGLE TRACK - lasers scan simultaneously
1) Select Config in the
Acquire menu
4b) The Config button
opens the pull down menu
to load/store Track
configurations
2) Select Single Track
4a) Select the
appropriate filters and
activate the Channels
4c) Transmitted light image
can also be generated.
Transmission channel is
usually set to white colour.
Carl Zeiss - Training Application and Support Center
3) Click Excitation to select
the laser and attenuation
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Zeiss LSM 510 META - Guided Tour
Applying a stored configuration and checking the settings
If you select Store by mistake, software
will ask you, if you want to overwrite the
configuration. ANSWER NO!
5) Chose a configuration in the
Track Configuration menu.
Select Apply
Each new login loads a predefined set of
correct configurations.
6) To check for correct
settings, click the Spectra
button
The Spectra button
opens a window to
display the activated
laser lines for excitation
(colored vertical lines)
and channels (colored
horizontal bars)
Carl Zeiss - Training Application and Support Center
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Zeiss LSM 510 META - Guided Tour
Multi Track Configuration
1) Select Multi Track for
sequential scanning
2) Select Config
3) Select a stored track from the
pull down menu, click on Apply
This button stores only the
highlighted single track or applies
a single track.
Carl Zeiss - Training Application and Support Center
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Zeiss LSM 510 META - Guided Tour
Cy5-Cy3-FITC Multi Track
Three laser lines and channels activated sequentially
Excitation
Detection
633 nm, using
the META
detector in
Channel mode
543 nm
488 nm
Carl Zeiss - Training Application and Support Center
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Zeiss LSM 510 META - Guided Tour
Setting the parameters for scanning
1) Select Scan
2) Select Mode
3) Select the Frame Size as predefined
number of pixels or enter your own
values (e.g 300 x 600). Use Optimal for
calculation of appropriate number of
pixels depending on N.A. and λ.
The number of pixels influences the
image resolution!
Carl Zeiss - Training Application and Support Center
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Zeiss LSM 510 META - Guided Tour
Setting the parameters for scanning
Note: When using a
Axioskop 2 FS, indicate
the Objective that is in
use in the Scan Control
window. This ensures
correct calculation of
pinhole, z-stack
optimization etc.
Carl Zeiss - Training Application and Support Center
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Zeiss LSM 510 META - Guided Tour
Adjusting the scan speed
Adjust the scan speed - a
higher speed with averaging
results in the best signal to
noise ratio. Scan speed 8
usually produces good results.
Use 6 or 7 for superior images.
Carl Zeiss - Training Application and Support Center
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Zeiss LSM 510 META - Guided Tour
Choosing the Dynamic Range (8/12 Bit per pixel)
Select the dynamic range - 8
bit will give 256 gray levels,
12 Bit will give 4096 levels.
Photoshop 5 will import 12
and 16 Bit images.
Publication quality images
should be acquired using
12 Bit.
12 Bit is also recommended
when doing quantitative
measurements or when
imaging low fluorescence
intensities.
Carl Zeiss - Training Application and Support Center
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Zeiss LSM 510 META - Guided Tour
Channel Settings - Adjusting the Pinhole
Pinhole size
= 1 Airy unit
Set pinhole size to 1 Airy unit for best compromise between depth discrimination and efficiency.
Pinhole adjustment changes the “Optical slice”.
When collecting multi channel images, adjust the pinholes so that each channel has the same
“Optical Slice”.
This is important for colocalization studies.
Carl Zeiss - Training Application and Support Center
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Zeiss LSM 510 META - Guided Tour
Image Acquisition
1) Find opens new image
window and automatically preadjusts detector sensitivity
2) Select Fast XY for continuous
fast scanning - useful for
finding and changing the focus
3) Single records a single image
4) Stop blanks the laser beam
and stops the scanning mirrors
5) Select Cont. for continuous
scanning with selected scan speed
Carl Zeiss - Training Application and Support Center
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Zeiss LSM 510 META - Guided Tour
Minimal Pixel Size determined by Nyquist Sampling
Magnification
NA
Pixel size
5
0.15
1.03 µm
10
0.3
0.51 µm
20
0.5
0.31 µm
Values are for scan zoom = 1.0
40
1.3 (oil)
0.12 µm
63
1.4 (oil)
0.11 µm
Adjusting the field size (“XY”) to 56 µm with
the 63× lens, would produce a pixel size of
0.1 µm
100
1.4 (oil)
0.11 µm
Brightness of image = Magnification2/NA2
Field size can be adjusted by changing the objective magnification, or by optical zooming. Changing
from 63 × to 100 × will reduce the field size, but will also reduce the amount of light available.
Carl Zeiss - Training Application and Support Center
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Zeiss LSM 510 META - Guided Tour
Optical Zooming
The level of zoom can be
changed either by using the
Zoom, Rotation & Offset
control in Mode menu of the
Scan Control, or by selecting
Crop in the image menu.
The image can also be
rotated by selecting and
dragging the bars
Carl Zeiss - Training Application and Support Center
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Zeiss LSM 510 META - Guided Tour
Selecting gain and offset – Choosing a lookup table
1) Select Palette
2) Select Range Indicator
Red = Saturation (maximum)
Blue = Zero (minimum)
Carl Zeiss - Training Application and Support Center
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Zeiss LSM 510 META - Guided Tour
Scan Control – Setting Gain and Offset
Detector gain determines the
sensitivity of the detector by
setting the maximum limit
Amplifier Offset determines the
minimum intensity limit
Amplifier Gain determines
signal amplification
Gain
Saturation at the maximum
→ reduce Detector Gain
Saturation at the minimum
→ increase Amplifier Offset
Gain set correctly
Offset set correctly
Carl Zeiss - Training Application and Support Center
Offset
Amplifier Gain increases the
whole signal, and the Amplifier
Offset will need to be decreased.
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Zeiss LSM 510 META - Guided Tour
Saturation of Signal Intensity with Laser Power
Photobleaching is linear!
Signal Intensity
• Fluorophore saturates
at 6% laser
transmission
• Photobleaching is
linear
2%
4%
6%
8%
Argon laser 488 nm % transmission
Laser transmission should not be set higher than the saturation level.
Carl Zeiss - Training Application and Support Center
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Zeiss LSM 510 META - Guided Tour
Adjusting the Laser Intensity
1) Set Pinhole to 1 Airy unit
2) Set Detector Gain high
3) When the image is saturated, reduce
AOTF transmission in the Excitation
panel to reduce the intensity of the laser
light at the specimen
image with
saturated
pixels
Carl Zeiss - Training Application and Support Center
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Zeiss LSM 510 META - Guided Tour
Adjusting Gain and Offset
Both Detector Gain and Amplifier
Offset saturated
gain and
offset not
correct
1) Increase the Amplifier Offset
until all blue pixels disappear, and
then make it slightly positive.
2) Reduce the Detector Gain until
the red pixels only just disappear.
gain and
offset
correct
no blue
no red
Carl Zeiss - Training Application and Support Center
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Zeiss LSM 510 META - Guided Tour
Adjusting the Laser, Gain and Offset using a Multi Track Configuration
Each channel is selected independently by
clicking on the colour button indicating the
channel i.e. Ch2-T1 (Channel 2, Track 1).
The laser power and all other parameters
are optimised as described in the previous
slides for each selected channel.
For accurate colocalisation, adjust
each Pinhole so that each channel
has the same Optical Slice
To adjust laser, gain or offset for a single track in a multi-track
configuration it is possible to temporarily deactivate the other
tracks in the Configuration control
Carl Zeiss - Training Application and Support Center
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Zeiss LSM 510 META - Guided Tour
Setting up Gain and Offset - Multi Track
1) Select Split XY in the Image window
2) In Palette, select Range indicator
3) Select each channel separately under Channels in the Scan control
window and adjust the Laser intensity, Detector Gain, and Amplifier Offset
as described previously.
Carl Zeiss - Training Application and Support Center
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Zeiss LSM 510 META - Guided Tour
Line Averaging
Averaging improves the image by
increasing the signal : noise ratio
Averaging can be achieved line by line,
or frame by frame
1) Select Line or Frame under Mode in
Scan Average within the Mode panel of
the Scan Control window
2) Select Number for averaging. The
more the better for the signal to noise
ratio (max 16) in this case, each line will
be scanned 4 times. But: Averaging
increases the exposure time of the
sample!!
Carl Zeiss - Training Application and Support Center
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Zeiss LSM 510 META - Guided Tour
Frame Averaging
1) Select Frame
2) Select the Number for averaging - The more the
better for signal to noise ratio (max 16).
Continuous averaging is possible in this mode.
Frame averaging helps reduce
photobleaching, but does not give quite
such a smooth image. There is also a
longer delay between each track when
using “Multi Track”.
Continuous averaging has a Finish button
which allows the scan currently in progress to
be completed before stopping
Carl Zeiss - Training Application and Support Center
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Zeiss LSM 510 META - Guided Tour
Collecting an Averaged Image
1) Under Scan Average select
the Number for the average.
In the Channels panel of the
Scan Control window select
Single. An averaged image will
be collected.
Range indicator set
to No Palette
Carl Zeiss - Training Application and Support Center
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Zeiss LSM 510 META - Guided Tour
Contents
• Starting the Zeiss LSM 510 microscope, software and laser
Selecting an objective and focusing the microscope
• Selecting an objective and focusing the microscope
• Configuring the laser scanning and detection for confocal image
acquisition
• Acquiring a Z- and Time - Series
• Data storage
Descriptions also include the LSM 510 META
Carl Zeiss - Training Application and Support Center
43
Zeiss LSM 510 META - Guided Tour
Scanning a Z-Series using Mark First/Last
1) Select Z Stack
2) Start scanning using Fast XY or XY cont
3) Keep your eye on the image and move the
focus to the beginning of the Z-Series, then
select Mark First
4) Move the focus back in the opposite direction
to the end of the Z-Series, then select Mark Last
5) X:Y:Z = 1:1:1 sets the Z-interval so that the
voxel has identical dimensions in X, Y, and Z.
6) Start will initiate the acquisition of the ZStack. The acquisition can be stopped at any
time.
NOTE
Focusing can be achieved manually (preferred),
or using Stage in the LSM menu if there is a
motorized scanning table.
Focus
Increment
Carl Zeiss - Training Application and Support Center
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Zeiss LSM 510 META - Guided Tour
Using Auto Z Brightness Correction
Auto Z provides an automatic gradual adjustment of Detector
Gain, Amplifier Offset, Amplifier Gain, and Laser intensity
setting between the first and last optical slice of a Z Stack.
1) After defining the Z position of the first and last optical slice
activate Auto Z.
2) Move to the First Slice and adjust the parameter for the
image acquisition in the Channels panel for each used
channel as described in the previous slides. Then click on
Set A to store the values.
3) Repeat the procedure after moving to the Last Slice. Click
on Set B to store the parameters for the last slice.
Note: Positions A and B do not have to be the first and last
slice of a stack and can also be defined simply by focussing
to the appropriate positions, adjusting the parameters and
pressing Set A or Set B.
4) The parameters for image acquisition will be gradually and
linearly adjusted between the first and last slice of the Z
Stack. Thus signal intensity and image quality is comparable
throughout the Z Stack.
Carl Zeiss - Training Application and Support Center
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Zeiss LSM 510 META - Guided Tour
Confocal Z Sectioning
Number of Sections for correct sampling
Optical thickness d depends on:
• Wavelength λ
• Objective lens, N.A.
• Refractive index n
• Pinhole diameter P
2
d ~ P n λ / (N.A)
~ 0.5 µm @ 63x1.4
The optical slice
thickness is displayed
in the Scan Control
For Z-sectioning it is optimal to have:
no missing information @ minimal number of sections
Slices overlap by the half of their thickness
„Nyquist-“ or Sampling- Theorem
Carl Zeiss - Training Application and Support Center
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Zeiss LSM 510 META - Guided Tour
Z Stack – Number of Slices and Increment
1) Select Z slice - the window
Optical Slice will appear
2) Select Optimal interval the
computer will calculate the
optimum number of sections
For more or less sections adjust Num Slices
Carl Zeiss - Training Application and Support Center
3) Select Start to
acquire the
complete
stack
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Zeiss LSM 510 META - Guided Tour
Z - Series using Z Sectioning
1) Select Z Stack
2) Select Z Sectioning
3) Select Line Sel
4) Select the large arrow
button and position the
XZ cut line
XZ cutline will be
displayed as diagram
within the XY image
Carl Zeiss - Training Application and Support Center
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Zeiss LSM 510 META - Guided Tour
Z Sectioning – Setting Range
Pull red
lines to
set limits
for ZSeries
1) Decide whether to Keep Interval
(number of slices will change) or Keep
Slice (Interval between slices will be
adjusted)
2) Select Range and position bars to
decide where the Z - Series begins and
ends
3) Select Start for image acquisition
Carl Zeiss - Training Application and Support Center
Drag
green line
to change
focus
position
Pressing Range produces an XZ image of
selected Z-range, plus 50% above and below
the selected stack.
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Zeiss LSM 510 META - Guided Tour
Viewing a Z - Series
In the image window
1) Select xy
2) Select Slice
3) Use scroll bar to
view individual
sections
Carl Zeiss - Training Application and Support Center
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Zeiss LSM 510 META - Guided Tour
Viewing a Z - Series using Gallery
1) Select Gallery
2) Select Data for
scale
Use Subset to
extract sections
Carl Zeiss - Training Application and Support Center
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Zeiss LSM 510 META - Guided Tour
Viewing a Z- Series using Orthogonal Sections
1) Select Ortho
2) Select mouse
(Select)
3) Using the mouse,
position the cut lines.
To save orthogonal
sections, select
Export and save as
contents of image
window.
Carl Zeiss - Training Application and Support Center
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Zeiss LSM 510 META - Guided Tour
Selecting and Saving a Region of Interest (ROI)
1) Select
Overlay and
define shape
of ROI
2) Extract
region
creates a ZStack from
the ROI
3) Save data
Carl Zeiss - Training Application and Support Center
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Zeiss LSM 510 META - Guided Tour
Using a ROI for faster image acquisition and data saving
1) Select EditROI from the LSM menu bar
2) Select Fit Frame Size to bounding Rectangle
3) Choose shape of ROI
4) Position and size the ROI
in the image with the mouse
5) Start Scan
To remove ROI and overlay select blue bin
or deactivate ROI. Closing the window only
removes overlay, ROI is still active.
Deactivate Use ROI in the LSM menu.
Carl Zeiss - Training Application and Support Center
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Zeiss LSM 510 META - Guided Tour
Multiple Regions of Interest
1) Un-select Fit Frame Size to bounding Rectangle, Choose shapes of ROIs
4) Position and size the ROIs with mouse
5) Start Scan
To remove ROIs and overlay select blue bin or deactivate ROIs. Closing the window
only removes overlay, ROIs are still active. Deactivate Use ROI in the LSM menu.
Carl Zeiss - Training Application and Support Center
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Zeiss LSM 510 META - Guided Tour
Time Series
• Set up scanning parameters
for image acquisition as
described in previous slides
• Select TimeSeries from the
LSM menu
• Enter the Number of cycles
• For a Time Delay between
image acquisition select min,
sec or ms and set time with
the slider
• Select Start T to start image
acquisition
• Instead of using Manual you
can select Time to start and
stop the series at a certain
system time!
Carl Zeiss - Training Application and Support Center
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Zeiss LSM 510 META - Guided Tour
Viewing a Time Series of a Z Stack
Z Sections
for any time
Carl Zeiss - Training Application and Support Center
Time points for
any Z Section
Both Z sections
and time series
57
Zeiss LSM 510 META - Guided Tour
Time Series – Physiology Experiments
1) If required, use
multiple regions of
interest
2) Set up Time Series
as before
3) Instead of using
StartT select
MeanROI to start
scanning
View and save
data by
selecting
Mean in the
image window
Carl Zeiss - Training Application and Support Center
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Zeiss LSM 510 META - Guided Tour
Imaging a large area using Tile Scan
This function is only
available with a
motorized stage
1) Select Stage on LSM
menu
2) Enter the Tile Numbers
3) Select Start
The maximum size is
4096 x4096 pixels
Carl Zeiss - Training Application and Support Center
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Zeiss LSM 510 META - Guided Tour
Any position can then
be marked and a
single image acquired
by selecting Move to
and then single
Carl Zeiss - Training Application and Support Center
Tiled Image
60
Zeiss LSM 510 META - Guided Tour
Contents
• Starting the Zeiss LSM 510 microscope, software and laser
Selecting an objective and focusing the microscope
• Selecting an objective and focusing the microscope
• Configuring the laser scanning and detection for confocal image
acquisition
• Acquiring a Z- and Time - Series
• Data storage
Descriptions also include the LSM 510 META
Carl Zeiss - Training Application and Support Center
61
Zeiss LSM 510 META - Guided Tour
Saving Data - Using Database
1) Select Save or Save as on image window or LSM menu
bar
2) Enter file name and notes if required
3) Select OK
Carl Zeiss - Training Application and Support Center
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Zeiss LSM 510 META - Guided Tour
Saving Data – Using Export
1) Select File from LSM menu
2) Select Export
3) Select Image type
4) Select Single image with raw data
(No overlay or Look up table etc.
is saved), Series with raw data, or
Contents of the image window
(Saves the image as shown on
the screen)
5) Select Save as type
Tif - Tagged image File” is OK for
8 bit - use “Tiff -16 bit” for 12 bit
acquired images
(Most other software will not
recognize 12 bit)
Carl Zeiss - Training Application and Support Center
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Zeiss LSM 510 META - Guided Tour
Shut Down Procedure
1. Go to: Acquire in the LSM menu - Laser – and deactivate HeNe Lasers by
clicking Off to switch off Lasers
Note: To turn off the Argon laser,
first click on Standby, then reduce
output power to 25%. Select Off.
2. Go to File - Exit to leave LSM 510 program
3. Shut down the computer
4. Wait until fan of Argon laser has switched off.
5. Turn off the remote control box
6. Switch off the mercury vapour lamp.
Carl Zeiss - Training Application and Support Center
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Zeiss LSM 510 META - Guided Tour
Please note:
This guided tour is intended merely
as a quick introduction into the
Zeiss LSM 510 software and does
not cover all aspects of the system.
Please consult the manual
for detailed instructions!
Carl Zeiss - Training Application and Support Center
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Zeiss LSM 510 META - Guided Tour
This guided tour is based on
work done by
Peter Jordan
ICRF
London
United Kingdom
edited and complemented by
Eva Simbürger, Solveig Hehl and René Hessling
Carl Zeiss Jena GmbH
Carl Zeiss - Training Application and Support Center
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