Confocal microscopy
Zeiss LSM 510 and Zeiss LSM 510 META
Visualisation of biological structures in 3D
Inverted Zeiss LSM 510 confocal microscope
Contents
• Starting the Zeiss LSM 510 microscope,
software and laser 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
Contents
• Starting the Zeiss LSM 510 microscope,
software and laser
• 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
Starting the LSM 510 software
1) Double click
the LSM 510
icon
2) Select “Scan
New Images”
3) Select “Start
Expert Mode”
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 for each session
1) Select Acquire
2) Select Laser
3) Switch
required laser/s
to Standby or On
Argon power
should be set to
about 6.1A
Turning on the lasers
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)
Contents
• Starting the Zeiss LSM 510 microscope,
software and laser
• 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
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
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
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
Contents
• Starting the Zeiss LSM 510 microscope,
software and laser
• 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
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
Configuration of the filters and storage of the track configurations
SINGLE TRACK - lasers scan simultaneously
4) The Config button opens the
pull down menu to load/store
Track configurations
1) Select Config in
the Acquire menu
2) Select
Single Track
3) Select the
appropriate filters
and activate the
Channels
Transmitted light
image can also be
generated.
Transmission
channel is usually
set to white colour.
3) Click Excitation to select the
laser and attenuation
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
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)
Each new login loads a predefined set
of correct configurations.
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.
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
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!
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.
Adjusting the scan speed
Adjust the scan
speed - a higher
speed with averaging
results in the best
signal to noise ratio.
Scan speed 8-9 usually
produces good
results. Use 6 or 7 for
superior images.
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.
Channel Settings - Adjusting the Pinhole
Pinhole size
= 1 Airy unit
0.8 “Airy units” produces the best signal : noise ratio
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.
Image Acquisition
1) Find
automatically preadjusts detector
sensitivity
2) Select Fast XY
for continuous fast
scanning - useful
for finding and
changing the
focus
3) Stop blanks the
laser beam and
stops the
scanning mirrors
Minimal Pixel Size determined by Nyquist Sampling
×
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
100
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
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.
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
Selecting gain and offset – Choosing a lookup table
1) Select Palette
2) Select Range Indicator
Red = Saturation (maximum)
Blue = Zero (minimum)
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
Offset
Amplifier Gain increases the whole
signal, and the Amplifier Offset will
need to be decreased.
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.
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
Adjusting Gain and Offset
Both Detector Gain and Amplifier Offset
saturated
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.
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
0.8 Airy units gives the best
signal:noise ratio
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.
Split
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!!
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
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
Contents
• Starting the Zeiss LSM 510 microscope,
software and laser
• 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
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 Z-Stack.
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
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.
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.
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
d ~ P n λ / (N.A)2
~ 0.5 µm @ 63x1.4
Optimal: (no missing information @ minimal
number of sections)
Slices overlap by the half of their thickness
„Nyquist-“ or Sampling- Theorem
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
3) Select Start
For more or less sections adjust Num Slices
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
Z Sectioning – Setting Range
1) Decide whether to Keep Interval (number of
slices will change) or Keep Slice (Interval
between slices will be adjusted)
Set
limits for
Z-Series
2) Select Range and position bars to decide
where the Z - Series begins and ends
3) Select Start for image acquisition
Vertical section of
sample
Viewing a Z - Series
In the image window
1) Select xy
2) Select Slice
3) Use scroll bar to
view individual
sections
Viewing a Z - Series using Gallery
1) Select Gallery
2) Select Data for
scale
Use Subset to
extract sections
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.
Selecting and Saving a Region of Interest (ROI)
1) Select
Overlay and
define shape of
ROI
2) Extract
region creates a
Z-Stack from
the ROI
3) Save data
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.
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.
Time Series
1)Set up scanning parameters
for image acquisition as
described in previous slides
2)Select TimeSeries from the
LSM menu
3)Enter the Number of cycles
4)For a Time Delay between
image acquisition select min,
sec or ms and set time with the
slider
5)Select Start T to start image
acquisition
Start and Stop of the Time Series
using Time as trigger uses the
system time!
Viewing a Time Series of a Z Stack
Z Sections
for any time
Time points for
any Z Section
Both Z sections
and time series
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
Contents
• Starting the Zeiss LSM 510 microscope,
software and laser
• 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
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
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)
This guided tour is based on
work done by
Peter Jordan
ICRF
London
United Kingdom
edited and complemented by
Eva Simbürger and Solveig Hehl
Carl Zeiss Jena GmbH