Download Report

Double click the LCS icon to start loading the
software.
Choose ”personal” and click ”OK”.
Initialize stage?
NO: This is mostly used, because you are not going
to use the motorized stage with the software (take
large images in an automated fashion).
YES: If you are going to acquire many images and
patch these together. You will have to make sure
that the objectives are in the down position and
that nothing is interferring with the movement of
the stage.
Click ”Beam” to launch the Beam Path Setting window.
Here you find a list of
pre-settings from Leica
as well as settings that
you have already saved
There are 2
(for sequential scanning
OTFS’s and nine
perhaps).
laser lines to
choose from.
You can regulate up to 4
PMT detector channels.
There is a list of pre-set instrument
parameter settings developped by Leica.
These are protected and cannot be
changed. These will automatically set the
choice of laser, laser intensity and pmt
settings.
These settings are often very general
and do not take into account crosstalk.
The emission curve of the
fluorescence dye will be displayed
in the spectrum.
Position the detection band directly
above the laser line.
Nevertheless, this is a good starting point for setting up your personal setting.
On our system there are 9 possible laser lines and two AOTFs.
Activate the AOTF and
bring the sliders up for
each laser line you need.
You will have to adjust
the % after the first scan.
Next, activate the PMTs and set the different detection windows. Place them a few nm
away from the lasers you are using. Otherwise you will have reflection.
Choose the color you want for the
different detectors. You can also get
the emission curve from the pull-down
menu under each PMT.
Adjust PMT gain to 600 V.
Check your sample on the microscope
1. Choose the MicCtrl and click on visual from the pull-down menu.
2. Choose an objective and put oil on if it’s an oil objective.
3. Choose the correct filter (on microscope) for the fluorescence you’re expecting
to find.
Empty position for scan
Switch to og between filters
4. Place your sample into the stage and focus your sample. On the right side of
the microscope there are two buttons for fast focusing. You can also change
the z-step on the microscope (0=slow, 3=fast)
5. Click MicCtrl again and choose Scan from the pull-down menu to switch to
scan mode.
6. You are now ready to scan.
7. Press the continuous button to scan your sample. The image will appear on
the right screen.
8. You will most probably have to adjust the focus, laser %, gain and offset. Use
the 7 knob control box. Make sure that the controls have been set as ”smart
gain” and ”smart offset”. You can retrieve the most useful setup from the
saved folder, choose MIC.
S.gain S.offset
z-pos
Q-LUT or over/under glow
It is very useful to switch between original color,
over/underglow and black/white with the Q-lut
icon on the display screen.
This table uses a range of green-red-orange-yellow-white-blue for pixel intensity.
Adjust the PMT gain so that blue (overexposed) pixels just disappear, or a few are
left in the brightest zones (these will bleach when doing averaging).
Adjust the offset so that green (underexposed) pixels just disappear except in the
blackest zones (like outside cells). Offset is the black level of an image and setting
the offset too low (- values) will cause the loss of low-intensity signal.
Important!
You need to find the comprimise between laser intensity and PMT gain.
High laser & low gain= clear image but bleaching and toxic for live cells/samples.
Low laser & high gain= noisy image but less bleaching and good for live
cels/samples.
Check for bleadthrough/crosstalk/cross emission – part 1
When emission spectra of two fluorochromes overlaps, the emission from one channel will extend
to another channel. Many commonly used fluorophore pair have more or less overlapping
emission spectra which pose the problem of emission bleeding through in most multi-labeling
application.
PMT 1
PMT 2
The cross-talking can be removed by narrowing the red detection band and/or by decreasing the
first laser intensity and lowering the voltage of the red PMT.
Most of the time, running a sequentail scan is the most safe. The only disadvantage is that there
is a short time delay when running a sequential scan.
Check for bleadthrough/crosstalk/cross emission – part 2
This sample has DNA labelled with Dapi, one G-protein transfected with YFP and one G-protein labelled
with Texas Red
Check for bleadthrough/crosstalk/cross emission – part 3
If you suspect bleadthrough, turn off all lasers except the first one from the left.
Now, observe the signal from each PMT. The first PMT shows the DNA-dapi signal.
In the second channel the dapi is bleading through.
Check for bleadthrough/crosstalk/cross emission – part 4
Check also for the possibility of bleadthrough with the second laser turned on and the
other lasers turned off. You can in this example observe some signal bleading through (or
crossexciting) in the third channel.
Running a sequencial scan – part 1
Set all parameters for the first recording method and save the instrument setting.
Turn on the first and last lasers. Turn on
the first and third PMTs.
Adjust laser %, gain and offset to get a
nice image.
Save the parameter. Your setting will
then show up in the user list.
Running a sequencial scan – part 2
Set all parameters for the second recording method and save the instrument setting.
Turn on the middle laser. Turn on the
second PMT.
Adjust laser %, gain and offset to get a
nice image.
Save the parameter. Your setting will
then show up in the user list.
Running a sequencial scan – part 3
Open the sequencial scan setting box.
Find you setting, highlight it and drag and drop (or click add) in the seq. scan setting
box. Find the next setting and do the same.
You can choose between 3 different sequencial scan modes.
This selection defines when the sequencial recording methods
are changed.
Running a sequencial scan
Highlight the instrument setting in the list box.
Click on the add button in the sequencial settings field.
You can choose between three
sequencial scan modes.
This selection defines when the
sequencial recording methods
are changed.
Correct format and pixel size
Choose the desired frame size by clicking on format. Try to do as
much as possible with a low scan format (512x512) and try not to
scan unnecessary. There are many changes that you can do without
having the continuous scan on.
When you have adjusted everything and are ready to scan your
final image, change scan format to 1024x1024 or 2048x2048,
depending on the voxel size (which depends on zoom and objective
magnification).
Working format: adjusting
laser intensiry, gain,
offset, zoom, averaging…
Final image with full resolution
Averaging – less signal to noise ratio
Click on the average button and define how often the recordings of a frame is
to be repeated.
Click on the line average and define how often the recordings of a line is to be
repeated. This is moslty used for live imaging.
The number will define how often the recording of an image is repeated. The higher
the number, the less signal-to-noise ratio you will get. Remember that this
repeated scanning will also bleach your sample, so this is a take-and-give situation.
4 frame
averaging
Setting up a z-stack - part 1
Start scanning and find the desired begin and end point of your
stack. You can either mark these by clicking in the small white
cases or by clicking on these icons.
Click on series to open
the series scan overwiew.
Setting up a z-stack - part 2
Optimized is the precise number of optical slices
you need to have a good 3D reconstruction (but
the number calculated does not correspond to a
fluorescent preparation).
Define yourself the distance between selecting
others from the list.
Click on the sect button and
determine the number of
Start scanning the stack by
clicking onto series icon.
recordings between begin point
and end point.
If you are doing sequential
scanning – go to next page!
Setting up a z-stack - part 2
…with sequential scanning
Define the begin and end points as described
before. Select the sect and define how many
sections you want.
Open the sequentail scan setting and add your
presaved settings.
Start the scan with the series button.
Setting up a timelapse
Select the right mode: xt, xyt, xzt or xyzt. The selection of one of these will
automatically activate the time button.
If you have selected an auqisition with z, you need to define the begin and end
positions and define the number of sections and averaging.
Click on the time button for setting up a time series
recording.
∆T: the recording time for a frame/stack.
Minimize: the smallest possible interval is used.
Frames: number of recordings of the image (stack).
Complete time: total recording time.
All of these parameters can be calculated
in relation to the other parameters.
Click series to start the scan.
Acquiring time-lapse
Select the right mode: xt, xyt, xzt or xyzt. The selection of one of these will
automatically activate the time button.
If you have selected an auqisition with z, you need to define the begin and end
positions and define the number of sections and averaging.
Click on the time button for setting up a
time series recording.
∆T: the recording time for a frame/stack.
Minimize: the smallest possible interval is used.
Frames: number of recordings of the image (stack).
Complete time: total recording time.
All of these parameters can be calculated
in relation to the other parameters.
You are now ready to scan.
With a bit depth of 8 bit it is possible to resolve 256 different intensity
values, with a bit depth of 12 bit the number is 4096 (the latter will
produce twice as large files).
Click on the mode button and select a scan mode in order
to define the dimentionally of the image recording.
Using the scan speed, you determine how many lines per second are
sampled by the scanner. Changing the scan mode also has an impact on
image quality. The lower the scan speed at a given scan format, the better
the signal-to-noise ratio of the image. Default is 400 Hz (lines per second).
When the bidirectional scanning is active, not only the foreward sweep but
also the reverse sweep of the scanner is used for sampling. We normally
use the bidiractional scan when working with quick acquisitions such as
calcium pulses.
Troubleshooting
1. Is your preparation in focus in the microscope?
2. Is there a filter in the way for the laser and emission
3. Is the laser scanning? Do you see the light on your preparation?
4. Are you at zoom 1?
5. Is the pmt activated and turned on (around 600V)?