sp5x user interface tcs sp5 x/user ma… · Λλ––––scan: scan: scan: new scan mode, that...
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SP5X USER INTERFACE:SP5X USER INTERFACE:SP5X USER INTERFACE:SP5X USER INTERFACE:
Λ2222----MAPPING GUIDELINE MAPPING GUIDELINE MAPPING GUIDELINE MAPPING GUIDELINE
Excitation emission scan: What is it?What is it?What is it?What is it?
Emission wavelength / nm
Excitation wavelength / nm
Λλ –––– scan: scan: scan: scan: new scan mode, that combines excitation and emission scan
λ2222 –––– map: map: map: map: result of Λλ – scan: a 2 dimensional fluorescence spectrum,
plots fluorescence intensity over excitation and emission
Page 2
λ2-mapping: Overview on general stepsOverview on general stepsOverview on general stepsOverview on general steps
Page 3
1. Define the λ2-
scan and acquire
the corresponding
data collection
2. Select ROIs on
the data collection
3. Calculate the λ2 contour plot
4. Calculate a three
dimensional display
Λλ –––– scan definitionscan definitionscan definitionscan definition
1) 1) 1) 1) Select scan mode
2) 2) 2) 2) Open window
3) 3) 3) 3) Enter excitation settings
4) 4) 4) 4) Enter detection settings settings
If you prefer to type in numbers:
5) 5) 5) 5) Activate check box: “Use advance settings”
6) 6) 6) 6) Enter relation between excitation and
detection settings settings
7) 7) 7) 7) Select PMT
Line indicating real ּגstartMinimum Gap >0
ΛΛΛΛ ((((excitation)excitation)excitation)excitation)
(emission(emission(emission(emission))))ּג
line Λ=ּגMinimum Gap=0
Excitation
Begin
Excitation End
Λ ExcitationStepsize
No. ofExcitationSteps
Minimum Gap
Maximum Λλ – distance
Λλ –––– scan definition: scan definition: scan definition: scan definition:
What do all this numbers mean?What do all this numbers mean?What do all this numbers mean?What do all this numbers mean?
ΛΛΛΛּג ---- band scan band scan band scan band scan (“Maximum Λλ – distance” activated)
Detection
Begin
Detection
EndDetection
Band Width
No. of Detection Steps
Detection
Step Size
ΛΛΛΛּג ---- triangle scan triangle scan triangle scan triangle scan (“Maximum Λλ – distance” not activated)
Line indicating real ּגstartMnimum Gap >0
line Λ=ּגMinimum Gap=off
Λλ –––– scan definition: scan definition: scan definition: scan definition:
What do all this numbers mean?What do all this numbers mean?What do all this numbers mean?What do all this numbers mean?
ΛΛΛΛ ((((excitation)excitation)excitation)excitation)
Excitation
Begin
Excitation End
Λ ExcitationStepsize
No. OfExcitationSteps
(emission(emission(emission(emission))))ּגMinimum Gap Detection
Begin
Detection
EndDetection
Band Width
No. of Detection Steps
λ DetectionStep Size
Λλ –––– scan definitionscan definitionscan definitionscan definition
And if this is getting too confusing:
Use the graphical definition!
Graphical experiment definitionGraphical experiment definitionGraphical experiment definitionGraphical experiment definition
� Click on “Graphical definition” => Corresponding window opens
� Each dot in the chart of this window corresponds to one image
Grey line: excitation wavelength = emission wavelength
� Select the PMT� Activate for band scan
� Deactivate for triangle scan� Select fluorescence mode
Graphical experiment definitionGraphical experiment definitionGraphical experiment definitionGraphical experiment definition
� Move the Sliders => Observe the changes in the chart
� All numbers in the “Acquisition tab” are updated automatically”
� The total number of images is displayed
Graphical experiment definitionGraphical experiment definitionGraphical experiment definitionGraphical experiment definition
� If you are now satisfied with the data format:
Close the graphical definition window
Graphical experiment definitionGraphical experiment definitionGraphical experiment definitionGraphical experiment definition
� What if you have spoilt your setting?
� Click “Reset Values to Defaults”.
� The system reloads the default setting.
These are the settings, LASAF starts with.
Λλ––––scan definition: scan definition: scan definition: scan definition:
Display in beamDisplay in beamDisplay in beamDisplay in beam path windowpath windowpath windowpath window
� Select constant power
� Make sure, both filter wheels are on empty position
Λλ––––scan definition: scan definition: scan definition: scan definition:
Display in beamDisplay in beamDisplay in beamDisplay in beam path windowpath windowpath windowpath window
� Adjust laser power (AOTF setting) and PMT Gain on-line
� Press “Live”
� Modify excitation wavelength and detection range while scanning
� If you want to transfer detection bandwidths changes from beam path window
to Λλ–scan definition: double click on spectral sliders, press ok
� Start data acquisition with “Capture“
� Wait until data preparation is finished, do not modify anything
� Data acquisition starts automatically
Λλ––––scan settings for first overview scan settings for first overview scan settings for first overview scan settings for first overview
ParameterParameterParameterParameter SettingSettingSettingSetting
General Format 256x256
Speed 400-600 Hz
AOTF <30% (if possible)
Accumulation 2
Bi-directional Acive
Rotation 0
Pinhole 1-2 Airy
PMT Gain 900-980
Excitation Begin 470
End 670
No. of Steps 21
Step Size 10
ParameterParameterParameterParameter SettingSettingSettingSetting
Detection Begin 480
End 730
Band width 20
No. of Steps
Step Size 10
Advanced
Settings
Detection
range
Minimum Gap 10
Max. Λλ––––
distance
off
Λλ––––scan settings for higher spectralscan settings for higher spectralscan settings for higher spectralscan settings for higher spectral resolutionresolutionresolutionresolution
ParameterParameterParameterParameter SettingSettingSettingSetting
General Format 256x256
Speed 400-600 Hz
AOTF <30% (if possible)
Accumulation 2-4
Bi-directional Acive
Rotation 0
Pinhole 1-2 Airy
PMT Gain 900-980
Excitation Begin Adapt according
overview dataEnd
No. of Steps Let it calculate
Step Size 5
ParameterParameterParameterParameter SettingSettingSettingSetting
Detection Begin Adapt according
overview dataEnd
Band width 10
No. of Steps Let it calculate
Step Size 5
Advanced
Settings
Detection
range
Minimum Gap 10
Max. Λλ––––
distance
Adapt according
overview data
If you see reflections:
� Make sure scan rotation is 0
� Match refractive index
� Optimize corring
� Increase “Minimum Gap” to 12 or 15nm
Data collectionData collectionData collectionData collection
� Data are saved in a data collection “LamdaLamda” that consists of a
series of experiments.
� Each experiment in the data collection is an excitation stack, that
can be analyzed with the already known standard tools.
� The excitation wavelength is given in the name.
� Each experiment in the data collection can consist of a different
number of images, and consequently can have a different size.
� Navigation on a data collection is available under
“Process/Tools/Excitation Emission Scan/Contour Plot/Preview”.
Data collection Data collection Data collection Data collection ––––
how to reload IPS from a previous datahow to reload IPS from a previous datahow to reload IPS from a previous datahow to reload IPS from a previous data setsetsetset
� Under “Configuration/
IPS_Masks/Confocal Apply”
check: “Scan Mode” and all
other parameters you want
to apply.
� Under “Acquire/Experiments” load the data set of interest with “Open”.
� Go to the data collection with the settings you want to apply.
� Highlight any experiment within this data collection.
� Click on “Apply”.
Data analysisData analysisData analysisData analysis
� Highlight the data collection “LambdaLambda”
� In the viewer you can scroll through the whole collection
� Go to “Process”, Select “Tools”
� Go to “Excitation Emission Scan”, Highlight “Contour Plot”
Data analysisData analysisData analysisData analysis
� If desirable, select region(s) of interest in the viewer, � all regions selected are combined to one one one one Λλ–plot
� A region drawn completely inside another region is
automatically subtracted (=> “bagel ROI”)
� If no region is selected the whole image is analyzed
� Select the interpolation mode
� Bilinear (Fast interpolation) � Nearest Neighbor ( Raw data display) � Between Points (Smooth interpolation)
Data analysis Data analysis Data analysis Data analysis –––– Navigation on the dataNavigation on the dataNavigation on the dataNavigation on the data collectioncollectioncollectioncollection
� Press “Preview”� If this button is grey out, make sure that the data collection
under “Experiments” is highlighted
� Activate the check box “Show image for current position”
� Move crosshair to excitation emission pair of interest, the
values of this pair are displayed in the lower right corner
� The corresponding image is displayed in the viewer
Data analysis Data analysis Data analysis Data analysis –––– Creation ofCreation ofCreation ofCreation of Λλ––––mapmapmapmap
� Press “Apply”� If this button is grey out, make sure that the data collection
under “Experiments” is highlighted
� A new data set is created under “Experiments” that contains
the Λλ–map.
� The Λλ–map is displayed in the big viewer.
Data analysis Data analysis Data analysis Data analysis ––––
View single excitation & emission spectraView single excitation & emission spectraView single excitation & emission spectraView single excitation & emission spectra
� Scale the LUT in the big viewer for convenient display of all
peaks.
� Move the cross hair in the small viewer to look on excitation
and emission spectra.� The tool works like a sectioning tool: Excitation and emission
spectra are measured along the cross hair lines.
� The cross hair position is displayed. This function can be used
to measure peak position.
� Remark: After having pressed “Apply”, this checkbox has no
function.
Data analysis Data analysis Data analysis Data analysis –––– Export of Export of Export of Export of Λλ––––mapmapmapmap
� Right mouse click on the Λλ–map.
� Select “Export …”
� Select the desired file format.
� Specify the destination folder.
� An image is exported , that uses the
current LUT setting.
Data analysis Data analysis Data analysis Data analysis –––– Raw Raw Raw Raw Data exportData exportData exportData export
� Press “Export” to export Λλ–plot. Data format is txt.
� Type in destination folder and name of exported data file.
� Two export files are created. One contains raw data, one
the interpolated data.
Data analysis Data analysis Data analysis Data analysis ––––Data import to ExcelData import to ExcelData import to ExcelData import to Excel
� Open new file.
� Select file type “*.txt”.
� Select: “Separated”.
� Select separator “Space”.
� Finish.
� The excitation wavelength will be displayed in decreasing
order in the last column, the central detection wavelength
will be displayed in increasing order in the last raw.
� Now you can calculate with different plots or extract
excitation or emission spectra.
Data analysis Data analysis Data analysis Data analysis ––––Data import toData import toData import toData import to ImageJImageJImageJImageJ
� Makes you directly an image.
Data analysis Data analysis Data analysis Data analysis –––– Logarithmic displayLogarithmic displayLogarithmic displayLogarithmic display
� Logarithmic display is advantageous, if you want to visualize
big and small peaks at once.
� Right mouse click between the two sliders in the LUT of the
big viewer.
� The LUT selection window appears.
� Select “SpectrumLog”.
� Press oK.
� Rescale the LUT until you get a convenient display.
� The small viewer is updated automatically.
� If you want to go back to linear display select LUT
“Spectrum”.
Data analysis Data analysis Data analysis Data analysis –––– 3D display3D display3D display3D display
� Highlight the Λλ–map (file “LambdaLambda_Contour…..”)
you want to display in 3D.
� Go to “Process”, Select “Tools”.
� Under “Excitation Emission Scans” select “3D View”
� A 3D Plot appears in the small viewer. You can turn the 3D
Plot by drawing the mouse over it.
� Alternatively, you can type in the viewing angle. This might
be beneficial, if you want to compare several 3D plots.
� Press “Reset” to go back to a standard setting.
Data analysis Data analysis Data analysis Data analysis –––– 3D display optimization3D display optimization3D display optimization3D display optimization
� Rescale peak height in the 3D plot by rescaling the LUT in the
big viewer.
� Toggle to logarithmic display by changing the LUT in the big
viewer to “SpectrumLog”. Rescale LUT.
� Caution: The use of other LUTs is possible for 2D display,
however, it will not be used in 3D. The linear Spectrum LUT
will be applied instead.
Data analysis Data analysis Data analysis Data analysis –––– 3D data3D data3D data3D data saving and exportsaving and exportsaving and exportsaving and export
� If you are satisfied with the display press “Apply” to save the
plot.
� A new file is generated that contains the 3D Plot.
� The 3D plot is now displayed in the big viewer.
� For data export right mouse click on the file, click on
“Export” and select the file type you want to use for export.
Specify the destination.
λ2 plot with Imaging SP HyD
Page 31
FeatureFeatureFeatureFeature AdvantageAdvantageAdvantageAdvantage BenefitBenefitBenefitBenefit
High QE - Lower laser power required - Less photobleaching => red dyes
do not past away before imaging
- Spectrum is “more true”
- Sample lives longer
- Faster scan possible - Save time
- alternatively: increase spectral
resolution (more images possible)
High
dynamics
- Possibility to measure bright and
dim structures in one λ2 scan
- Display big and small fluorescence
peaks in one plot
Low dark
noise
- Low signal is not hidden behind
background
- Longer accumulation is possible
- Visualize weak fluorescence peaks
- Resolve smaller differences in
fluorescence intensity
Λλ––––scan settings for scan settings for scan settings for scan settings for HHHHyDyDyDyD with high spectral resolutionwith high spectral resolutionwith high spectral resolutionwith high spectral resolution
ParameterParameterParameterParameter SettingSettingSettingSetting
General Format 256x256
Speed 400-600 Hz
AOTF <30% (if possible)
Accumulation 2
Bi-directional Acive
Rotation 0
Pinhole 1 Airy
HyD Gain Standard, 20-50
Excitation Begin Adapt according
overview dataEnd
No. of Steps Let it calculate
Step Size 5
ParameterParameterParameterParameter SettingSettingSettingSetting
Detection Begin Adapt according
overview dataEnd
Band width 10
No. of Steps Let it calculate
Step Size 5
Advanced
Settings
Detection
range
Minimum Gap 10
Max. Λλ––––
distance
Adapt according
overview data
If you see reflections:
� Make sure scan rotation is 0
� Match refractive index
� Optimize corring
� Increase “Minimum Gap” to 12 or 15nm
AAAApplicationpplicationpplicationpplication: Lambda: Lambda: Lambda: Lambda----SquareSquareSquareSquare
Page 33Nov. 2010Courtesy : Alberto Diaspro, Ph.D. University of Genoa
λ2 2 2 2 ––––MappingMappingMappingMapping
What is it What is it What is it What is it
good for?good for?good for?good for?
λ2 –Mapping: Discover hidden informationDiscover hidden informationDiscover hidden informationDiscover hidden information
Page 34
Benefits of Benefits of Benefits of Benefits of λ2222 ----MapsMapsMapsMaps
• Full spectral analysis of images
• Understand samples with very
complex fluorescence
• Depict multifaceted features at
one look
• Complete spectrum in each pixel
• Extract excitation and emission
spectra
Page 35Nov. 2010
λ2 -Mapping: What is it good for?What is it good for?What is it good for?What is it good for?
λ2222 ---- ApplicationsApplicationsApplicationsApplications
• “Dye Hunting”: Screen for fluorescent proteins and discover new dyes
• Characterization of natural fluorescence
• Find the best label for autofluorescent samples
• Dye identification and localization in cells and structures
• Optimization of excitation and emission range
Sample: courtesy of Matthias Weiss,
Cellular Biophysics Group, Bioquant,
Heidelberg, Germany.
Dye identification and experiment Dye identification and experiment Dye identification and experiment Dye identification and experiment
optimization for best data qualityoptimization for best data qualityoptimization for best data qualityoptimization for best data quality
485 Emission/ nm 695 Excitation/ nm
470
620
Sample: fixed cells with triple staining:
GalNacT2_GFP (golgi), LAMP-546 (endosomes),
Calnexin 594 (ER). The λ2–map reveals three
fluorescence peaks. The IPS for image acquisition
were adjusted accordingly.
Find the best fluorescent label!Find the best fluorescent label!Find the best fluorescent label!Find the best fluorescent label!
λ2 2 2 2 plotsplotsplotsplotsOverview image Overview image Overview image Overview image
Primary mouse Primary mouse Primary mouse Primary mouse hepatocyteshepatocyteshepatocyteshepatocytes: labeled with : labeled with : labeled with : labeled with DiODiODiODiO
The label is less suitable due to the overlap
with autofluorescence.
Strong Strong Strong Strong autofluorescenceautofluorescenceautofluorescenceautofluorescence of primary mouse of primary mouse of primary mouse of primary mouse
hepatocyteshepatocyteshepatocyteshepatocytesSample: Courtesy of René Meyer, Klingmüller Group, Systems
Biology of Signal Transduction, DKFZ, Heidelberg, Germany
Primary mouse Primary mouse Primary mouse Primary mouse hepatocyteshepatocyteshepatocyteshepatocytes: labeled with : labeled with : labeled with : labeled with DiDDiDDiDDiD
The label is better suitable because it is
excited and it emits in a different spectral
range.750
470
500
670
Characterization of Fluorescent ProteinsCharacterization of Fluorescent ProteinsCharacterization of Fluorescent ProteinsCharacterization of Fluorescent Proteins
Sample: Sample: Sample: Sample: Mixture of fixed cells expressing four different
fluorescent proteins. Excitation emission peaks are given in
brackets. All cells show a small autofluorescence peak at
(512, 533) nm.
Courtesy of Kees Jalink, Department of Cell biology, The Netherlands Cancer Institute
Amsterdam, The Netherlands
YFP
(515, 530)
GFP
(470, 511)
mCherry
(577, 605)
mKATE
(582, 620)
700
470
480
620
λ2222----Plots of single cellsPlots of single cellsPlots of single cellsPlots of single cells
λ2222----topographytopographytopographytopography
of cell mixtureof cell mixtureof cell mixtureof cell mixture
Explore Natural Fluorescence Explore Natural Fluorescence Explore Natural Fluorescence Explore Natural Fluorescence
λ2 plots of defined
regions of interest
ROI definition in
selected images of
the λ2 data collection
Scaling of Scaling of Scaling of Scaling of λ2222 plots: plots: plots: plots:
Excitation range: 470 to 670 nm,
Detection range: 500 to 780 nm
Different structures possess
different fluorescence
characteristics.
Overview image of algae
High Quality Imaging High Quality Imaging High Quality Imaging High Quality Imaging –––– atatatat New New New New WavelengthsWavelengthsWavelengthsWavelengths
Page 40Nov. 2010
Primary culture of rat cortical neuronsPrimary culture of rat cortical neuronsPrimary culture of rat cortical neuronsPrimary culture of rat cortical neurons
Structure Structure Structure Structure labellabellabellabelExcitation Excitation Excitation Excitation
[nm][nm][nm][nm]
Emission Emission Emission Emission
[nm][nm][nm][nm]
nucleusnucleusnucleusnucleus DAPIDAPIDAPIDAPI 405405405405 424424424424----465465465465
NestinNestinNestinNestin (neuronal (neuronal (neuronal (neuronal
stem cells)stem cells)stem cells)stem cells)cy2cy2cy2cy2 495495495495 504504504504----545545545545
DCX (immature DCX (immature DCX (immature DCX (immature
neurons)neurons)neurons)neurons)cy3cy3cy3cy3 560560560560 559559559559----624624624624
Beta IIIBeta IIIBeta IIIBeta III----tubulintubulintubulintubulin cy5cy5cy5cy5 652652652652 653653653653----716716716716