SP5X USER INTERFACE TCS SP5 X/User Ma… · Λλ––––scan: scan: scan: new scan mode, that combines excitation and emission scan λ 2222 ––––map: map: map: result of

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


� 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


� If you are now satisfied with the data format:

Close the graphical definition window


� 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


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



Speed 400-600 Hz


Accumulation 2-4


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


Detection Begin Adapt according

overview dataEnd

Band width 10


Step Size 5

Advanced

Settings

Detection

range

Minimum Gap 10


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



Speed 400-600 Hz


Accumulation 2


Rotation 0

Pinhole 1 Airy

HyD Gain Standard, 20-50

Excitation Begin Adapt according

overview dataEnd


Step Size 5


Detection Begin Adapt according

overview dataEnd

Band width 10


Step Size 5

Advanced

Settings

Detection

range

Minimum Gap 10


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

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SP5X USER INTERFACE TCS SP5 X/User Ma… · Λλ––––scan: scan: scan: new scan mode, that combines excitation and emission scan λ 2222 ––––map: map: map: result of