From Eye to Insight

LIGHTNING
Manual20190919_EN

Depending on the licese, available method and wizard are different.
Image optimization duringConfiguresettings

image acquisition

optimization(Afteracquisition)
Lightning Process×○

Lightning Expert○○

Lightning○×＊LAS X Small is not available for Lightning＊Call up Seq. and Apply are not available for Lightning wizard.
So, Call up Seq. or Apply in “TCS SP8”, then go to Lightning wizard.

1for

image
image

Lightning wizard
The wizard uses the Lightning or Lightning Expert license
1. Click TCS SP8 in the menu bar at the top left.
2. Select Lightning entry.
The Lightning wizard starts with Acquire step.
The Lightning wizard contains 5 steps.
1. You can choose between 4 scan modes in the Acquisition
Mode dialog of the Lightning wizard.
xyz, xyzt, Mark and Find, Tilescan
2. In the Lightning wizard, sequential
acquisition is enable by default.

TheBetween Frames mode is preset as thescan mode in the Sequential Scan dialog. You can also acquire
image using the Between Stacks or Between Lines mode. In
the Pinhole are of the XY dialog, the average defined wavelength
for the respective sequence is automatically applied as theEmission  [nm] emission wavelength.
3. You have 2 options for configuring image acquisition parametersetting:
A. Coupling Speed and Resolution in the Lightning Grade dialog
1. Make sure that coupling is enabled in the Lightning Grade dialog. The chain has tohighlighted in red. This is enable by default.
2. Drag the slider to the correct position to define whether the image is to be acquired atmaximum speed (Speed direction) or with maximum resolution (Resolution direction)
Note: You can define the individual value ranges or the parameters in the Configuration
steps, See Page 10, “Lightning Configuration”
B. Without coupling Speed and Resolution in the XY dialog:
1. Click the chain to disable coupling in the Lightning Grade dialog.
2. Configure your instrument parameter setting in the XY dialog.
4. Enter the required data for Mounting Medium and Refractive Index,and define the image optimization strategy
AdaptiveOn the basis of the SNR, the setting for optimum image aredetermined automatically

GlobalThis strategy is recommended as default for all image

LowSignalToNoise

This strategy is optimized for high noisy image.
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5. There are 2 buttons for live mode at the lower edge of the screen:
Live

Enable normal live mode

Fast Live

Enable fast live mode, 512x512 pixel、600Hz、Bidirectional

6. If you execute the image acquisition by clicking Start, a data record in the original (ex.
Series002) and data record for the optimized images with the _Lngending (ex. Series002_Lng) are created.

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Lightning Process
The Lightning Process and Lightning Expert license gives you the option to optimize
previously acquired images in the LASX outside of the lightning wizard.
This is done in the Process step:Lightning Process
Lightning Expert
Lightning

Process menu
You can change Strategy or Refractive index of mounting medium
In the tab Process / Lightning, select the tool Lightning
process. (Lightning Process license)
Select Lightning menu (Lightning Process license)
At the bottom of the Process display window, a dialog appears

the Lightning setting, and you can adjust setting here.

Strategy: Select a strategy for which application the automatic image optimization is to beexecuted.
AdaptiveOn the basis of the SNR, the setting for optimum image aredetermined automatically (default)

GlobalThis strategy is recommended as default for all image

LowSignalToNoise

This strategy is optimized for high noisy image.

If necessary, adapt the Lightning setting, for this purpose, see next page “Lightning ExpertSetting”.
Expert Setting: Opens the dialog the deconvolution parameter, see next page “Lightning
Expert Setting”
Open from

Opens a file browser for selecting the file with stored setting

Get defaults

Reset the setting to the default values.

Save to fileOpens a file browser for selecting the storage location anddesignation of the file.

Click Apply to start the image optimization.
4Expert SettingsApply to all channels

Apply parameters to all channel

Type

Automatically selected from Confocal, STED and Multiphoton

Number of iterations

Lightning is based on the iterative algorithm.
general, the following applies:

InThe higher number ofiterations, the better the results.

Large numbers ofiteration, however, tend to lead to more artifacts, whichmeans that there is always an optimal number ofiteration. In the Lightning automatic mode, the optimal
number of iteration isdetermined sothat gooddeconvolution quality is ensured while also keeping
artifact to a minimum.
iterations yourself.

5You can define the number ofContrast Enhancement

The contrast of the measurement data is increased before
the deconvolution. You can adjust the contrast with theslider (0 - 1)

Cut off (%)

Before deconvolution, voxel values below a certain
threshold value set to 0.

You can adjust the thresholdvalue with the slider (0 - 1).
Regularization Method

Regularization reduces the effect of noise. These are 2
methods, both of which presume good smoothness in theoriginal image in the definition smoothness: Total
Variation and Good’ Roughness.

Regularization

This enables you to set the degree of regularization. TheParameterlower the value, the more severely the noise is reduced.

Optimization

These are used to optimize the processing noisy images.
These are 5 optimization level available (None, Low,Medium, High, VeryHigh) for smoothing the initial image.
If an image is very noisy, it is advisable to set theoptimization to High (default setting).

Post-Filter

Select a smoothing filter for the image after image
optimization. None, Bilateral (Edge-preserving filter),Gaussian (Gaussian filter)

Excitation Wavelength (nm)Wavelength of the excitation laser.

Emission Wavelength (nm)Detected wavelength which lie at a distance of
15% from the left edge of the detection range

pinhole (AU)The width of the pinhole.

This is usuallyconfigured to 1 AU.
Normalization

Select how the signal value are scaled from 2
methods

Range

Scaled in a 16-bit range. The signals remain
available, since they are standardization by
sum total of intensity.

Photon Count

Scaled in terms of the detected number ofphotons that survive.

Hence, the result isquantifiable in regards to the detected photons.
Objective

Adapt the following preset values individually6for your experiments.
Numerical Aperture

Numerical Aperture NA

Immersion Reflective Index

Refractive index of the immersion medium forthe objectiveMagnification

Objective magnification

Z-Offset (m)z-position during the start of the acquisition
relative to the coverglass.

Objective Design Parameters

The value of the objective characteristic and thecoverglass are preset here and cannot bechangedImmersion

ReflectiveIndex

Refractive index of the immersion medium for

Designthe objective

Cover Slip Thickness DesignThickness of the coverglass

Cover Slip Reflective Index

Refractive index of the coverglass for theDesignobjective

Cover Slip
Refractive Index

Refractive index of the utilized immersion
medium for the objective

Thickness (m)Thickness of the coverglass

Mounting Medium
Mounting Medium

Select the utilized mounting medium from thedropdown menu here

Refractive index
Saving and Loading Setting

Refractive index of the mounting medium
You can save the setting made and reload themfor additional experiments.

To do so, thefollowing button are available
Open from fileOpens a file browser for selecting the file thestored settings.

Save to fileOpens a file browser for selecting the locationand designation of the file

＊Batch Processing
Select all raw data by pressing the Control key while making your selection, then Apply.

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STED
If you selected the STED method under the type STED Setting, the following STED
parameters are displayed.

Depletion Wavelength [nm]Depletion wavelength

Depletion Power [%]Intensity of the depletion laser

Saturation Factor

The saturation factor indicated how intensely thefluorescence is suppressed by the depletion laser. Ifthe Auto check box for the automatic configuration isdisable, you can adjust the saturation factor in theinput fields.

Excitation Laser Mode

Dropdown menu for selecting of the laser mode for
excitation: Pulsed (Pulsed) or continuous (CW)

Depletion Laser Mode

Dropdown menu for selecting of the laser mode for
excitation: Pulsed (Pulsed) or continuous (CW)

Axial Percentage [%]Indicates the ratio of the intensities of the axial andlateral STED laser (0: only lateral; 1: only axial)

Drift Correction

In the event of cell movements, this compensates alinear drift in the xy-direction for the image capture ofz-stacks.

Gated

When the function is enabled, the detection is carriedout within a defined time gate. Only the photon thatreach the detector during this time gate are detected.

Gate Start [ns]Start of the time gate

Gate End [ns]End of the time gate

8Multi-Photon
If you selected the Multi-Photon method under the type of Multi-Photon Settings, thefollowing multi-photon parameters are displayed.

Internal Detector

You can set with which detectors the multiphoton system shouldwork. When the function is activated, the detection performed
with internal detection (descanned detection), when deacvated,with external detection (non-descanned detection).

Photon CountYou select whether the excitation should be done with 2 or 3
photons.

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Lightning Configuration
In this dialog, you can configure the specific value ranges for the parameters of
the Lightning Grade slider.
Dragging the grab points on the sliders change the limit values. Alternatively, you can enter
that fill within the specified value ranges into the input fields or change them by clicking thearrow keys.

Pinhole AU

Image resolution in the xy-direction.

The value specified here

correspond to a multiplication factor that is applied to theparameters configured in the Acquire step for image resolution.
You can use this function to acquire images with the maximum
conductive resolution for image optimization.
XY

Image resolution in the xy-direction.

The value specified here

Oversamplingcorrespond to a multiplication factor that is applied to theparameters configured in the Acquire step for image resolution.
You can use this function to acquire images with the maximum
conductive resolution for image optimization.

Z

Image resolution in the z-direction.

The value specified here

Oversamplingcorrespond to a multiplication factor that is applied to theparameters configured in the Acquire step for image resolution.
You can use this function to acquire images with the maximum
conductive resolution for image optimization.

Line Average /In PMT or HyD detectors with Standard method, the slider in theAccumulation

Lighting Grade dialog affects the Line Average function.
In HyD detector with Counting method, the slider affects the Line
Accumulation function, which is more suitable for counting photons.

Speed

You can select the scan speed for image acquisition

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Tips
＊ Optimization ： There are used to optimize the processing of noisy images.

Sixoptimization levels are available for smoothing the initial imaging. If an image is very noisy,
it is advisable to set the optimization High or Very High.
For image with high S/N no regularization is necessary. For image with low S/N, no or low
regularization can cause noise to interpret as signal and appear as an artifact in the image.
High S/N

None > Very Low > Low > Normal > High > Very HighLow S/N(④) (②)
Result of LIGHTNING at High S/N. Intensity distribution at High S/N before (①) and after
LIGHTNING(②).

Result of LIGHTNING at Low S/N.

before( ③ ) and after LIGHTNING( ④ ).

Intensity distribution at Low S/NW/O regularization (Blue), w/ regularization(Orange).

①②③④＊Iteration: The higher number of iterations, the better the results. Large number of iteration,
however, tend to lead to more artifacts.

You can identify the iteration number fromProperties.

＊Cut Off: Before LIGHTNING, the set threshold value is subtracted from all voxels. This
improved the result of the deconvolutions.

＊Batch process:Select files while pressing CTRL key and Apply LIGHTNING.

11＊Reflactive index of mount mediaMounting Medium

Refractive Index

Water

1,333

100% PBS

1,335

Glycergel®

1,38

50% Vectashield ® + 50% PBS

1,39

50% PBS + 50 % Glycerol

1,406

80% Glycerol + 20 % Water

1,451

100% Vectashield ®

1,452

Mowiol®

1,46

ProLong® Gold / Diamond

1,47

Kaiser‘s Glycerol Jelly

1,47

Fructose (80,2%)

1,49

DPX

1,525

CFM-3

1,52

ProLong® Glass

1,52

TDE

1,33 – 1,5212