From Eye to Insight

LIGHTNING
Manual20220217_EN_ST

Confidential - Company ProprietaryDepending on the licese, available method and wizard are different.
Image

optimization

duringimage acquisition

Configuresettings

optimization

for(Afterimage
imageacquisition)
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 “STELLARIS”, then go to Lightning wizard.
＊Lighting does not available for acquisition with TauSense (e.g. TauSeparation, TauSTED)＊Lightning and Dynamic Signal Enhancement can be processed at the same time.
Then, only Lightning and DSE processed image is created, not created Lightning or DSE
processed image.

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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 among 4 scan modes in the Acquisition Modedialog 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. The
Pinhole in the XY dialog: The average defined wavelength for therespective sequence is automatically applied as the Emission [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 11, “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

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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

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 “LightningExpert Setting”
Open fromOpens 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.

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Expert Settings

Apply to all channels

Apply parameters to all channel

Type

Automatically selected from Confocal, STED, Passthroughand Multi-photon＊ Passthrough: This method automatically activated foracquired image with TauSense

Number of iterations

Lightning is based on an iterative algorithm. In general,
the following applies: The higher number of iterations, thebetter the results.

Large numbers of iteration, however,tend to lead to more artifacts, which means that there isalways an optimal number of iterations. In the Lightning
automatic mode, the optimal number of iterations isdetermined so that good deconvolution quality is ensured
while also keeping artifact to a minimum. You can define
the number of iterations yourself.
Optimization

Use this slider to adjust the intensity of the deconvolution or
the sensitivity of the resolution (0-5). The default value is 1.
With a value =o, a minimum deconvolution is still calculated.
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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 thresholdvalue set to 0. You can adjust the threshold value with theslider (0 - 1).

Regularization Parameter

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

Smoothing

These are used to optimize the processing noisy images.
These are 7 optimization level available (Automatic, None,Very Low, Low, Medium, High, Very High) for smoothing
the initial image. If an image is very noisy, it is advisable toset the optimization to High.

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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 of photons
that survive. Hence, the result is quantifiable inregards to the detected photons.
Any fuzzy structures resulting from the out-of-Deblurring Settings

focus blur, for example, are removed afterdeconvolution.

This is especially effective for

STED, MP and DLS images.

This function isdeactivated by default.
EnableActivate the function by checking the checkbox

Object Size (nm)Using this parameter, the effect of the Deblurring
is adjusted.

The lower the value, the finer thestructures that remain after image optimization.
Don not set the value smaller than the size of thestructures that you want to identify.
Objective

Adapt the following preset values individually foryour experiments.

Numerical Aperture

Numerical Aperture NA

Immersion Reflective Index

Refractive index of the immersion medium for theobjective

Magnification

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 bechanged7

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Immersion

ReflectiveIndex

Refractive index of the immersion medium for the

Designobjective

Cover Slip Thickness Design

Thickness of the coverglass

Cover

Refractive index of the coverglass for the objective

Slip

Reflective

Index

DesignCover 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 SettingRefractive 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 location anddesignation 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. If theAuto check box for the automatic configuration is disable,
you can adjust the saturation factor in the input 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 and lateral
STED laser (0: only lateral; 1: only axial)

Drift Correction

In the event of cell movements, this compensates a linear
drift in the xy-direction for the image capture of z-stacks.

Gated

When the function is enabled, the detection is carried out
within a defined time gate. Only the photon that reachthe detector during this time gate are detected.

Gate Start [ns]Start of the time gate

Gate End [ns]End of the time gate

9Confidential - Company Proprietary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 withinternal detection (descanned detection), when deacvated, withexternal detection (non-descanned detection).

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

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DLSLight-Sheet Settings
If the system has the Lightning DLS license, an additional dropdown menu is displayed.
You can adjust the following parameters for Lighsheet acquisitions here:
If only the Lightning DLS license is available, but not the Lightning Expert or Lightning
Process license, then under Expert Settings only the parameters for Deblurring Settingsand Light-Sheet Settings are displayed and can be edited.

Illumination NA

Numerical aperture of the illumination objective

Illumination

Axial shift of the light sheet relative to the axial focus position

Position

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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 the parameters
configured in the Acquire step for image resolution. You can use thisfunction to acquire images with the maximum conductive resolution forimage optimization.
XY

Image resolution in the xy-direction.

The value specified here

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

Z

Image resolution in the z-direction.

The value specified here

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

Line Average /In HyD detectors with Analog or Digital 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＊Iteration: The higher number of iterations, the better the results. Large number of iterations,however, tend to lead to more artifacts.

You can identify the iteration number fromProperties.

＊Smoothing： There are used to optimize the processing of noisy images. Six optimizationlevels 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＊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.

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Confidential - Company Proprietary＊Reflactive index of mount media
Mounting Medium

Refractive Index

Water

1,333

100% PBS

1,335

Glycergel®

1,38

50% Vectashield ® + 50% PBS

1,3950% PBS + 50 % Glycerol1,406

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 – 1,52

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