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ride

Introduction 2

DIFFRACTION ...........................................................................2

ULGH WORK ROUTINE .......................................................................3

Working with ride 4

ACQUIRE IMAGE ......................................................................4


IMAGE PREPARATION .............................................................6
DECONVOLUTION ....................................................................8
VISUALIZATION ......................................................................12

Appendix 23
Eride1202
ride • Introduction

ride

Introduction
The ride module utilizes mathematic methods to eliminate noise and diffraction interferences from your
images which come from sources outside the focus level.

Diffraction
In fluorescence and bright-field microscopy, diffraction from areas above or
below the focus level leads to over-exposure, distortion and blurring. This
diffraction can be led back to the mechanism of the spreading of light. Even
an ideal, single point light source cannot be observed as a point, but rather
as an image spread over an area, i.e. Airy disk pattern.
Point Spread Function In this case, the Point Spread Function (PSF) of the source is decisive,
(PSF) which in turn is dependent on the system´s optical features. Generally, the
following is valid for this Point Spread Function (PSF).:
g(x) = f(x) * h(x) + n(x)
x :spacepoint:
f(x) : ideal image
h(x) : point spread function
n(x) : noise function
g(x) : observed image
* : convolution

The noise function n(x) and the point spread function h(x) must be
determined in order to be able to reconstruct the ideal image. While an
estimation of the noise function is highly possible, the point spread function
is so highly dependent on the optical features of the entire construction as
well as the specimen, that it eludes a measurment in practical use.
Deconvolution An alternative approach, which is also helpful in ride, is the utilisation of
mathematic functions and algorithms for the improvement of image quality.
The ride module offers various proceedings to attain this; each with different
preferences and characteristics. These instructions can only give you advice
for the choice and selection of methods. The final decision for choosing a
method, however, must be made by you.
These images of a spirogyra
illustrates ride´s efficiency.
The image on the left shows
the original and the image
on the right shows the
results after blind
deconvolution.

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Work Routine

Work Routine
The three core elements of ride are: Acquire, 3D-Deconvolution and Simple,
as well as Extended Visualization.
Requirements ride can process simple images as well as three-dimensional stacks,
whereby stacks are its specialty. The acquisition of such image stacks can
be done directly from ride. When using an automated microscope, the
necessary parameters are directly saved with the images, thus eliminating
any further interventions. When using other microscopes or image stacks
from other sources, the z-positions of the images must first be defined.
Acquisition ride´s acquisition function can take over the control of the microscope. ride
supports you with the acquisition of image stacks which are directly
optimized for deconvolution.
3D-Deconvolution ride offers various proceedings for the removal of diffraction from image
stacks. The term deconvolution in the context of ride covers all of these
procedures. The Blind Deconvolution filter is not available in the module´s
simplest construction stage. The results of deconvolution are, however, an
image stack which you can directly compare with the starting image stack.
Numerous attempts with different filters and parameters are often necessary
in order to reach a good result.
Visualization The different procedures required for visualisation are used to extract a 2D
image, 3D image, a stereo image or a video from an image stack.
Simple Visualization constructs projections of the sum of the minimal as well
as the maximal intensity in directions X, Y, or Z. In addition, the Extended
Visualization enables you to set the projection direction via rotation around
the X and Z axis, 3D views via isoprojection and projection via α-Blending,
and to create stereo images and videos.

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Acquire Image Stack
Attention: The Acquire Image Stack command is only available if an application controlled
z-drive has been installed on the microscope.
 Make sure the camera and the stage control are ready for use.
 Mark an available image buffer in the image-manager.
 Select the Oper > 3D-Images > Acquire command.
 The command is also accessible on the 3D-Images button bar.
 Move the stage to the highest level until the specimen is no longer clearly
visible.
 Optimum results are achieved if the Z-region, in which acquisitions are
made, covers half of the specimens thickness both above and below the
specimen.
 Click on the Top button, to set this stage position to be the upper edge of the
acquisition region.
 Move the stage to the lowest level until the specimen is no longer clearly
visible.
 Click on the Bottom button, to set this stage besition as the lower edge of the
acquisition region.
 The Z-Distance field located in the Z-Positions group provides the space
between the upper and lower stage position. It is automatically set and
cannot be altered.
 Click on the Help button, to gain access to Ride´s online help with regards to
the acquisition function.
 Click on the Calculate Z-count button to open the distance calculator.
 Enter the Z-Distance, Emission Wavelength, Refractive index of immersion
medium and Numerical Aperture values for the objective being used.
 The Z-Distance can be taken from the Acquire Image Stack dialog box.
 The Emission Wavelength depends on the type of fluorochrome used in
flourescent microscopy. Set it to 540 nm when using a brightfield setup.
 The Refractive index of immersion medium is to be taken from the
manufacturer´s instructions. Typical values are:
Air = 1, Water = 1.333, Glycerol = 1.5, Oil = 1.515.
 The Numerical Aperture can be found on the objective.
 Click on the Calculate Count / Z-Increment button to carry out the calculation.
 Transfer the value from the Count field to the Acquire Image Stack dialog
box´s count field.
 To begin acquisition, flick on the Acquire button located in the Acquire Image
Stack dialog box.

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These instructions will describe the Working with ride
deconvolution of this image of a spirogyra.
Acquire Image
Image Preparation
Deconvolution
Visualization
Apparative Requirements: You will
require a microscope with a computer-
controlled Z-Drive to automatically
acquire a stack.

Meaning of Z-Distance: The Point


Spread Function (PSF) describes the
The Acquire Image Stack dialog box. The spreading of light in 3 dimensional
software supports you during the acquisition space. To establish this, the
of the stack which will be processed via information, with regards to other levels
deconvolution.
in the space, e.g. other frames, must be
given. The utilized algorithms deliver
optimal results if the distance of the
image levels in Z-distance fulfill
requirements which can be set by the
The help-window used to choice of the Z-distance.
calculate the distance and
the required number of Determining specimen thickness:
images You must estimate the thickness of the
specimen in order to create the optimal
acquisition conditions for the upcoming
deconvolution. To do this, set the upper
edge of the specimen to a clear visibility
and note the Z-Value. Then, set the
lower edge of the specimen to a clear
visibility and note the Z-Value once
again. The difference between the two
Z-Values is the specimen´s thickness.

Image Count: The number of images in


a stack is only limited by the available
memory capacity. Please note that a
single image acquired with one of
today´s digital cameras may require
The acquired stack is then marked by a special symbol
more than 3 MB of memory - even in the
in the image-manager.. black/white mode.

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Set Size
Attention: The reduction of the data size via the Rize and Crop Frame command is not
necessarily required. However, it is recommended with larger data (e.g. high-resolution
images and/or many frames) in order to reduce the required calculation time and memory
space.

 Select the Resize command located in the Oper > 3D-Images menu, in order
to zoom the entire stack and to change the resolution simultaneously.
 The command is also accessible in the 3D-Images button bar.
 The Resize dialog box is opened.
 Select Maintain XY-ratio from the Ratio list.
 By using this function, the images located in the stack are equally zoomed
in the x and y positions. The z position should remain unchanged.
 Enter the value 0.5 into x column located in the Factor field.
 The value in column y located in the Factor field changes to 0.5.
 The values in column x and y located in the Size field halve themselves.
 The values in column z located in the Factor and Size fields remain
unchanged.
 Click the Execute button to carry out the preset alterations.
 The program creates a new, smaller stack named ’Spirogyra 19 [1]’ in the
next image buffer.
Crop Frame
 Select the Set Frame command located in the Image menu.
 The command is also accessible in the Standard button bar.
 Set the frames so that the dark fringe is left out.
 By using the mouse, move the frame to the far left.
 By keeping the left mouse key depressed, adjust the frame size so that only
the region desired is framed in.
 It is possible that you will have to move the frame once again, in order to
position it properly.
 Fix the frame by depressing the right mouse key.
 Select the Crop frame command located in the Oper > 3D-Images menu.
 A new stack is created in the destination image buffer. It contains only the
framed in region from each image in the stack.
 The Crop frame command works differently than the Enable Frame
command located in the standard button bar. When using the Enable
Frame command, it defines a rectangular region in the current image
without modifying it. When using stacks, the frame only corresponds to the
upper most image. Therefore, when working with stacks, always use the
Crop frame command located in the Oper > 3D-Images.

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The Resize dialog box which enables a Working with ride
stack to be zoomed in all three directions.
The relation between the different axes is Acquisition
selected from the Ratio list. Image Preparation
Deconvolution
Visulaization
Calculating a deconvolution is a rather
intricate procedure which respectively
needs a lot of time. Especially at first
you will need to experiment with the va-
The frame enables the upper part
of the image which does not rious filters and settings in order to find
contain any information to be the best method for the stack in questi-
eliminated. The position and size of on. You can reduce the size of a stack
the frame is not yet optimized in the with the Resize and Crop frame com-
illustrated example.
mand to speed up this process.
Ratio: Zoom axes independently:
This command enables you to determi-
ne the zoom factor for each of the three
axes independently. Remember that
this tends to produce distortions.
Ratio: Maintain XY-ratio: This com-
mand enables you to keep the proporti-
ons in the XY-plane. This setting, in
many instances is optimal since the re-
solution in the XY direction normally is
very large and normally much higher
than in the Z direction.
Ratio: Zoom axes homogeneously:
An even zoom in all directions is only
usefull if the resolution in the Z direction
is also very high.

The extracted stack. The com-


parison of the entries in the image
manager which were achieved with
the Resize and Crop frame
commands show a voxel count of
1.792.000 (25 images x 320 x 224
pixel) instead of 8.336.000 voxels
(25 images in stack x 640 x 512
Pixel).

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Selecting Filters and Settings
Attention: The 3D-Deconvolution command is only available if a calibrated image is ac-
tive, or a x, y, and z equidistantly calibrated stack is active.
Note: An explanation to the individual filters can be found in this handbook´s appendix.
 Select the 3D-Deconvolution command located in the Oper > 3D-Images
menu.
 Alternatively click on the 3D-Deconvolution button located in the 3D-Imag-
es button bar.
 The 3D-Deconvolution dialog box opens.
 In the image document, the view of the active frame is replaced by the
depiction chosen in the Projection tab.
 Select the No Neighbor option in the Filter Selection group.
 The explanation of the various filters and notes with regards to their choice
can be found in the appendix.
 Enter the corresponding parameters of the image in the Numerical Aperture,
Emission Wavelength and Refractive Index fields located in the Microscope-/
Acquisition Parameters group.
 The example of the spirogyra contains an aperture of 0.75, an emission
wavelength of 540 nm and a refractive index of 1.0.
 Clear the Transmitted Brightfield check box, if it is marked.
 The entry, whether having to do with an acquisition in transmitted light,
transmitted bright light, or flourescence procedures, is of decisive
importance for the deconvolution results.
 Enter the value 85 in the Haze Removal field located in the Filter-Parameters
group.
 A noise suppression of 85% is, in many instances, a good starting value.
 Click on the Execute button to start the deconvolution.
 The deconvolution is calculated based on the set parameters.
 The non-treated stacks and the new stacks are represented parallel in the
image document after the calculation has been completed.
Visualization of the Results
 Select the Projection tab to show the reached quality.
 Click on the Maximum Intensity Projection button to get an overview of the
result.
 The projection for the non-treated and filtered stacks are shown
simultaneously.
Optimizing the Parameters
 Select the Filter tab to start a new calculation.
 Enter the value 99 into the Haze Removal field.
 Click on the Execute button to start the deconvolution again.
 After the calculation has been completed, all of the non-treated stacks and
the new stacks with the current projection settings are shown parallel in the
image document.

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The Filter tab of the 3D-Deconvolution Working with ride
dialog box.
The Tiling group is only active with the Image Aquisition
Inverse Filter and Blind Deconvolution Image Preparation
options. Deconvolution
The Haze Removal field is active with the
No Neighbor and Nearest Neighbor Visualization
options.
The Transmitted Brightfield and Phase Haze Removal: This function determi-
Object check boxes are replaced by the nes how strong the stack is to be "deha-
Microscope list and the Haze Removal zed". 85% is is a good starting value.
field is replaced by the Iterations field. Increase the Haze Removal step by
step until the result can no longer be
changed, or artifacts begin appearing.
Should artifacts - false copies of the
specimen signal - appear, reduce the
Haze Removal once again.
Voxel: A voxel is created, when a pixel,
which has a surface via calibration, is
given a thickness. This thickness is in
accordance to the Z-distance of two sin-
gle images.
The Projection tab of the 3D- Projection Methods:
Deconvolution dialog box. You will find an
explanation to the numbered buttons in 1: Sum Projection: The mean of the
the side column. voxels along the projections axis is de-
The Frame Selection group is only active termined, to which only voxels with an
if Slice View method has been selected.
The Zoom Z field located in the Viewport intensity higher than 0 are contributed.
group is only active when entry X or Y has The profile is spread over the entire dy-
been selected from the Direction list. namic range. The sum production high-
lights the areas of medium intensity.
2: Maximum Intensity Projection:
The Voxels along the projection axis
with the highest intensity are searched
for and used in the depiction. This pro-
jection highlights structures of higher in-
tensity.
3: Minimum Intensity Projection: The
voxels along the projection axis with the
lowest intensity are searched for and
used for the depiction. This projection
highlights structures of lower intensity
and is especially suitable for transmit-
ted brightfield acquisitions.
4: Slice View: This projection shows
single frames along the projections
axis. The Frame Selection group beco-
mes active and enables the selection of
frames and their presentation as a vi-
deo-clip. An explanation on how to do
this can be found on page 13.

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Saving Results
 Close the dialog box by clicking OK.
 The deconvolutioned stack will be written to the destination image buffer.
 It contains the filename extention ’Non’ (No Neighbor).
 Only when clicking OK is the result written to an image buffer and thus
made available outside the 3D-Deconvolution dialog box.
Varying the Filter
 Select the starting stack from the image-manager.
 Select an empty image buffer as your destination image buffer.
 Select the 3D-Deconvolution command located in the Oper > 3D-Images
menu.
 The 3D-Deconvolution dialog box opens.
 Select the Inverse Filter option in the Filter Selection group.
 Enter the image´s corresponding parameters into the Numerical Aperture,
Emission Wave Length and Refractive Index fields located in the Microscope/
Acquisition Parameters group.
 The correct parameters from the first round are adapted by the procedure
being described.
 Clear the Transmitted Brightfield check box, if selected.
 Select the Activate check box in the Tiling group.
 The Overlap field is activated.
 Enter value 12 in the Overlap field.
 Click the Execute button to start the deconvolution.
 The deconvolution is calculated via the set parameters.
 After calculation has been completed, the non treated stack and the new
stack are shown parellel in the image document.
Checking Results
 Select the Projection tab to check the achieved quality.
 Click the Maximum Intensity Projection button to gain an overview of the
result.
 The projection for the non-treated and filtered stack are shown
simultaneously.
Saving Results
 Click on the OK button.
 The deconvolutioned stack is written to the destination image buffer.
 It gets the filename extension ’InvF’ (Inverse Filter).

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Maximum intensity projection for the Working with ride
original stack. The unclear contrasts as well
as the haze covering the image both Image Acquisition
influence the aquisition. Image Preparation
Deconvolution
Visualization
Projection: The dialog box´s projection
tab serves solely for the assessment
and comparison of the actual
parameters and the aimed results. The
created projection is not saved when
leaving the dialog box. Please use the
Simple Visualization and the Extended
Visualization commands to save the
created projections.
Maximum intensity projection for the stack
after utilizing the No Neighbor filters. The
Haze Removal here was set to 90%. The
haze has disappeared from the image.
Hence, finer structures are now visible and
the contrasts have been clearly improved.

Maximum intensity projection for the stack


after utilizing the Inverse Filter. Calculation
was done by Tiling with a Overlap value of
12. The greater efficiency of the Inverse
Filter in comparison to the No Neighbor is
not so evident when viewing the simple
features of the spirogyra.

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Comparing the Results
 Change the depiction to 2x1 in the Viewport-Manager.
 Select the stack from the image-manager, which were created via No Neigh-
bor and Inverse Filter.
 Select the Simple Visualization command located in the Oper > 3D-Images
menu or click the Simple Visualization button located on the 3D-Images button
bar.
 The Simple 3D-Visualization dialog box opens.
 The button of the last type of projection remains depressed in the Mode
group.
 Click on the Slice View button.
 The upper most frame is shown in both image documents.
 The Frame Selection group´s button enables you to mark single frames. You
can also animate the selected frames in the form of a small video.
 Click on the Play button.
 The Simple 3D-Visualization dialog box is faded out.
 The individual frames are shown one after the other in the image
document.
 Press [Esc], to stop play and to once again show the dialog box.
Constructin the projection
 Clilck on the Maximum Intensity Projection button to return to simple
projection.
 Click OK, to close the Simple 3D-Visualization dialog box.
 The projections shown in the image document are written to a destination
image buffer and the image buffer following thereafter.
 The projections are no longer stacks, but simply two-dimensional images.

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. Working with ride
Reverse Image Acquisition
Play Image Preparation
First Frame Deconvolution
Prior Frame Visualization
Next Frame
Frames: A frame, with correspondence
Last Frame to a stack, is a single image from a
Options stack. Thus, a single frame of a stack is
in all actuality a complete image.
Frames, in this case, must be clearly
differentiated from frames for markation
or skeletons of a two dimensional
The buttons in the Frame Selections group used to control playback in the Slice View image, such as the Crop frame does
mode. so.

Slive View: The Slice View mode does


The Options dialog box located in the Frame not create a video. In actuality, the
Selection group. The Frame rate is based on the time
in which a single frame is shown. program changes the presentation of
the frames as they are released via
buttons, and as they are set in the Op-
tions dialog box. This procedure only
effects the presentation of the individual
images on the screen. In order to
properly create an AVI-video, use the
Extended 3D-Visualization
command. With the help of the Anima-
tion tab located in this dialog box, you
can create video data.

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Visualizing the results
 Mark the results stack in the image-manager.
 Select the Simple Visualization command located in the Oper > 3D-Images
menu or click the Simple Visualization button located in the 3D-Images button
bar.
 The Simple Visualization dialog box opens.
 Click on the Sum Projection button.
 A ray goes through the stack along the selected projection direction (X, Y
or Z). For each voxel, the medium intensity is shown. In addition to this, the
shown intensity region is spread. This procedure enhances regions of
medium intensity.
 Click OK.
 The dialog box is closed.
 The selected projection is created and the created 2D image is written to
the destination image buffer.
 Decide on the next available image buffer as the destination image buffer.
 Select the Oper > 3D-Images > Simple Visualization command.
 Click the Maximum Intensity Projection button.
 A ray penetrates the stack for each Voxel from the back to the front along
the selected projections direction (X, Y or Z).The highest intensity found is
represented for each Voxel. This procedure highlights regions of higher
intensity.
 Click OK.
 The dialog box is closed.
 The selected projection is created and the resulting 2D-image is written to
the destination image buffer.
 Decide on the next available image buffer as the destination image buffer.
 Select the Oper > 3D-Images > Simple Visualization command.
 Click the Maximum Intensity Projection button.
 A ray penetrates the stack for each Voxel from the back to the front along
the selected projections direction (X, Y or Z).The highest intensity found is
represented for each Voxel. This procedure highlights regions of higher
intensity.
 Click OK.
 The dialog box is closed.
 The selected projection is created and the resulting 2D-image is written to
the destination image buffer.
 Set the depiction to 3X3 in the viewport-manager.
 Place the projections in the viewport-manager so that they can be best
compared.

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Working with ride
Image acquisition
Original No Neighbor Inverse Filter Image preparation
Deconvolution
Visualization
Saving images and database: The
typical process of work with ride in-
volves numerous test-runs with various
filters and parameters. Thus, an acute
danger of overwriting already targeted
results exists. Make it a rule to immedi-
ately protect devonvolutioned stacks
with the Image > Protect Image com-
mand. It is even better to write the cre-
ated stack to a database. Use the
Database > Define Fields... com-
mand to expand an existing database
by fields for the filter selection, micro-
scope and acquisition parameters,
haze removal, iteration and tiling. Add a
field for projection methods to be also
able to save projections with the re-
quired entries.

Illustration of the created projections. The Sum Projections are shown in the upper
most row. The Maximum Intensity Projections are shown below that, and finally at the
bottom, the Minimum Intensity Projections are shown. The columns from left to right
contain the projections from the original stack; the stack after using the No Neighbor
filter (haze removal 90%); and the stack after using the Inverse filter (Tiling activated,
Overlap 12).

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Smoothing the Stack
 Mark the stack which was created via the inverse filter.
 You will find this stack based on its file name extension invF.
 Select the Oper>3D-Images>Smooth… command or click the Smooth button.
 The Smooth 3D-Image dialog box is opened.
 Enter the value 2 into the Radius field and confirm it by clicking on the Execute
button.
 The Smooth 3D-Image dialog box is closed.
 A new stack is created in the destination image buffer.
Create the Iso-View
 Select the Oper > 3D-Images > Extended Visualization command or click on
the Extended Visualization button located in the 3D-Images button bar.
 The Extended 3D-Visualization dialog box opens.
 Click on the Iso-Surface Projection button.
 The Background and Iso-Value fields located in the Iso-surface group are
activated.
 Enter the values 25 in the Tilt X field, 0 in the Tilt Z field, and 125% in the Size
field.
 The Tilt X und Tilt Z fields control the lining up of the ray.
 Enter the value 150% in the Resize field located in the Display group.
 Select Black from the Background pick list located in the Iso-surface group.
 Enter the value 128 in the Iso-Value field located in the Iso-surface group.
 Double click the Iso-surface color field located in the Colors group of the Op-
tions tab.
 The Select Color or Face dialog box opens.
 Select a bright, clear green from the Basic colors list.
 The fluorochrom´s emission wave length of 523 nm is in the green area.
 Click on the OK button in order to take on the chosen color from the Iso-sur-
face field.
 Click on the Execute button to calculate the projection.
 The projection is calculated and shown in the image document.
 The image does not allow a clear view of the structures of the spirogyra.
 In the Projection tab, in the Iso-Value field, located in the Iso-surface group,
change the value to 1024 and click on the Execute button.
 The structure of the spirogyra becomes clear.
 Click OK.
 The Extended 3D-Visualization dialog box closes.
 The projection is then written as a 2D-image to the destination image
buffer.

16
The Radius value located in the Smooth 3D- Working with ride
Image dialog box is the mean witdth of the
Gaussian distribution is used for smoothing. Image Acquisition
Image Preparation
Deconvolution
Visualization
Iso-Projection: During an Iso-
The Extended 3D-Visualization dialog projection, a voxel, which has the same
box. The Transfer tab is only active if the intensity or higher as the fixed Iso-
Projection by blending mode has been
selected. value, is searched for along the ray
The Tilt X and Tilt Z fields change the from front to back. This voxel is
direction of the view-axis along which the interpreted as part of the surface to be
projection is carried out. The rotation shown.
around the Y-axis only takes place during
an animation.
Background: Ride requires an entry
for the background outside of the image
area, should the Iso-surface stretch to
the edge of the image.

Black or White: A black background


means that all the intensities outside of
the image area are seen as 0. A white
background means that all of the voxels
have maximum intensity. As soon as
the ray reaches a voxel above the
threshold, it will be interpreted as part of
the outer surface. Hence, the surface is
always touched by the ray from
"outside". Thus, a self-contained image
emerges.

None: No background means that


every reaching of the threshold is
interpreted as part of the surface.
Hence the surface can be "touched"
from inside. Thus an open image
emerges.

Shading: In order to intensify the 3D-


effect of the projection, every point on
A comparison of the two iso-projections. In the illustration to the left, the Iso-value is the surface is shaded based on the
so low, that even the signals not belonging to the specimen are visible. When using a
higher Iso-value, as in the illustration to the right, the noise signals are no longer used local gradient.
for the creation of the iso-surface. The result is now a pseudo-3D portrayal.
Animation: The Animation tab of the
An iso-projection Extended 3D-Visualization dialog
Y of a simple box enables you to set the region and
structure. Ray S the step from one frame to the next for
X runs through the the rotation on the Y-axis. ride then
three identical
stacks. creates a video.

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False-Color Presentation of the Projection
 Mark the smoothed stack in the Image-Manager.
 Select the Oper > 3D-Images > Extended Visualization command or click the
Extended Visualization button located in the 3D-Images button bar.
 The Extended 3D-Visualization dialog box is opened.
 Click the Projection by blending button.
 The Transfer tab is activated.
 Click the Transfer tab.
 The Opacity curve graph shows a histogram of the stack´s intensities.
 In the graphs, the opacity curve remains a constant line with two control
points to the left and right.
 Click the LUT… button.
 The Load Palette dialog box opens.
 Select the Rainbow2.LUT palette and click the Open button.
 The chosen palette is inserted as a colored bar underneath the opacity
curve located in the Extended 3D-Visualization tab.
 Set the opacity for low intensities to 0.
 To do this, pull the opacity curve´s left control point all the way to the
bottom, via the mouse.
 Add two additional control points to the opacity curve.
 To do this, click any two points you wish on the opacity curve.
 The new control points appear where you have clicked.
 Define the ascent of the opacity curve.
 Pull the left point of the two new control points in the blue region all the way
to the bottom.
 Pull the right point of the two new control points in the blue region all the
way to the top.
 Pull the outer most right control point all the way to the top.
 Hence, the opacity for for lower intensity of 0 is quickly able to rise to 1 in
the interesting areas.
 Click the Execute button to create a projection via the blending with these
settings.
 The projection results ares shown in the image document.
 Continue to alter the opacity curve until a satisfactory image has been
reached.
 To do this, alter the opacity curve by moving the already existing control
points and/or adding new control points.
 Click again on the Execute button to assess the projection.
 Click the OK button to run the projection and to write the results to the
destination image buffer.

18
The Transfer tab of the Extended 3D- Working with ride
Visualization dialog box. The Opacity
curve graph shows the histogram of the Image acquisition
stack´s intensity distribution and the Image preparation
opacity curve with the control points. Deconvolution
The colored bar beneath the opacity
curve is the actual LUT. Visualization
Opacity: The opacity marks the "non-
transparency" of the voxels. Intensities
with opacity 0 are regarded as
transparent and are not shown in the
projection. Intensities with opacity 1 are
regarded as solid. With the help of the
opacity curve, you can accurately fade
out certain intensity areas.

When selecting a LUT and replacing


the opacity curve of the projection via
blending, you have efficient tools to
create impressive images.

Edit LUT: Instead of using one


of the available LUTs, you can
also change these to fit your
needs. To do this, use the Edit
LUT… command located in the Oper >
3D-Images menu. This command is
identical to the Edit LUT command,
located in the Oper menu, with the
exception that it effects the whole stack.
This command is described in the
The opacity curve after the reduction of the opacity for lower intensities and the analySIS-Reference Handbook.
addition of two control points (left), as well as after the moving of the new control points
(right). Change 16-Bit-Display: You
can change the display-
behavior to take full advantage
of the dynamic area of the
utilized 16 Bit gray value images. Use
the Change 16-Bit-Display… command
located in the menu Oper > 3D-Images.
With the exception that this command
effects the whole stack, it is identical to
the Change 16-Bit-Display… command
located in the Oper menu. This
command is described in the analySIS
reference handbook.

The projection via blending with the settings shown here on the left. The results of the
iso-projection is once again shown on the right as comparison.
Note that both of the pojections are shown in false colors; these colors do not give any
information about the actual colors observed.

19
ride • Appendix

ride

Appendix
The Various Filters
The ride module offers you a variety of filters, based on the software-pack-
age being used. The Blind Deconvolution Filter is only available in the fol-
lowing ride versions: ride 3D-Blind WF, ride 3D-Blind CF, and ride
Complete.
Name Use on single Required Microscope and
images Parameters Acquisition Parameters for
possible? the setup
No Neighbor yes haze removal Fluorescence (preset)
Transmitted Brightfield
Phase object
Nearest Neighbor yes (identical to haze removal Fluorescence (preset)
No Neighbor) Transmitted Brightfield
Phase object
Inverse Filter yes tiling Fluorescence (preset)
Transmitted Brightfield
Phase object
Blind Deconvolution no tiling Microscope type
iterations

No Neighbor
The No Neighbor filter is based on a purely theoretical point spread function
which is applied to the images. The correction function is calculated based
on the difference to the starting images.
Specification The No Neighbor filter works non-iteratively with the image being viewed.
2D The No Neighbor filter only works with the data of a singel image and thus
can be used unlimitedly for single images.
Application No Neighbor is the fastest of all filters. Use it for the first attempts with a
stack.

Nearest Neighbor
The Nearest Neighbor filter is based on a point spread function. This
function uses data from the viewed image and the two directly neighboring
images. The point spread function is used on the neighboring images. The
scaled sum of both neighboring images is removed from the image being
viewed.
Specification The Nearest Neighbor filter works non-iteratively with the image being
viewed and its two neighbors.
2D The behavior of Nearest Neighbor is identical with No Neighbor when work-
ing with single images.

20
The Various Filters

Application The Nearest Neighbor filter uses only two neighboring images for correction.
Thus, the filter works faster and better than the No Neighbor filter. The qual-
ity loss, with comparison to Inverse Filter and Blind Deconvolution is minimal
when working with stacks with few images.

Inverse Filter
The Inverse Filter approaches the point spread function via a linear term
g(x) = f(x) * h(x) . The linear inverse function is calculated out of this value,
h – 1(x) , with the root-mean-square deviation being minimized. The deconvo-
lution ultimately takes place via this inverse filter.
Specification The Inverse Filter works non-iteratively on the entire stack.
2D The use of the Inverse Filters is also possible on single images.
Application The Inverse Filter processes the information from all of the images in the
stack. Hence, a very good quality can be achieved, however with a very high
processing effort. The increase in quality in comparison to the No Neighbor
and Nearest Neighbor filters becomes evident when stacks using many im-
ages are used.

Blind Deconvolution
Attention The Blind Deconvolution is not a part of all ride-modules. It is only available
in the following modules: ride 3D-Blind WF (for fluorescent and transmitted-
light acquisitions), ride 3D-Blind CF (for confocal, Two Photon und Spin-
ning-Disk-Microscopes) and ride Complete (for all Microscopes).

The Blind Deconvolution filter does not make any presumptions about the
point spread function, but rather extracts it from the stack. An estimated
point spread function h(x) is used as a starting point. Then, an assuption is
made as to which ideal image f(x), via this point spread function, would have
led to the observed image g(x). Then, an estimation has to be made as to
which point spread function caused the original image f(x) to transform into
the observed g(x) image. This alternate assessment can be repeated as of-
ten as desired. Special mathematic processes are used to ensure that these
iterations converge to reasonable values.
Specification The Blind Deconvolution filter works iteratively on the entire stack.
2D It is not possible to apply the Blind Deconvolution filter to single images.
Application The iterative procedure which is used by the Blind Deconvolution filter,
promises the best results from all other filters. The quality of the results can
be directly influenced by the Iterations parameters. Using iterative
procedures on an entire stack with high resolution can be timely. Therefore,
first use the Resize and Crop frame commands to optimize the settings
which are rather timely. Then you can carry out the final deconvolution with
the found values on the original stack.

21
ride • Appendix

Parameter
Transmitted Brightfield (No Neighbor, Nearest Neighbor, Inverse Filter): Select the Transmitted
Brightfield chekc box in the Microscope/Acquisition Parameters group,
when working with this method rather than with fluorescence. When the
Transmitted Brightfield check box is activated, ride expects a light
background. Hence, the background will be illuminated.
Phase Object (No Neighbor, Nearest Neighbor, Inverse Filter): Select the Phase Object
check box in addition to the Transmitted Brightfield check box, should the
object being studied be acquisitioned with phase or modulations contrast, or
with a DIC. In this case, ride interprets low intensity differences as belonging
to the object.
Haze Removal (No Neighbor, Nearest Neighbor): When using the No Neighbor and Nearest
Neighbor methods, you give an objective for deconvolution with the Haze
Removal parameters. The higher the value for the Haze Removal, the more
noise is removed from the stack. When the value is too high, the program
interprets actual image information as noise as well. Typically, there is an
area in which the increase of the parameters does not have any more
influence on the visual quality of the image. This is a sign that the filter
method has been exhausted. An additional increase in Haze Removal can,
by all means, lead to artifacts.
Tiling, Overlap (Inverse Filter, Blind Deconvolution): The Inverse Filter and Blind
Deconvolution methods, which both need timely processing, access all the
voxels of a stack. The influence of the point spread function is, at some
distance to the starting point of the XY-direction, only very minimal. A
possibility to speed-up this procedure is to laterally divide the stack into
numerous stacks which can be independently processed from one another.
When deconvoluting these smaller stacks, only the image information of the
smaller volumes is accessed. The resulting processed stacks are then
adapted to one another, in order to avoid visible cracks in the edges of the
tiles. To do this, the parameters in a certain area between two tiles is cross-
dissolved. The width of this area is determined by the Overlap parameter..
Effect of Tiling
I
Intensity
profile T1

Intensity
X profile T2

Intensity
profile with
T1 T2 Tiling

22
The Various Filters

ride tries to process the entire stack at once, should you erase the marking
of the Activate control box in the Tiling group. The lack of random access
memory and disc space can hinder this. The tiling is automatically activated
should you not have enough resources available. You are informed via the
Blind 3D-Deconvolution information box that this function is being used.
The Blind 3D-Deconvolution
Status dialog box shows the
status of this current
method. The entry in the
sub-volume line shows that
the tiling is active. If the tiling
were not being used, there
would only be one sub-
volume.

Should the tiling be activated by force, you can reduce the amount of Voxels
via the Resize or Crop frame, to continue working without tiling or activate
the tiling and setting the Overlap parameters deliberately at a high level. In
this manner, you achieve a soft transition from tile to tile.

23
ride • Appendix

24
Index

Frames 13
1XPHULFV
3D-Deconvolution 8
+
Haze removal 9
$
Acquire image stack 4
Animation 17 ,
Apparative requirements 5 Image Count 5
Image stack, acquisition 4
Inverse Filter 21
% Iso-Surface projection 16
Background 17 Iso-View 16
Blind 3D-Deconvolution Status 23
Blind Deconvolution 21
0
Maximum intensity projection 9
& Minimum intensity projection 9
Calculate Z-count 4
Change 16-Bit-Display 19
Crop frame 6 1
Nearest Neighbor 20
No Neighbor 20
'
Database 15
Deconvolution 2 2
Determining specimen thickness 5 Opacity 19
Diffraction 2 Optimize parameter 8

( 3
Edit LUT 19 Point Spread Function 2
Extended visualization 16 Projection 11
Projection by blending 18
Projection methods 9
)
False-Color presentation 18
Filters 20 5
Blind deconvolution 21 Ratio
Inverse Filter 21 Maintain XY-ratio 7
Nearest Neighbor 20 Zoom axes homogeneously 7
No Neigbar 20 Zoom axes independently 7
Parameters 22 Resize 6, 7

25
Index

6
Select filter 8
Set frame 6
Set size 6
Shading 17
Simple visualization 12
Slice view 9
Smooth 16
Sum projection 9

9
Visualize results 8, 14
Voxel 9

:
Work Routine 3

26