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<html>
<head>
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<title>Image Registration</title>
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<h1 class="title toc-ignore">Image Registration</h1>
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<p><br></p>
<div id="spatial-data-alignment" class="section level1">
<h1>Spatial Data Alignment</h1>
<p>Spatial genomic technologies often generate diverse images and
spatial readouts, even though the tissue slices are from adjacent
sections of a single tissue block. Hence, the alignment of images and
spatial coordinates across tissue sections are of utmost importance to
dissect the correct spatial closeness across these sections.</p>
<p>VoltRon allows users to <strong>align spatial omics datasets of these
serial sections</strong> for data transfer and 3 dimensional stack
alignment. The order of the tissue/sample slices should be provided by
the user. VoltRon provides a fully embedded <strong>shiny
application</strong> to either automatically or manually align images.
The automatic alignment is achieved with the <strong>OpenCV</strong>’s
C++ library fully embedded in the VoltRon package.</p>
<table>
<tbody>
<tr style="vertical-align: center">
<td style="width:43%; vertical-align: center">
<img src="https://bimsbstatic.mdc-berlin.de/landthaler/VoltRon/Package/images/manualregistration.png" class="center">
</td>
<td style="width:43%; vertical-align: center">
<img src="https://bimsbstatic.mdc-berlin.de/landthaler/VoltRon/Package/images/autoregistration.png" class="center">
</td>
</tr>
</tbody>
</table>
<p><br></p>
<div id="alignment-of-xenium-and-visium" class="section level2">
<h2>Alignment of Xenium and Visium</h2>
<p>In this use case, we will align <strong>immunofluorescence
(IF)</strong> and <strong>H&amp;E images</strong> of the <strong>Xenium
In Situ</strong> and <strong>Visium CytAssist</strong> platforms
readouts. Three tissue sections are derived from a single
formalin-fixed, paraffin-embedded (FFPE) breast cancer tissue block. A 5
<span class="math inline">\(\mu\)</span>m section was taken for Visium
CytAssist and two replicate 5 <span class="math inline">\(\mu\)</span>m
sections were taken for the Xenium replicates. More information on the
spatial datasets and the study can be also be found on the <a
href="https://www.biorxiv.org/content/10.1101/2022.10.06.510405v1">BioArxiv
preprint</a>.</p>
<p>You can download the Xenium and Visium readouts from the <a
href="https://www.10xgenomics.com/products/xenium-in-situ/preview-dataset-human-breast">10x
Genomics website</a> (specifically, import <strong>In Situ Replicate 1/2
and Visium Spatial</strong>). Alternatively, you can <strong>download a
zipped collection of three Visium and Xenium readouts</strong> from <a
href="https://bimsbstatic.mdc-berlin.de/landthaler/VoltRon/SpatialDataAlignment/Xenium_vs_Visium/10X_Xenium_Visium.zip">here</a>.</p>
<p>VoltRon includes built-in functions for converting readouts from both
Xenium and Visium platforms into VoltRon objects. We will import both
Xenium replicates alongside with the Visium CytAssist data so that we
can register images of these assays and merge them into one VoltRon
object.</p>
<pre class="r watch-out"><code>library(VoltRon)
Xen_R1 &lt;- importXenium(&quot;Xenium_R1/outs&quot;, sample_name = &quot;XeniumR1&quot;)
Xen_R2 &lt;- importXenium(&quot;Xenium_R2/outs&quot;, sample_name = &quot;XeniumR2&quot;)
Vis &lt;- importVisium(&quot;Visium/&quot;, sample_name = &quot;VisiumR1&quot;)</code></pre>
<p>Before moving on to image alignment, we can inspect both Xenium and
Visium images. We use the <strong>vrImages</strong> function to call and
visualize reference images of all VoltRon objects.</p>
<pre class="r watch-out"><code>vrImages(Xen_R1)
vrImages(Xen_R2)
vrImages(Vis)</code></pre>
<table>
<tbody>
<tr style="vertical-align: center">
<td style="width:33%; vertical-align: center">
<img src="https://bimsbstatic.mdc-berlin.de/landthaler/VoltRon/Package/images/xeniumr1.png" class="center">
</td>
<td style="width:33%; vertical-align: center">
<img src="https://bimsbstatic.mdc-berlin.de/landthaler/VoltRon/Package/images/xeniumr2.png" class="center">
</td>
<td style="width:33%; vertical-align: center">
<img src="https://bimsbstatic.mdc-berlin.de/landthaler/VoltRon/Package/images/visium.png" class="center">
</td>
</tr>
</tbody>
</table>
<p><br></p>
<p>Although images of the first Xenium replicate and the Visium assay
are workable, we have to adjust the brightness of the second Xenium
replicate before image alignment. You can use
<strong>modulateImage</strong> function to change the brightness and`
saturation of the reference image of this VoltRon object. This
functionality is optional for VoltRon objects and should be used when
images require further adjustments.</p>
<pre class="r watch-out"><code>Xen_R2 &lt;- modulateImage(Xen_R2, brightness = 800)
vrImages(Xen_R2)</code></pre>
<p><img width="40%" height="40%" src="https://bimsbstatic.mdc-berlin.de/landthaler/VoltRon/Package/images/xeniumr2_new.png" class="center"></p>
<p><br></p>
<div id="automated-image-alignment" class="section level3">
<h3>Automated Image Alignment</h3>
<p>In order to achieve data transfer and integration across these two
modalities, we need to first make sure that spatial coordinates of these
three datasets are perfectly aligned. To this end, we will make use of
the <strong>registerSpatialData</strong> function which calls a
<strong>shiny app</strong> embedded into VoltRon. The function takes a
single list as an input where the order of VoltRon objects in the list
should be the same as the <strong>order of serial sections</strong>.</p>
<p>We will make use of the <strong>registerSpatialData</strong> function
to <strong>automatically register two Xenium assays onto the Visium
assay</strong>. The Visium CytAssist image (or the <strong>image on the
center</strong> of the list) would be taken as the image of reference,
and hence all other images (or spatial datasets) are to be aligned to
the Visium data. Then, registerSpatialData will return a list of VoltRon
objects whose assays include both the original and registered versions
of spatial coordinates. The shiny app will provide <strong>two
images</strong> for this task:</p>
<ul>
<li>An image that shows the matched points across two images, and</li>
<li>A slideshow with of the reference and registered images that
demonstrates the alignment accuracy.</li>
</ul>
<p>We will select <strong>FLANN</strong> method for automated alignment
which incorporates the <strong>SIFT</strong> method for automated
keypoints selection and utilizes the <strong>Fast library for
Approximate Nearest Neighbors (FLANN) algorithm</strong> for matching
keypoints. <strong>NOTE:</strong> For better alignment performance,
users can incorporate image manipulation tools above each image and sync
images into the same orientation by rotating, flipping (horizontally and
vertically) and negating these images. We always negate DAPI images to
align them onto H&amp;E images.</p>
<pre class="r watch-out"><code>xen_reg &lt;- registerSpatialData(object_list = list(Xen_R1, Vis, Xen_R2))</code></pre>
<p><img width="100%" height="100%" src="https://bimsbstatic.mdc-berlin.de/landthaler/VoltRon/Package/images/XeniumVisiumRegistration_FLANN.gif" class="center"></p>
<p><br></p>
<p>You can save and use the same parameters later, and reproduce the
alignment without choosing parameters the second time.</p>
<pre class="r watch-out"><code>mapping_parameters &lt;- xen_reg$mapping_parameters
xen_reg &lt;- registerSpatialData(object_list = list(Xen_R1, Vis, Xen_R2),
mapping_parameters = mapping_parameters)</code></pre>
<p>You can find a presaved set of parameters <a
href="https://bimsbstatic.mdc-berlin.de/landthaler/VoltRon/SpatialDataAlignment/Xenium_vs_Visium/mapping_parameters.rds">here</a>.</p>
<pre class="r watch-out"><code>mapping_parameters &lt;- readRDS(&quot;mapping_parameters.rds&quot;)
xen_reg &lt;- registerSpatialData(object_list = list(Xen_R1, Vis, Xen_R2),
mapping_parameters = mapping_parameters)</code></pre>
<p>If the pre-saved parameters are available, the registration can also
be performed without using the shiny app. By using <strong>interactive =
FALSE</strong>, we can register images and VoltRon objects directly.</p>
<pre class="r watch-out"><code>mapping_parameters &lt;- xen_reg$mapping_parameters
xen_reg &lt;- registerSpatialData(object_list = list(Xen_R1, Vis, Xen_R2),
mapping_parameters = mapping_parameters,
interactive = FALSE)</code></pre>
<p>In case there are only two images, <strong>the first image will be
taken as the image of reference</strong>. Hence, in order to align the
first Xenium Replicate to the Visium dataset, we can create a list of
two VoltRon objects as given below.</p>
<pre class="r watch-out"><code>xen_reg &lt;- registerSpatialData(object_list = list(Vis, Xen_R2))</code></pre>
<p><br></p>
</div>
<div id="manual-image-alignment" class="section level3">
<h3>Manual Image Alignment</h3>
<p>Given the diverse types of tissue sections and their complex
morphology, we need an alternative alignment strategy if automated
registration may fail. VoltRon allows <strong>manually choosing
keypoints (or landmarks)</strong> on images that are locations on the
tissue with structural/morphological similarity. Similar to the
automated mode, <strong>the image on the center</strong> will be taken
as reference and the users will be able to observe the quality of the
registration and remove/reselect keypoints as they see fit.</p>
<pre class="r watch-out"><code>xen_reg &lt;- registerSpatialData(object_list = list(Xen_R1, Vis, Xen_R2))</code></pre>
<p><img width="100%" height="100%" src="https://bimsbstatic.mdc-berlin.de/landthaler/VoltRon/Package/images/XeniumVisiumRegistration.gif" class="center"></p>
<p><br></p>
<p>You can save and use the same keypoints later, and reproduce the
manual alignment without choosing keypoints for the second time.</p>
<pre class="r watch-out"><code>mapping_parameters &lt;- xen_reg$mapping_parameters
xen_reg &lt;- registerSpatialData(object_list = list(Xen_R1, Vis, Xen_R2),
mapping_parameters = mapping_parameters)</code></pre>
<p>You can find a presaved set of parameters with selected manual
keypoints <a
href="https://bimsbstatic.mdc-berlin.de/landthaler/VoltRon/SpatialDataAlignment/Xenium_vs_Visium/mapping_parameters_manual.rds">here</a>.</p>
<pre class="r watch-out"><code>mapping_parameters &lt;- readRDS(&quot;mapping_parameters_manual.rds&quot;)
xen_reg &lt;- registerSpatialData(object_list = list(Xen_R1, Vis, Xen_R2),
mapping_parameters = mapping_parameters)</code></pre>
<p>If the pre-saved keypoints are available with parameters, the
registration can also be performed without using the shiny app. By using
<strong>interactive = FALSE</strong>, we can register images and VoltRon
objects directly.</p>
<pre class="r watch-out"><code>mapping_parameters &lt;- xen_reg$mapping_parameters
xen_reg &lt;- registerSpatialData(object_list = list(Xen_R1, Vis, Xen_R2),
mapping_parameters = mapping_parameters,
interactive = FALSE)</code></pre>
<p>In case there are only two images, <strong>the first image will be
taken as the image of reference</strong>. Hence, in order to align the
first Xenium Replicate to the Visium dataset. We can create a list of
two VoltRon objects as given below.</p>
<pre class="r watch-out"><code>xen_reg &lt;- registerSpatialData(object_list = list(Vis, Xen_R2))</code></pre>
<p><br></p>
</div>
<div id="combine-voltron-object" class="section level3">
<h3>Combine VoltRon object</h3>
<p>Now that the VoltRon objects of Xenium and Visium datasets are
accurately aligned, we can combine these objects to create <strong>one
VoltRon object with three layers</strong>. Since all sections are
derived from the same tissue block, we want them to be associated with
the same sample, hence we define the sample name as well. VoltRon will
recognize that all layers are originated from the same sample/block, and
choose the majority assay as the main assay.</p>
<pre class="r watch-out"><code>merge_list &lt;- xen_reg$registered_spat
VRBlock &lt;- merge(merge_list[[1]], merge_list[-1], samples = &quot;10XBlock&quot;)
VRBlock</code></pre>
<pre><code>10XBlock:
Layers: Section1 Section2 Section3
Assays: Xenium(Main) Visium
Features: RNA(Main) </code></pre>
<p>Here, we can quickly check the change in spatial coordinate systems
in the new tissue block. The <code>registerSpatialData</code> function
syncronizes the coordinate systems of all VoltRon objects in the list
before merging. Both Xenium sections have now two coordinate system
where the registered system <strong>main_reg</strong> is the default
one.</p>
<pre class="r watch-out"><code>vrSpatialNames(VRBlock, assay = &quot;all&quot;)</code></pre>
<pre><code> Assay Layer Sample Spatial Main
Assay1 Xenium Section1 10XBlock main,main_reg main_reg
Assay2 Visium Section2 10XBlock main main
Assay3 Xenium Section3 10XBlock main,main_reg main_reg</code></pre>
<p><br></p>
</div>
<div id="datalabel-transfer-across-layers" class="section level3">
<h3>Data/Label Transfer Across Layers</h3>
<p>The combined VoltRon object of Visium and Xenium datasets can be used
to transfer information across layers and assays. This is accomplished
by aggregating and summarizing, for example, gene counts of cells from
the Xenium assay aligned to Visium spots. Either labels or cell types
can be summarized to generate:</p>
<ul>
<li>pseudo cell type abundance assays or</li>
<li>pseudo gene expression assays.</li>
</ul>
<p><br></p>
<div id="data-transfer-cells-spots" class="section level4">
<h4>Data Transfer (Cells-&gt;Spots)</h4>
<p>We must first determine the names of the assays where labels are
transfered <strong>from</strong> one <strong>to</strong> the other. For
the sake of this tutorial, we can select Assay1 of <strong>Xenium as the
source</strong> assay and the Assay2 of <strong>Visium as the
destination</strong> assay.</p>
<pre class="r watch-out"><code>SampleMetadata(VRBlock)</code></pre>
<pre><code> Assay Layer Sample
Assay1 Xenium Section1 10XBlock
Assay2 Visium Section2 10XBlock
Assay3 Xenium Section3 10XBlock</code></pre>
<p>The <strong>transferData</strong> function detects the types of both
the <strong>source (from)</strong> and the <strong>destination
(to)</strong> assays and determines the how the data should be
transfered. We can first transfer data from the Xenium assay to the
Visium assay (hence <strong>Cells -&gt; Spots</strong>), the raw count
data of each cell in the source Xenium assay will be aggregated into
spots in a newly create pseudo Visium assay. The new assay with
aggregated counts will be named <strong>Visium_pseudo</strong>.</p>
<pre class="r watch-out"><code>VRBlock &lt;- transferData(VRBlock, from = &quot;Assay1&quot;, to = &quot;Assay2&quot;)</code></pre>
<p>VoltRon supports multiple feature type within each assay. Now, the
Visium assay includes two spot-type features:</p>
<ul>
<li>the original Visium spot feature counts,</li>
<li>a pseudo Visium feature count matrix with aggregated Xenium raw
counts.</li>
</ul>
<pre class="r watch-out"><code>vrMainAssay(VRBlock) &lt;- &quot;Visium&quot;
VRBlock</code></pre>
<pre><code>VoltRon Object
10XBlock:
Layers: Section1 Section2 Section3
Assays: Visium(Main) Xenium
Features: RNA(Main) RNA_pseudo </code></pre>
<p>We can now visualize both the original and aggregated counts of a
gene, such as ERBB2 and ESR1 that marks ductal carcinoma in situ (DCIS)
regions, to validate the correlation of gene signatures across adjacent
tissue sections, and to validate the accuracy of the automated image
alignment. Here, PGR is also expressed at a small DCIS region found on
adipocyte niche of the tissue.</p>
<pre class="r watch-out"><code>library(patchwork)
vrMainFeatureType(VRBlock, assay = &quot;Visium&quot;) &lt;- &quot;RNA&quot;
g1 &lt;- vrSpatialFeaturePlot(VRBlock,
features = c(&quot;ERBB2&quot;, &quot;ESR1&quot;, &quot;PGR&quot;), crop = FALSE,
norm = FALSE, ncol = 3)
vrMainFeatureType(VRBlock, assay = &quot;Visium&quot;) &lt;- &quot;RNA_pseudo&quot;
g2 &lt;- vrSpatialFeaturePlot(VRBlock,
features = c(&quot;ERBB2&quot;, &quot;ESR1&quot;, &quot;PGR&quot;), crop = FALSE,
norm = FALSE, ncol = 3)
g1 / g2</code></pre>
<p><img width="100%" height="100%" src="https://bimsbstatic.mdc-berlin.de/landthaler/VoltRon/Package/images/registration_TACSTD2.png" class="center"></p>
</div>
<div id="data-transfer-spots-cells" class="section level4">
<h4>Data Transfer (Spots-&gt;Cells)</h4>
<p>A similar transfer can be achieved on the opposite direction. We can
select Assay2 of <strong>Visium as the source</strong> assay and Assay1
of <strong>Xenium as the destination</strong>, thus we can transfer
whole transcriptome counts of the Visium assays to Xenium to create new
feature sets for Xenium data with more features originally available in
the Xenium panel.</p>
<pre class="r watch-out"><code>vrMainFeatureType(VRBlock, assay = &quot;Visium&quot;) &lt;- &quot;RNA&quot;
VRBlock &lt;- transferData(VRBlock, from = &quot;Assay2&quot;, to = &quot;Assay1&quot;)</code></pre>
<p>We now set the main feature set of the Xenium assays.</p>
<pre class="r watch-out"><code>vrMainFeatureType(VRBlock, assay = &quot;Xenium&quot;) &lt;- &quot;RNA_pseudo&quot;
vrMainFeatureType(VRBlock, assay = &quot;all&quot;)</code></pre>
<pre><code> Assay Feature
1 Assay1 RNA_pseudo
2 Assay2 RNA
3 Assay3 RNA</code></pre>
<pre class="r watch-out"><code>library(patchwork)
g1 &lt;- vrSpatialFeaturePlot(VRBlock,
assay = &quot;Assay1&quot;, features = c(&quot;ERBB2&quot;, &quot;ESR1&quot;, &quot;PGR&quot;),
crop = TRUE, norm = FALSE, alpha = 1, n.tile = 300, ncol = 3)
g2 &lt;- vrSpatialFeaturePlot(VRBlock,
assay = &quot;Assay2&quot;, features = c(&quot;ERBB2&quot;, &quot;ESR1&quot;, &quot;PGR&quot;),
crop = TRUE, norm = FALSE, alpha = 1, ncol = 3)
g1 / g2</code></pre>
<p><img width="100%" height="100%" src="https://bimsbstatic.mdc-berlin.de/landthaler/VoltRon/Package/images/registration_spot2cell.png" class="center"></p>
</div>
<div id="label-transfer-cells-spots" class="section level4">
<h4>Label Transfer (Cells-&gt;Spots)</h4>
<p>The <strong>transferData</strong> function can also transfer
<strong>metadata features</strong> across layers and assays. In this
case, we will transfer cell type labels that were trained on the Xenium
sections onto the Visium sections. We will use the cluster labels
generated at the end of the Xenium analysis section of workflow from <a
href="spotanalysis.html">Cell/Spot Analysis</a>. You can download the
VoltRon object with clustered and annotated Xenium cells along with the
Visium assay from <a
href="https://bimsbstatic.mdc-berlin.de/landthaler/VoltRon/SpatialDataAlignment/Xenium_vs_Visium/VRBlock_data_clustered.rds">here</a>.</p>
<pre class="r watch-out"><code>VRBlock &lt;- readRDS(&quot;VRBlock_data_clustered.rds&quot;)
vrSpatialPlot(VRBlock, assay = &quot;Xenium&quot;, group.by = &quot;CellType&quot;, pt.size = 0.4)</code></pre>
<p><img width="100%" height="100%" src="https://bimsbstatic.mdc-berlin.de/landthaler/VoltRon/Package/images/cellspot_spatial_xenium_annotated.png" class="center"></p>
<p>Here, we can see that both Xenium layers are clustered and annotated
where we can use these cell annotations and transfer them to the Visium
assay to create an assay of <strong>estimated cell type
abundances</strong>. If the features argument is specified, and if its a
single metadata feature with, e.g. cell types, then the each spot at the
new pseudo Visium will be collection of abundances of the categories
within that metadata feature.</p>
<pre class="r watch-out"><code>VRBlock &lt;- transferData(VRBlock, from = &quot;Assay1&quot;, to = &quot;Assay2&quot;, features = &quot;CellType&quot;,
new_assay_name = &quot;Visium_CellType&quot;)
VRBlock</code></pre>
<pre><code>VoltRon Object
10XBlock:
Layers: Section1 Section2 Section3
Assays: Visium(Main) Xenium
Features: RNA_pseudo(Main) RNA Visium_CellType </code></pre>
<p>By visualizing the transferred labels on the Visium spots, we can see
abundance of some DCIS and invasive tumor subtypes.</p>
<pre class="r watch-out"><code>vrMainFeatureType(VRBlock) &lt;- &quot;Visium_CellType&quot;
vrSpatialFeaturePlot(VRBlock, assay = &quot;Visium&quot;,
features = c(&quot;IT_1&quot;,&quot;DCIS_2&quot;),
crop = TRUE, alpha = 1, ncol = 3)</code></pre>
<p><img width="100%" height="100%" src="https://bimsbstatic.mdc-berlin.de/landthaler/VoltRon/Package/images/registration_CellType.png" class="center"></p>
<p><br></p>
</div>
<div id="label-transfer-rois-" class="section level4">
<h4>Label Transfer (ROIs-&gt;…)</h4>
<p>VoltRon allows users to annotate regions of interests (ROIs) in a
given assay and transfer the annotations to these ROIs across other
assays within the same tissue block. Let us annotate two specific tumor
regions in the Visium section. In the process, a new assay called
<strong>ROIAnnotation</strong> will be added to the VoltRon object.</p>
<pre class="r watch-out"><code>VRBlock &lt;- annotateSpatialData(VRBlock, assay = &quot;Visium&quot;,
label = &quot;annotation&quot;, use.image.only = TRUE)</code></pre>
<p><img width="80%" height="80%" src="https://bimsbstatic.mdc-berlin.de/landthaler/VoltRon/Package/images/registration_ROIAnnotation.png" class="center"></p>
<p><br></p>
<p>You can observe the changes in the object and check the assay ID of
this new ROI type assay using <code>SampleMetadata</code> function.</p>
<pre class="r watch-out"><code>VRBlock</code></pre>
<pre><code>VoltRon Object
10XBlock:
Layers: Section1 Section2 Section3
Assays: Xenium(Main) Visium ROIAnnotation
Features: RNA(Main) </code></pre>
<pre class="r watch-out"><code>SampleMetadata(VRBlock)</code></pre>
<pre><code> Assay Layer Sample
Assay1 Xenium Section1 10XBlock
Assay2 Visium Section2 10XBlock
Assay3 Xenium Section3 10XBlock
Assay4 ROIAnnotation Section2 10XBlock</code></pre>
<p>The metadata of the ROI assay will include the annotation of the ROIs
as well.</p>
<pre class="r watch-out"><code>Metadata(VRBlock, assay = &quot;ROIAnnotation&quot;)</code></pre>
<pre><code> Assay Layer Sample annotation
InvasiveTumor_Assay4 ROIAnnotation Section2 10XBlock InvasiveTumor
DuctalCarcinoma_Assay4 ROIAnnotation Section2 10XBlock DuctalCarcinoma</code></pre>
<p>Now we can transfer the ROI labels from the
<strong>annotation</strong> metadata column and define the same metadata
column in the remaining assays.</p>
<pre class="r watch-out"><code>VRBlock &lt;- transferData(object = VRBlock, from = &quot;Assay4&quot;, to = &quot;Assay1&quot;,
features = &quot;annotation&quot;)
VRBlock &lt;- transferData(object = VRBlock, from = &quot;Assay4&quot;, to = &quot;Assay3&quot;,
features = &quot;annotation&quot;)</code></pre>
<p>Let us observe the changes across all assays.</p>
<pre class="r watch-out"><code>vrSpatialPlot(VRBlock, group.by = &quot;annotation&quot;, assay = &quot;Xenium&quot;, crop = TRUE)
vrSpatialPlot(VRBlock, group.by = &quot;annotation&quot;, assay = &quot;Visium&quot;, crop = TRUE)</code></pre>
<p><img width="100%" height="100%" src="https://bimsbstatic.mdc-berlin.de/landthaler/VoltRon/Package/images/registration_ROI_xenium.png" class="center"></p>
<p><img width="50%" height="50%" src="https://bimsbstatic.mdc-berlin.de/landthaler/VoltRon/Package/images/registration_ROI_visium.png" class="center"></p>
<p><br></p>
<p>You can also use the <strong>addSpatialLayer</strong> function to
overlay annotation segments to the spatial plot of the Xenium data.</p>
<pre class="r watch-out"><code>vrSpatialPlot(VRBlock_new2, group.by = &quot;CellType&quot;, assay = &quot;Assay1&quot;, crop = TRUE) |&gt;
addSpatialLayer(VRBlock_new2, assay = &quot;ROIAnnotation&quot;, group.by = &quot;annotation&quot;, spatial = &quot;main&quot;, alpha = 0.4)</code></pre>
<p><img width="50%" height="50%" src="https://bimsbstatic.mdc-berlin.de/landthaler/VoltRon/Package/images/registration_ROI_xenium_overlay.png" class="center"></p>
<p><br></p>
</div>
</div>
</div>
<div id="alignment-of-xenium-and-he" class="section level2">
<h2>Alignment of Xenium and H&amp;E</h2>
<p>In this use case, we will align <strong>immunofluorescence
(IF)</strong> of the <strong>Xenium In Situ</strong> platform to an
<strong>H&amp;E images</strong> generated from the same sections as the
Xenium. VoltRon provides built-in utilities to import images as spatial
datasets where <strong>tiles</strong> are the spatial points. We will
import both Xenium and H&amp;E images into two separate VoltRon objects
and overlay H&amp;E images.</p>
<p>You can download the Xenium readout and the H&amp;E image of the same
tissue section from the <a
href="https://www.10xgenomics.com/products/xenium-in-situ/preview-dataset-human-breast">10x
Genomics website</a> (specifically, import <strong>In Situ Replicate
1</strong> and <strong>Supplemental: Post-Xenium H&amp;E image
(TIFF)</strong>).</p>
<pre class="r watch-out"><code>library(VoltRon)
# import Xenium
Xen_R1 &lt;- importXenium(&quot;Xenium_R1/outs&quot;, sample_name = &quot;XeniumR1&quot;)
# import H&amp;E image and build a VoltRon object
Xen_R1_image &lt;- importImageData(&quot;Xenium_FFPE_Human_Breast_Cancer_Rep1_he_image.tif&quot;,
sample_name = &quot;XeniumR1image&quot;,
channel_names = &quot;H&amp;E&quot;)
Xen_R1_image</code></pre>
<pre><code>VoltRon Object
XeniumR1image:
Layers: Section1
Assays: ImageData(Main) </code></pre>
<p>Lets take a look at the image of the Xen_R1_image object</p>
<pre class="r watch-out"><code>vrImages(Xen_R1_image)</code></pre>
<p><img width="70%" height="70%" src="https://bimsbstatic.mdc-berlin.de/landthaler/VoltRon/Package/images/importdata_HE.png" class="center"></p>
<p><br></p>
<div id="automated-image-alignment-1" class="section level3">
<h3>Automated Image Alignment</h3>
<p>We can use the <strong>registerSpatialData</strong> function to
warp/align images across multiple VoltRon objects and define these
aligned images additional channels of existing coordinate systems of
assays in one of these VoltRon objects.</p>
<p>First we align the H&amp;E image to the DAPI image of the Xenium
replicate. Similar to the first use case, we need to negate the DAPI
image and change the alignment of the image to match it with the H&amp;E
image. We can also scale the resolution of the H&amp;E image to
9103.71x6768.63.</p>
<pre class="r watch-out"><code>xen_reg &lt;- registerSpatialData(object_list = list(Xen_R1, Xen_R1_image))</code></pre>
<p><img width="92%" height="92%" src="https://bimsbstatic.mdc-berlin.de/landthaler/VoltRon/Package/images/registration_HE_function.png" class="center"></p>
<p><br></p>
<p>Now we create a new channel for the existing coordinate system of the
Xenium data. Here, the spatial key of the registered H&amp;E image will
be <strong>main_reg</strong>. We choose the destination of the
registered image which is the first Assay of the Xenium data
(i.e. <strong>Assay1</strong>). The original DAPI coordinate system, and
we give a name for the new image/channel which is
<strong>H&amp;E</strong>.</p>
<pre class="r watch-out"><code>Xen_R1_image_reg &lt;- xen_reg$registered_spat[[2]]
vrImages(Xen_R1[[&quot;Assay1&quot;]], channel = &quot;H&amp;E&quot;) &lt;- vrImages(Xenium_reg, name = &quot;main_reg&quot;, channel = &quot;H&amp;E&quot;)</code></pre>
<p>We can now observe the new channels (H&amp;E) available for the
Xenium assay using <strong>vrImageChannelNames</strong>.</p>
<pre class="r watch-out"><code>vrImageChannelNames(Xen_R1)</code></pre>
<pre><code> Assay Layer Sample Spatial Channels
Assay1 GeoMx Section1 prolonged case 4 main scanimage,DNA,PanCK,CD45,Alpha Smooth Muscle Actin,H&amp;E</code></pre>
<p>We can call the registered H&amp;E image of the Xenium data or later
put the aligned H&amp;E when calling <strong>vrSpatialPlot</strong> or
<strong>vrSpatialFeaturePlot</strong>.</p>
<pre class="r watch-out"><code>vrImages(Xen_R1, channel = &quot;H&amp;E&quot;, scale.perc = 5)</code></pre>
<p><img width="70%" height="70%" src="https://bimsbstatic.mdc-berlin.de/landthaler/VoltRon/Package/images/registration_HE.png" class="center"></p>
<p><br></p>
</div>
</div>
<div id="alignment-of-visium-and-visium" class="section level2">
<h2>Alignment of Visium and Visium</h2>
<p>In the next use case, we will align <strong>H&amp;E images</strong>
associated with Visium data generated from tissue block sections of
<strong>adult humans with postmortem dorsolateral prefrontal cortex
(DLPFC)</strong>. Two pairs of adjacent sections was obtained from the
tissue block of the third donor. Each pair are composed of two 10 <span
class="math inline">\(\mu\)</span>m serial tissue sections, and pairs
are located 300 <span class="math inline">\(\mu\)</span>m apart from
each other. Hence, we align each pair individually. The datasets can be
downloaded from <a
href="https://research.libd.org/spatialLIBD/">here</a>.</p>
<pre class="r watch-out"><code>library(VoltRon)
DLPFC_1 &lt;- importVisium(&quot;DLPFC/151673&quot;, sample_name = &quot;DLPFC_1&quot;)
DLPFC_2 &lt;- importVisium(&quot;DLPFC/151674&quot;, sample_name = &quot;DLPFC_2&quot;)
DLPFC_3 &lt;- importVisium(&quot;DLPFC/151675&quot;, sample_name = &quot;DLPFC_3&quot;)
DLPFC_4 &lt;- importVisium(&quot;DLPFC/151676&quot;, sample_name = &quot;DLPFC_4&quot;)</code></pre>
<p><br></p>
<div id="automated-image-alignment-2" class="section level3">
<h3>Automated Image Alignment</h3>
<p>We will again use the registerSpatialData function to
<strong>automatically register two Visium assays (two H&amp;E
images)</strong>. This time, we will use the
<strong>BRUTE-FORCE</strong> method for automated alignment which we
found to be more accurate compared to FLANN when aligning two H&amp;E
images. The shiny app also provides two tuning parameters that used by
the the BRUTE-FORCE workflow:</p>
<ul>
<li><strong># of Features</strong> option specifies the number of
maximum image features spotted within each image which later be used to
match to the other image.</li>
<li><strong>Match %</strong> specifies the percentage of these features
matching at max which in turn used to compute the
registration/transformation matrix.</li>
</ul>
<p>We will use <strong>1000 features</strong> for this alignment, set
<strong>Match %</strong> to 20% of the features to be matched across
images. The quality of the alignment will be determined by the fine
tuning of these parameters where users will immediately observe the
alignment quality looking at the slideshow.</p>
<pre class="r watch-out"><code>DLPFC_list &lt;- list(DLPFC_1, DLPFC_2)
reg1and2 &lt;- registerSpatialData(object_list = DLPFC_list)</code></pre>
<p><img width="100%" height="100%" src="https://bimsbstatic.mdc-berlin.de/landthaler/VoltRon/Package/images/VisiumDLFPCRegistration.gif" class="center"></p>
<p><br></p>
<p>We can now apply a similar alignment across the second pair of
VoltRon objects. We will use <strong>800 features</strong> for this
alignment, set <strong>Match %</strong> to 50% of the features to be
matched across images.</p>
<pre class="r watch-out"><code>DLPFC_list &lt;- list(DLPFC_3, DLPFC_4)
reg3and4 &lt;- registerSpatialData(object_list = DLPFC_list)</code></pre>
<p><br></p>
</div>
<div id="d-spot-clustering" class="section level3">
<h3>3D Spot Clustering</h3>
<p>We can now combine all sections into one VoltRon object. There are
two pairs of serial tissue sections, but both pairs (thus 4 sections)
are from the same tissue block. Hence, we can combine these two lists
into one list and merge VoltRon objects even though sections were
aligned separately.</p>
<pre class="r watch-out"><code>merge_list &lt;- c(reg1and2$registered_spat, reg3and4$registered_spat)
SRBlock &lt;- merge(merge_list[[1]], merge_list[-1], samples = &quot;DLPFC_Block&quot;)
SRBlock</code></pre>
<pre><code>VoltRon Object
DLPFC_Block:
Layers: Section1 Section2 Section3 Section4
Assays: Visium(Main) </code></pre>
<p><br></p>
<p>Aligning spots along the z dimension allows us to cluster these spots
using both the gene expression similarities and spatial adjacency (both
along the x-y direction and in the z direction). We first generate a
spatial neighborhood graph and use this graph along with the gene
expression neighborhood graph <strong>(under development)</strong>.</p>
</div>
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