FROM IMAGES TO ANSWERS Deconvolution of Widefield and Confocal images Quantatitive and Qualitative Deconvultion, 3D filters and 3D Analysis. Autoquant Userconference New England 2007 Media Cybernetics

Overview 3D Rendering and Visualization –Explore the depth of your images by visualizing and interacting with them in three dimensions. 3D Image Processing –Process the Image to facilitate Rendering and Analysis. 3D Image Analysis –Find the data you are looking for in your image.

3D Rendering: Creating 3D Volumes 1.Volume created though rendering of image stack 2.Voxel sizes determined through spatial calibration 3.Application of projections 4.Application of lookup tables 5.Interpolation of image volume 6.3D display of voxel data 7.Contrast adjustment

3D Rendering & Visualization: Rendering parameters 1.Image Parameters 2.Volume 3.Light 4.Material 5.Palette 6.VOI (Volume of Interest) 7.Slicer

3D Rendering: Defining a Voxel Voxel- A “Volume Element”. A means for modeling data in three dimensions “A three-dimensional element that is the smallest nondivisible element of a digital volume” - As defined by the US Government (Really!) Volume- 3D representation of 2D image planes. A collection of voxels

3D Rendering: The Perfect 3D Stack-1

3D Rendering: The Perfect 3D Stack-2

3D Rendering: Image Parameters Voxel Size Voxel: The dimensions of one voxel effect the visualization and measurements Sub sampling: Use it for larger images, to decrease loading time and increase performance. However, the image quality in this case will be lower. XY-Z ratio of 10 XY-Z ratio of 1

3D Rendering: Volume Composition Blend: Blending averages the values of the voxels in a straight line through the volume of the object. Sum: This method displays the sum of the value of the voxels in a straight line. It is most useful for dark images Maximum Intensity Projection (MIP) takes the value of the brightest voxel in the same straight line as used by the Blend option

3D Rendering: 3D Projections Example Blend Projection Sum Projection Maximum Intensity Projection 3D Projections of Detached Mammalian Retina Courtesy of Ken Linberg/Steve Fisher Lab, Brian Matsumoto- UCSB

3D Rendering: Volume Global Transparancy Transparency = 1 Transparency = 0 Transparency = 0.923

3D Rendering: Volume Number of Slices and Interpolation # Slices = 11 Interpolation = On # Slices = 11 Interpolation = Off # Slices = 0 Interpolation = On The number of slices drawn for 3D and 2D multi- texture map rendering can be used to improve performance but can result in reduced image quality. Linear interpolation on texture maps is used to increase the visible resolution.

3D Rendering: Volume Lighting

3D Rendering: Color Mapping Color mapping assigns an image intensity value to the display intensity value and color

3D Rendering: Volume of Interest Use the Volume of interest to modify the visible portion of the data volume.

3D Rendering: Orthogonal and Oblique Sectioning Reveal detail in single plane of reference Resolve spatial arrangements Pollen Grain- Courtesy of Olympus America

3D Rendering: Volume Slicer

3D Rendering: Sectioning- ‘Slicing’ Bovine Kidney Tissue- Glomerulus (Alexa 568), Proximal and Distal Tubules (Alexa 488). Courtesy of Brian Matsumoto x/y projection x/z y/z

Image Processing: Understanding Filters Examples of 3D filters

Image Processing: Median Filter 2D&3D Median: Select this filter if you want to remove impulse noise from an image. The Median filter replaces the center pixel with the median value in its neighborhood. It will also blur the image.

Image Processing, Gaussian Filter Gauss: Use this filter to soften an image by eliminating high-frequency information using a Gauss function. This has the effect of blurring sharp edges. The operation of the Gauss filter is similar to the LoPass filter, but it degrades the image less than the LoPass filter.

Image Processing: Sobel Filter 2D Sobel: Select this filter if you want to enhance just the principal edges in an image. The Sobel applies a mathematical formula to a 3 x 3 neighborhood to locate and highlight its edges.

Image Processing: Erosion Filter Erode: Select this morphological filter if you want to modify the size of objects in your image. The Erode filter erodes the edges of bright objects, and enlarges dark ones.

Image Processing: Distance Filter Distance Map: This filter creates a distance map of the current sequence. The distance map is created as a new floating point image, where the amplitude of every pixel corresponds to the shortest Euclidean distance to the edge of binary object. The binarization is based on the Threshold value, which is defined in percentage of the maximum pixel value of the image class. For floating point images the minimum and maximum are defined by the dynamic range.

Image Processing: Thinning Filter 50 Iterations40 Iterations 30 Iterations Thinning 30 Iteration Threshold 50 Thinning: Select this filter to reduce an image to its skeleton. The thinning is based on the distance map of the volume and produces a 26- connected medial axis of the objects above the threshold. If the Stop after option is on the pixels farther than the Iterations distance from the object’s edge are not skeletonized

Image Processing: 3D Skeletons

Image Analysis: Apply Measurements to 3D Images Manual Measurements –Individual point. –Point-to-Point distance. –Point-to-Surface distance line. –Line. –Angle. –Point-to-Line distance. –Circumference of an object bisected by plane. –Surface distance between two points. Volume Measurements –Volume. –Surface Area. –Bounding box measurements. –Centroid information. –Automatic update of measurements from frame-to-frame playback of 4D sequences. –Set filter ranges for any measurements. –Clean borders.

Image Analysis: Manual Measurements

Image Analysis: Iso-Surface Filters Iso-Surface Filter value impacts volume measurements.

Image Analysis: Surface Value Surface value impacts volume measurements.

Quantatitive and Qualitative Processing, Visualization and Analysis. 3D Filters Manual Measurements Volume Measurements Data AnalysisDeconvolution 3D Rendering and Slice Views 3D Rendering Iso_Surface