parallel (min-max) 1) Manually crop xy to 500x500 using ImageJ 2) Select substack: slices 1-150 3) Use Image-> Stacks-> Reslice...[/]: default settings - Output spacing = 1, Start at = top, everything else unticked 4) Use Image-> Adjust-> Canvas Size...: - Width = 500 - Height = 300 (i.e. pad image by ~150 pixels on the bottom) - Position = Top-Center - Zero Fill = unticked 5) Go File-> New-> Script... - Select Langauge IJ1 Macro - Paste the following ***macro*** for image translation: *** xTranslate=0; yTranslate=0.325; /*Translate a stack of images so that the first image is moved n pixels in the X direction, the second moved 2n pixels, the third 3n pixels and so on.. the variable "n" is represented by xTranslate and yTranslate */ for(i=1;i<=nSlices;i++){ setSlice(i); run("Translate...", "x="+i*xTranslate+" y="+i*yTranslate+" interpolation=Bilinear slice"); } *** - Adjust the 'yTranslate' value as needed according the alpha tilt of microscope and piezo step size: yTranslate = sin(alpha)/[z-piezo/(sCMOSpixel/mag)] = sin(30) / [0.25/(6.5/40)] = 0.325 (in this case) - Run macro on stack 6) Reverse the re-slice operation at step 3) 7) Select sub-stack: slices 145-170 8) Use Image-> Scale... - X Scale = 1 - Y Scale = cos(alpha) = cos(30) = 0.866 - Z Scale = 1 - Width, Height, Depth = 500, 500, 26 - Interpolation = Bilinear - 3 boxes = ticked - Title = default... 9) Use Image-> Adjust-> Canvas Size...: - Width = 500 - Height = 500 (i.e. pad image to orginal size for comparison) - Position = Center - Zero Fill = unticked 10) Save 16bit tiff to 8-bit tiff using ImageJ min-max scaling 0-255 11) Use ImageJ file-> save as-> image sequence-> format: PNG name: img start at: 0 digits: 4 12) Use output directly perpendicular (min-max) 1) Manually crop xy to 500x500 using ImageJ 2) Select substack: slices 10-160 3) Use Image-> Stacks-> Reslice...[/]: default settings - Output spacing = 1, Start at = top, everything else unticked 4) Use Image-> Adjust-> Canvas Size...: - Width = 500 - Height = 300 (i.e. pad image by ~150 pixels on the bottom) - Position = Top-Center - Zero Fill = unticked 5) Go File-> New-> Script... - Select Langauge IJ1 Macro - Paste the following ***macro*** for image translation: *** xTranslate=0; yTranslate=0.325; /*Translate a stack of images so that the first image is moved n pixels in the X direction, the second moved 2n pixels, the third 3n pixels and so on.. the variable "n" is represented by xTranslate and yTranslate */ for(i=1;i<=nSlices;i++){ setSlice(i); run("Translate...", "x="+i*xTranslate+" y="+i*yTranslate+" interpolation=Bilinear slice"); } *** - Adjust the 'yTranslate' value as needed according the alpha tilt of microscope and piezo step size: yTranslate = sin(alpha)/[z-piezo/(sCMOSpixel/mag)] = sin(30) / [0.25/(6.5/40)] = 0.325 (in this case) - Run macro on stack 6) Reverse the re-slice operation at step 3) 7) Select sub-stack: slices 145-170 8) Use Image-> Scale... - X Scale = 1 - Y Scale = cos(alpha) = cos(30) = 0.866 - Z Scale = 1 - Width, Height, Depth = 500, 500, 26 - Interpolation = Bilinear - 3 boxes = ticked - Title = default... 9) Use Image-> Adjust-> Canvas Size...: - Width = 500 - Height = 500 (i.e. pad image to orginal size for comparison) - Position = Center - Zero Fill = unticked 10) Save 16bit tiff to 8-bit tiff using ImageJ min-max scaling 0-255 11) Use ImageJ file-> save as-> image sequence-> format: PNG name: img start at: 0 digits: 4 12) Use output directly