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=Slide FISH ([[Daniel:Notebook/HiResChrPaint/2014-9-19|Started 09/19/2014]])= [[Daniel:Notebook/HiResChrPaint|Back to Calendar]] ==Microscopy== For today's microscopy, I tried something new in order to help determine some of the properties of the microscope/sample. I took an image series for both the DMD and the PPRL samples using different resolutions in the following order (values are for one side, total image is number of pixels squared): 2048 pixels, 2048 pixels, 1024 pixels, 512 pixels, 256 pixels, 2048 pixels. All other properties, gain, x,y,z locations, etc. were kept constant. This will allow me to see (a) if the resolution of the image seems to matter and (b) fluorophore quenching from multiple rounds of imaging. ===DMD-488 and Cot1-546=== Note that all thresholds are set the same in this series as well. They are {min,max}: Bright Field {50,200}; 488 {40,60}; 546 {20,80}. <gallery widths=500px heights=500px perrow=3 caption="Image series for DMD-488 (green) and Cot1-546 (red)"> File:20140923_DMD488_Cot1546.lif_Series_2048pxSnapshot.jpg|2048 pixels (1) File:20140923_DMD488_Cot1546.lif_Series_2048px_2Snapshot.jpg|2048 pixels (2) File:20140923_DMD488_Cot1546.lif_Series_1024px_3Snapshot.jpg|1024 pixels (3) File:20140923_DMD488_Cot1546.lif_Series_512x_4Snapshot.jpg|512 pixels (4) File:20140923_DMD488_Cot1546.lif_Series_256px_5Snapshot.jpg|256 pixels (5) File:20140923_DMD488_Cot1546.lif_Series_2048px_6Snapshot.jpg|2048 pixels (6) </gallery> ====Comparison==== The following gallery is the comparison between the 2048 pixel images, 1,2 and 6, showing the degradation between the image series. The '''Difference''' method subtracts the two pixel images (image 2 or 6 subtracted from image 1) and the '''Minimum''' method chooses the lower of the two pixel values and displays that in the comparison. Using the difference method, spots that are those that are different between the two images. Using the minimum method, pixels that show up are those that are the same. All images were thresholded (as above) and viewed as maximum projection prior to image comparison. <gallery widths=600px heights=600px perrow=2 caption="Image comparisons for 2048 pixel images, DMD-488 (green) and Cot1-546 (red)" > File:20140923_DMD488_Cot1546.lif_Series_2048px_1-2Combined_DifferenceSnapshot1.jpg|1-2 Comparison: Difference File:20140923_DMD488_Cot1546.lif_Series_2048px_1-2Combined_MinimumSnapshot1.jpg|1-2 Comparison: Minimum File:20140923_DMD488_Cot1546.lif_Series_2048px_1-6Combined_DifferenceSnapshot1.jpg|1-6 Comparison: Difference File:20140923_DMD488_Cot1546.lif_Series_2048px_1-6Combined_MinimumSnapshot1.jpg|1-6 Comparison: Minimum </gallery> ===Padlock Probes-488 Cot1-546=== Note that all thresholds are set the same in this series as well. They are {min,max}: Bright Field {50,200}; 488 {40,70}; 546 {40,70}. <gallery widths=500px heights=500px perrow=3 caption="Image series for PPRL-488 and Cot1-546"> File:20140923_PPRL488_Cot1546.lif_Series_2048px_1Snapshot_All1.jpg|2048 pixels (1) File:20140923_PPRL488_Cot1546.lif_Series_2048px_2Snapshot_All1.jpg|2048 pixels (2) File:20140923_PPRL488_Cot1546.lif_Series_1024px_3Snapshot_All1.jpg|1024 pixels (3) File:20140923_PPRL488_Cot1546.lif_Series_512px_4Snapshot_All1.jpg|512 pixels (4) File:20140923_PPRL488_Cot1546.lif_Series_256px_5Snapshot_All1.jpg|256 pixels (5) File:20140923_PPRL488_Cot1546.lif_Series_2048px_6Snapshot_All2.jpg|2048 pixels (6) </gallery> ====Comparison==== The following gallery is the comparison between the 2048 pixel images, 1,2 and 6, showing the degradation between the image series. The '''Difference''' method subtracts the two pixel images (image 2 or 6 subtracted from image 1) and the '''Minimum''' method chooses the lower of the two pixel values and displays that in the comparison. Using the difference method, spots that are those that are different between the two images. Using the minimum method, pixels that show up are those that are the same. All images were thresholded (as above) and viewed as maximum projection prior to image comparison. <gallery widths=600px heights=600px perrow=2 caption="Image comparisons for 2048 pixel images, DMD-488 (green) and Cot1-546 (red)" > File:20140923_PPRL488_Cot1546.lif_Series_2048px_1-2Combined_DifferenceSnapshot_All1.jpg|1-2 Comparison: Difference File:20140923_PPRL488_Cot1546.lif_Series_2048px_1-2Combined_MinimumSnapshot1.jpg|1-2 Comparison: Minimum File:20140923_PPRL488_Cot1546.lif_Series_2048px_1-6Combined_DifferenceSnapshot1.jpg|1-6 Comparison: Difference File:20140923_PPRL488_Cot1546.lif_Series_2048px_1-6Combined_MinimumSnapshot1.jpg|1-6 Comparison: Minimum </gallery> [[Category:HRCP]] [[Category:Microscopy]] [[Category:20140919]]
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