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1 !" L } (NIH) o/Mac NIH Image ! Scicon Image (Win ) ImageJ (Mac OS, Win OS) TIFF, GIF, JPEG pl " , !" $ !" aw !"J AVA 8 OS ? !" !"HP !" PDF !" !"HP !"HP ~ !" !" #p01 ImageJ (Image Processing and Analysis in Java) !" " 7e !" 9 !" !" > !" " ^ !" IJ ^ !" ^ !" Z !" j ij 8 %nj ^ !" 8 l , !"8 | !" D D D (3D+ ) " P !"Tool Bar ( !" !" " ^ : !" B yQ - !" j ^ Analyze!Measure !" shift W !" J . D . !" [ & !" U !" !" - S Photoshop D l !)]

2 " Progress bar ", _ " !ImageJ RGB " " !"RGB " Red, Green, Blue 8 bit !" " " !" bit (0~255) l X 256 Binary !" 6 bit (0~65,535)d l X 64 K Binary !"32 bit (0~4,294,967,296)d X 4G Binary !" 9 Binary RC pl L ", _ !" " !File!Open Smaples!Blobs !" 8 bit " 255 aw !" aw l Lookup Table (LUT) !ImageJ W LUT z !Image Lookup Tables !Image Lookup Tables Red !Image Lookup Tables Green !Image Lookup Tables Blue ", _ " 7e !"Process Menu " 7e !File!Open Smaples!Blobs !Process Smooth: " !Process Sharpen: " Smooth Sharpen I Smooth Sharpen 9 s yQ n !

3 Process Find Edges: - n !"Enhance Contrast: n !File Open Samples Enhance Me !Process Enhance Contrast Saturated Pixel 0 OK ", _ ; | 8 !"$ Process Smooth Process Sharpen , , { !" 8 !Plugins Macros !" E3 !" Recorder G !" s Create l l l !" !File Save As E3 .text \ !Plugins Macros RC !Plugins Macros $ Save ^ OK !" D I !" d " D , !" , 1 ", _ " Cs Merge 1 !" " !File!Open Smaples!Fluorescent Cells !" " yQ Green, Red, Blue .4 !}

4 Image!Color!RGB Split _ _ " 0 q z 8 " yQ " ^ % % q " ", _ Merge 2 !" " Cs !Image!Coloer!RGB Redd , Gree:, Blue: B M E yQ " & yQ "Keep source images & # " "OK " C| "$ " ' & s ", _ %nj 9 !File!Open Smaples!M51 Galaxy !" " !Image!Adjust!Brightness/Contrast !"Auto !"%nj D # | !Analyze!Surface !"Draw Wireframe !"Shade !"Draw Axis !"OK ", _ !" " !File!Open Smaples!Lena n < + " C 5 !" " yQ Green, Red, Blue .4 !Image!Color!

5 RGB Split !" " !Image!Adjust!Brightness/Contrast !"Auto !" " Cs !Image!Coloer!RGB .h N " _ IJ ^ !" " !File!Open Smaples!Leaf !" Gray Scale U !"Image!Type!8-bit !" ^ 50 mm . m !"m 50 mm ^ !Analyze!Set Scale Distance in Pixels aw Known Distance l n50 &6 Unit of Length nmm &6 Global Check & ' ^ ^ !" D m !" m !Analyze!Measure Length _ ^ !" & !" " z R} l m "Analyze Tools Scale "Width in mm z 20 & 20 mm " K v _ ^ !" " 9 !Process!Binary!Make Binary !" n . ^ !Analyze!Analyze Particles Show Outlines ^ Display Results Check OK l !

6 " ^ ' O A ^ !" o A 2 Area 2,000 mm2 _ ^ 3 !" ' ^ !Image!Adjust!Threshold !" 8 8 D " U9 ^ !" . !Analyze!Analyze Particles !" ^ ' O A ^ !" A 62 249 l mm2 _ Z. !" Z ^ !File!Open Smaples!Dot Blot !Image!Adjust!Threshold !" + Shift ' ^ !Analyze!Analyze Particles !" ^ ^ A 7 ^ !" Z ' a _ %]`j !"Kodak 21 %]`j OD &~ !"&~ " H Mean Gray Value ^ !" / Background ^ 18 ^ ' 19 t L . T s !Image!Adjust!Brightness!Contrast ^ _ %]`j !

7 Analyze! @ K & !"Function l Rodhard ^ !"Unit &6 !"@! &6 Save ! j !Gloval Calibration l Check & OK !" - \ _ ^ !" " !File!Open Smaples!Gel !" m !Analyze!Plot Profile !"$ ^ IJ yQ !" IJ ^ ^ ? !Analyze!Measure !" . + q " % !" % > LJ _ ( > = ) !" ImageJ $ % > !Process Subtract Background !" " Dot Blot . OD Plot Profile @ K !" Profile i 9 !"i 9 t . # Profile m !" K _ 1) !" " !

8 File!Open Smaples!Gel !" > !Process!Subtract !"Rolling Ball Radius ^ j !" RC Light Background Check & L RC Check V !" p J !Analyze!Gel!Select First Lane !" p Lane 8 !Analyze!Gel!Select Next Lane !" p Lane 8 ^ _ ( l ) !"B !Analyze!Gels!Plot Lanes !" m O !"B u B ^ !" ' a !Analyze!Gels!Label Peaks ^ percent s _ ^ ^ 1 !Analyze!Measure l Analyze!Particles ^ ^ ^ !Analyze!Set Measurements !"Aread O !"Mean Gray Valued ) iN Analyze!Calibrate " ; ( ) !

9 "Standard Deviation ( g)d g !"Modal Gray Valued ) !"Min & Max Gray Valued ) X b !"Centroid u d ) uk !"Center of Mass ( u)d ) ' XY j iN !"Perimeter FJ d VFT !"Bounding Rectangle d ) b p !"Fit Ellipse ( *)d * _ ^ ^l 2 !"Circularity *pj d D "* r2 * !Feret s Diameter r d J) IJ !"Integrated Density `j d " ) C !"Median Y d " ) Y !Skewness j d .h a x !"Kurtosis cj d .h !"Area Fraction d l Image > Adjust > Threshold . !"Limit to Threshold 2 d o ^ Image > Adjust > Threshold 2 !"Display label d "E A " l Result table / _ ^ ^ 3 !"Invert Y Coordinates Y d XY= " f = s @ !

10 "Redirect To d " Measure ^ Analyze Particle Z s a Redirect To " ? " O ^ !"Decimal Places b d result _ b @!