Project Astounding Confocal Fred Martin BIOSC. 3  Dr. Giorgi 5/10/2010
Z-Stack Metadata: Specimen used :  Invitrogen Side #6 Muntjac Cells (part#F36925) <ul><li>Slide contains* muntjac skin fib...
Frame 001 Frame 004 Frame 007  Frame 010 Frame 013 Frame 016  Z-stack: total 19 slices showing every 3 rd  slice (StepSize...
Max projection of 3  channel, 19 slice z- stack Format- 512x512 StepSize 0.50  µm Zoom 1.7 Pinhole 67.9  µm   PMT 1 (Offs....
Orthoview of 3  channel, 19 slice z- stack
Frame 1 @ 0.33sec   Frame 2 @ 0.67sec   Frame 3 @ 1.00sec   Frame 4 @ 1.33sec   Frame 5 @ 1.67sec   Frame 6 @ 2.00sec   Fr...
Mark and Find Function: 3 random x,y,z coordinates identified & recaptured later Top row series: manual images before defi...
Two frames from Movie of 3D volume rendering of z-stack: rotating at -45deg y axis viewing angle Image 1: Frame 9 of 20  @...
Movie of 3D volume rendering of z-stack, rotating at - 45deg. y axis viewing angle
Original Separation 1 Separation   2 Dye Separation Series:  3 channels, Auto- separate,  weak  strength Original
Muntjac fibroblast slide display signal in all 3 channels at 40x oil, test image (unmounted slide to reboot & subsequently...
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Project SP5

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Leica SP5 Confocal

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Project SP5

  1. 1. Project Astounding Confocal Fred Martin BIOSC. 3 Dr. Giorgi 5/10/2010
  2. 2. Z-Stack Metadata: Specimen used : Invitrogen Side #6 Muntjac Cells (part#F36925) <ul><li>Slide contains* muntjac skin fibroblast cells stained with a combination </li></ul><ul><li>of fluorescent stains: </li></ul><ul><li>The prominent filamentous actin are labeled with Alexa Fluor® 488 phalloidin. Em –green </li></ul><ul><li>Mitochondria are labeled with an anti-OxPhos Complex V inhibitor protein mouse monoclonal </li></ul><ul><li>antibody in conjunction with Alexa Fluor 555 goat anti-mouse IgG. Em –orange </li></ul><ul><li>C) Nuclei are labeled with the far-red fluorescent TO-PRO®-3 nucleic acid stain.   Em- red </li></ul>* Source: Invitrogen website Sections 19 Line-Accumulation 1 Zoom 1.7 Format-Height 512 pixels Voxel-Height 446.1 nm Format-Width 512 pixels Voxel-Width 446.1 nm Channels 3 StepSize 0.50 µm Resolution 8 bits Size-Depth -9.0 µm Line-Average 1 Size-Height 227.9 µm Frame-Average 1 Size-Width 227.9 µm Frame-Accumulation 1 Pinhole [airy] 1.00 SequentialMode 0 Pinhole [m] 67.9 µm 981.2  PMT 3 (HV) 648.9  PMT 2 (HV) 505.8  PMT 1 (HV) -0.3  % PMT 3 (Offs.) -0.2  % PMT 2 (Offs.) -0.1  % PMT 1 (Offs.)
  3. 3. Frame 001 Frame 004 Frame 007 Frame 010 Frame 013 Frame 016 Z-stack: total 19 slices showing every 3 rd slice (StepSize 0.50 µm)
  4. 4. Max projection of 3 channel, 19 slice z- stack Format- 512x512 StepSize 0.50 µm Zoom 1.7 Pinhole 67.9 µm PMT 1 (Offs.) -0.1  % -0.2  % -0.3  % PMT 2 (Offs.) PMT 3 (Offs.) PMT 1 (gain) 505.8 648.9  981.2  PMT 2 (gain) PMT 3 (gain)
  5. 5. Orthoview of 3 channel, 19 slice z- stack
  6. 6. Frame 1 @ 0.33sec Frame 2 @ 0.67sec Frame 3 @ 1.00sec Frame 4 @ 1.33sec Frame 5 @ 1.67sec Frame 6 @ 2.00sec Frame 7 @ 2.33sec Frame 8 @ 2.67sec Frame 9 @ 3.00sec Frame 10 @ 3.33sec Frame | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | Lambda Scan from 550-610nm: Image Series above correlate to each wavelength/intensity reading in chart/graph to right Structure is fibroblast actin labeled with Alexa Fluor 488: weaker, post-peak EM signal
  7. 7. Mark and Find Function: 3 random x,y,z coordinates identified & recaptured later Top row series: manual images before defining xyz positions with “Mark” function Bottom row series: Auto images captured with “Find” function by recalled xyz positions
  8. 8. Two frames from Movie of 3D volume rendering of z-stack: rotating at -45deg y axis viewing angle Image 1: Frame 9 of 20 @ 2.5 sec of 5.0 sec Image 2: Frame 20 of 20 @ 5.0 sec of 5.0 sec
  9. 9. Movie of 3D volume rendering of z-stack, rotating at - 45deg. y axis viewing angle
  10. 10. Original Separation 1 Separation 2 Dye Separation Series: 3 channels, Auto- separate, weak strength Original
  11. 11. Muntjac fibroblast slide display signal in all 3 channels at 40x oil, test image (unmounted slide to reboot & subsequently could not locate this area of slide: all nuclei were bleached elsewhere

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