Techniques In Confocal Microscopy & Image Processing

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Techniques in Confocal Microscopy & Image Processing

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Techniques In Confocal Microscopy & Image Processing

  1. 1. Confocal Microscopy and Image Processing Technique developed by: Debanjan Das
  2. 2. Introduction  Culture, dilution and staining of Chinese Hamster Ovary (CHO) cells  Zebrafish embryo staining  Confocal microscopy and image processing(using Image J) of the above
  3. 3. Flow Chart: Culture & Staining of CHO cells Cells DMSO (3) Nocodazole (2) Fix 3.7% HCHO,15min20min,37°C 0.1% Triton X-100,5 min2 X PBSAllexa 568 Phalloidon (1:100), 30 mins 3 X PBS Blocking Sol,30 min 1° Ab (α tubulin 1:500), 30 min 3 X PBS 2° Ab (Alexa 488,1:100), n=4 No 2° Ab, n=1, DMSO 30min 3 X PBSStore in dark
  4. 4. CHO Cells – Confocal Microscopy 1. Raw Image 2. Red Channel (F Actin) 3. Green Channel (Tubulin) 4. Z Project 5. Merged & Processed 6. Control
  5. 5. Processed, Magnified Image in Merged Channels – Normal CHO cell Green Stains ~ Tubulin Red Stains ~ F-Actin
  6. 6. Nocodazole treated CHO cells - Confocal Microscopy 3. Green Channel (Tubulin)2. Red Channel (F Actin)1. Raw Image 4. Z Project 5. Merged & Processed 6. Control
  7. 7. Nocodazole treated CHO cells – Merged, Magnified & Processed Image Red Staining ~ F- Actin Green Staining ~ Tubulin Nocodazole typically depolymerizes microtubules; here, we can see tubular structure is destroyed (green staining) to a more homogenous, scattered tubulin dimers spread out in the cell cytoplasm
  8. 8. Quantification of F – Actin in normal CHO and Nocodazole treated cells Normal CHO ~ F-Actin Nocodazole treated CHO ~ F Actin
  9. 9. Quantification of tubulin in normal CHO and Nocodazole treated cells Normal CHO ~ Tubulin Nocodazole treated CHO ~ tubulin
  10. 10. Data Table # Label Area (pixels) Mean Integrated Density %Area 1 red3 24567 38.360 942383 96.410 2 red3 6699 38.976 261101 92.939 3 red3 3755 68.898 258713 95.632 4 red3 4028 78.927 317918 90.119 5 red3 5624 7.1123755E-4 4 0.036 6 green3 3821 102.828 392905 90.788 7 green3 24044 46.472 1117378 79.458 8 green3 6494 67.450 438023 93.902 9 green3 3527 120.500 425005 92.855 10 green3 5301 0.011 56.000 0.396 11 red4 21760 28.989 630793 97.854 12 red4 49250 25.675 1264517 96.904 13 red4 14344 14.403 206596 94.088 14 red4 16872 12.942 218363 93.706 15 red4 6600 0.313 2064 24.970 16 green4 20506 84.030 1723127 99.127 17 green4 46605 75.045 3497482 98.710 18 green4 16605 40.912 679348 93.285 19 green4 13845 37.702 521988 88.935 20 green4 10550 0.022 232 0.976 Key: Red3 > 1 thru 4 > F-Actin,Red channel, normal cell Red3 > #5> background Green3 > 6 thru 9 > Tubulin, Green channel Green3 > 10 > background Red4 > 11 thru 14 > Red Channel, F-Actin, Nocodazole Red4 > 15 > background Green4 > 16 thru 19 > Green Channel, tubulin, Nocodazole Green4 > 20 > background Unit of Area: grayscale/pixels
  11. 11. Quantification  Formula: Weighted Mean Intensity of 4 cells (grayscale/pixels) – Background Correction  F-Actin: Normal Cell > Mean Intensity = 56.29 Nocodazole treated cell > Mean Intensity = 20.189 % Decrease in area > 64% after drug treatment  Tubulin: Normal Cell > Mean Intensity = 84.30 Nocodazole treated cell > Mean Intensity = 59.40 % Decrease in area > 29.53 % after drug treatment  Conclusion: Nocodazole causes depolymerization in microtubules; hence, we noticed a 29.53 % decrease in area in tubulin content after drug treatment. Interestingly, percentage decrease of 64% was more significant in F-Actin then tubulin, proving that nocodazole not only affects microtubules, but also attacks F- Actin. This phenomenon also proves that tubulin and F-Actin are not too biologically different and made of moieties where effects of nocodazole can be easily observed.
  12. 12. Processing of Given Image Stacks – Exercise in Image J Raw Image Processed Image
  13. 13. Model Image of ZebraFish Embryo (3 day old) Source:www.nimr.mrc.ac.uk/ devbiol/xu/zebra_image/
  14. 14. Anatomy of a 72 hr old embryo (dorsal view) Source: zfin.org/zf_info/ anatomy.html Hair cells (tether cells) bind seeding particles, thereby localizing otolith formation. Tether cells usually occur in pairs at the anterior and posterior ends of the ear. tether cells initially appear immature and do not acquire the characteristics of mature hair cells until approximately 21.5 h.
  15. 15. Zebrafish Embryo Staining Methods - Flowchart Day 1: Gently remove Chorions From Embryo’s Anthesthetize Embryos (1/20 volume 20X Tricaine) Transfer To 20 ml Scintillation Vial Repeat steps 1 and 2 for all time points Move embryos to moving shaker PBS 0.1% Triton X-100 (2X 30min) Transfer embryos to 24 well dish PBT rinse (PBS + 1% Triton, 1hr on shaker)
  16. 16. Zebrafish Embryo Staining Methods – Flowchart (continued) PBT Rinse (PBS + 0.1% Triton X-100 2X 30 min) Blocking Solution (1 hr on shaker) Primary Antibody incubation (1:100 tubulin, 1:250 HCS) Incubate overnight (dark, in fridge) Day 2: PBT rinse (PBS + 0.1% Triton X-100 3X 30 min) ~ done by TA Secondary Antibody Incubation (Alexa 488 for tubulin 1:500, Alexa 568 for HCS-1 1:500 Store in dark at 4 degrees C, overnight
  17. 17. Zebrafish Embryo Staining Methods – Flowchart (continued) Day 3: PBT rinse (PBS + 0.1% Triton X-100 3X 30min) PBS rinse (1X 30 min) 70% glycerol in PBS on shaker (30 min) Remove egg yolk with sharp tweezers, under microscope Place drop VectaShield Mounting Medium Pick up single embryo, place in slides and place cover slips Stpre Coverslip in fridge, away from light
  18. 18. Zebrafish Embryo – Raw Images Red Channel Green Channel
  19. 19. Zebrafish Embryo – Processed & Merged Images (selected slices)
  20. 20. Zebrafish Embryo – Processed & Merged Images (Z project) Control Tether Cells Lateral Line System
  21. 21. Conclusion  Cells and embryos are successfully stained and photographed under confocal microscope (using 2D & 3D imagery)  Positive results were obtained when compared with controls  Images were effectively processed using Image J software

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