Techniques in Confocal Microscopy & Image Processing

1. Confocal Microscopy and Image
Processing
Technique developed by:
Debanjan Das

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. 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. 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. Processed, Magnified Image in Merged Channels –
Normal CHO cell
Green Stains ~
Tubulin
Red Stains ~
F-Actin

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. 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. Quantification of F – Actin in normal
CHO and Nocodazole treated cells
Normal CHO ~ F-Actin Nocodazole treated CHO ~ F Actin

9. Quantification of tubulin in normal CHO and
Nocodazole treated cells
Normal CHO ~ Tubulin Nocodazole treated CHO ~ tubulin

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. 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. Processing of Given Image Stacks – Exercise in
Image J
Raw Image Processed Image

13. Model Image of ZebraFish Embryo (3 day old)
Source:www.nimr.mrc.ac.uk/ devbiol/xu/zebra_image/

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. 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. 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. 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. Zebrafish Embryo – Raw Images
Red Channel Green Channel

19. Zebrafish Embryo – Processed & Merged Images
(selected slices)

20. Zebrafish Embryo – Processed & Merged Images (Z
project)
Control
Tether Cells
Lateral Line System

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