Confocal Microscopy and Image Processing

Technique developed by: Debanjan Das

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

Flow Chart: Culture & Staining of CHO cells

CellsDMSO (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 PBSBlocking 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

CHO Cells – Confocal Microscopy

1. Raw Image 2. Red Channel (F Actin) 3. Green Channel (Tubulin)

4. Z Project 5. Merged & Processed 6. Control

Processed, Magnified Image in Merged Channels – Normal CHO cell

Green Stains ~

Tubulin

Red Stains ~

F-Actin

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

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

Quantification of F – Actin in normal CHO and Nocodazole treated cells

Normal CHO ~ F-Actin Nocodazole treated CHO ~ F Actin

Quantification of tubulin in normal CHO and Nocodazole treated cells

Normal CHO ~ Tubulin Nocodazole treated CHO ~ tubulin

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 cellRed3 > #5> backgroundGreen3 > 6 thru 9 > Tubulin, Green channelGreen3 > 10 > backgroundRed4 > 11 thru 14 > Red Channel, F-Actin, NocodazoleRed4 > 15 > backgroundGreen4 > 16 thru 19 > Green Channel, tubulin,NocodazoleGreen4 > 20 > background

Unit of Area: grayscale/pixels

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.

Processing of Given Image Stacks – Exercise in Image J

Raw Image Processed Image

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

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.

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)

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

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

Zebrafish Embryo – Raw Images

Red Channel Green Channel

Zebrafish Embryo – Processed & Merged Images (selected slices)

Zebrafish Embryo – Processed & Merged Images (Z project)

Control

Tether CellsLateral Line System

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