Hosuk:LabNotes/2013-10-29: Difference between revisions
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>Hosuki78 (Created page with "*LabNote ===Stage adapter detachment and reinstall test=== *Use 10/22 sample, which was used for the experiment yesterday ([[Hosuk:LabNotes/2013-10-28|10...") |
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*There was no offset at other direction. | *There was no offset at other direction. | ||
*Composite image, Left:Before, Right:After (Blue : 1st rolony, Red : ACTB, Green : RAB7A) | *Composite image, Left:Before, Right:After (Blue : 1st rolony, Red : ACTB, Green : RAB7A) | ||
[[File:DetachTest_Before(left)_After(Right)_Red(AACTB)_Green(RAB7A)_Blue(1stRolony).png| | [[File:DetachTest_Before(left)_After(Right)_Red(AACTB)_Green(RAB7A)_Blue(1stRolony).png|800px]] | ||
[[File:DetachTest_Zoonin.png| | [[File:DetachTest_Zoonin.png|500px]] | ||
===Adding ACTB probe only=== | |||
*Image the sample with 3 dye probes | |||
*Strip, and image again | |||
*Add ATTO488, ACTB probe only, imaging | |||
====Result==== | |||
*ACTB were found more than before… WHY? | |||
**This is maybe because those dye compete each other while annealing. Or one dye lost it's intensity because some percentage(it'll be very little though) of the dye's emission energy is used for excite another dye, so Cy5 signal is always brighter than other two dye. | |||
{| {{table}} | |||
| ATTO488 channel after adding 3 dye-labeled probes ([[Media:S2_20131022_3Dye_DetachTest_2After_ACTB.tif|Raw]]) | |||
|| | |||
| ATTO488 channel after stripping ([[Media:S2_20131022_Add1ProbeOnlyTest_1Strip_ACTB.tif|Raw]]) | |||
|| | |||
| ATTO488 channel after adding ATTO488 probe only ([[Media:S2_20131022_Add1ProbeOnlyTest_2AddACTBOnly_ACTB.tif|Raw]]) | |||
|- | |||
| [[File:S2_20131022_3Dye_DetachTest_2After_ACTB.png|450px]] | |||
|| | |||
| [[File:S2_20131022_Add1ProbeOnlyTest_1Strip_ACTB.png|450px]] | |||
|| | |||
| [[File:S2_20131022_Add1ProbeOnlyTest_2AddACTBOnly_ACTB.png|450px]] | |||
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|} |
Latest revision as of 21:55, 29 October 2013
Stage adapter detachment and reinstall test[edit]
- Use 10/22 sample, which was used for the experiment yesterday (10/28)
Procedure[edit]
- Image – 3 channel along Z stacking
- Save location (Mark and find function)
- Lower objective, detach stage adapter, move stage to left side far from original position
- Reinstall adapter
- Move stage back to saved position, and image
Result[edit]
- 7 pixel, 281.25nm x 7 = 1.96875um off to upper direction.
- There was no offset at other direction.
- Composite image, Left:Before, Right:After (Blue : 1st rolony, Red : ACTB, Green : RAB7A)
File:DetachTest Before(left) After(Right) Red(AACTB) Green(RAB7A) Blue(1stRolony).png File:DetachTest Zoonin.png
Adding ACTB probe only[edit]
- Image the sample with 3 dye probes
- Strip, and image again
- Add ATTO488, ACTB probe only, imaging
Result[edit]
- ACTB were found more than before… WHY?
- This is maybe because those dye compete each other while annealing. Or one dye lost it's intensity because some percentage(it'll be very little though) of the dye's emission energy is used for excite another dye, so Cy5 signal is always brighter than other two dye.
ATTO488 channel after adding 3 dye-labeled probes (Raw) | ATTO488 channel after stripping (Raw) | ATTO488 channel after adding ATTO488 probe only (Raw) | |||
File:S2 20131022 3Dye DetachTest 2After ACTB.png | File:S2 20131022 Add1ProbeOnlyTest 1Strip ACTB.png | File:S2 20131022 Add1ProbeOnlyTest 2AddACTBOnly ACTB.png |