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FRAP
- FLIP
- FLAP
Photo-activation
Caged
compounds
雷射掃描共軛焦顯微鏡應用
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| Fluorescence
Recovery After Photobleaching (FRAP) |
| 在分子的局部區域,
以能量充足的雷射光源集中激發局部區域,
讓其造成分子局部的螢光漂白.
再觀察螢光是否擴散回收
? 螢光回復機制
? 回收速率如何 ? 回收比率如何
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| 光漂白後的螢光回收率技術
- Fluorescence Recovery After
Photobleaching (FRAP) - 可用以判定一定時間內的螢光回復機制,
分子間動態反應,
分子間相互關係的化學變化,
利用此技術,
我們可以量化螢光回收比率,
分子擴散速率,
擴散機制. |
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Leica
FRAP 技術,
可以同時設定不同的局部區域,
以不同的雷射波長,
同時掃描.,
亦可指定漂白時序.
配合長時間及 3D
掃描,
您可觀察整個時區內的螢光回復機制,
分子動態反應機制及分子間的相互關係機制.
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HeLa
cell: nuclear protein, coexpression
with GFP
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進階可程式掃描
(
Advanced Time-Lapse ), 例如定義指定漂白局部
(
ROI ), Zoom-in 影像顯示,
指定 Bleaching
Point ( Beampark ), 時序定義,
雷射波長,
雷射強度...
等.
各種參數都可輕易的編輯設定.
使用人員無須有編寫軟體訓練,
即可簡易使用. |
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R.
.D.Phair
and T.Mistell 2000, nature
404, p 605-609
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| FRAP
應用範例 |
Protein
moving within cell nucleus -
FRAP
of nucleoplasmic regions:
images
taken before and during recovery after
bleaching. Indicated area is shown
enlarged in pseudocolor in the lower
panels.
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Bleaching:
A
defined area of a living
cell
is bleached by a single,
high-powered
spot laser
pulse
of 250ms (beampark)
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Recovery
of fluorescence in the bleached area
is
recorded by Sequential
imaging scans.
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It
is shown that proteins move
rapidly
throughout the nucleus
a)a)
GFP-HMG-17
b) GFP-SF2/ASF
c) GFP-fibrillarin
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Fluorescence
recovery after photobleaching ( 應用選讀
)
A)
Plot of fluorescence intensity in a region of
interest versus time after
photobleaching a fluorescent protein. The
prebleach (F i ) is compared with the asymptote
of the recovery (F 8) to calculate
the
mobile and immobile fractions. Information from
the recovery curve (from F o to F 8) can be used
to determine the diffusion
constant
of the fluorescent protein.
B)
Cells expressing VSVG–GFP were incubated at 40
ｰC to retain VSVG–GFP in the
endoplasmic
reticulum (ER) under control conditions (top
panel) or in the presence of tunicamycin (bottom
panel). Fluorescence recovery
after photobleaching (FRAP) revealed that VSVG–GFP
was highly mobile in ER membranes at 40 °C
but was immobilized
in the presence of tunicamycin.
Lippincott-Schwartz,
et al. - JUNE
2001 VOLUME
2
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| FLIP
: Fluorescence Loss In Photobleaching
光漂白過程中螢光損失的動態機制. |
··Fluorescence
in one area of the cell is repeatedly
bleached with high laser power while
images of the entire cell are collected
with low laser power.
Using
FLIP you
can measure the dynamics of 2D or 3D molecular
mobility e.g.
diffusion,
transport
or any other kind of movement of fluorescently
labeled molecules
in
living cells.
The time
course of fluorescence loss
is monitored here.
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Lippincott-Schwartz,
et al. - JUNE
2001 VOLUME
2 Science /
上圖 :
重複在一局部區域作光漂白
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Leica
LCS 軟體可在同一掃描中同時作
FRAP
及 FLIP. |
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| PA
: PhotoActivation ( 光活化
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PA is a
photo-induced alteration of the excitation or
emission spectrum of a fluorophore (e.g.
fluorescent proteins). Three different
proteins are described
•PA-GFP:
irradiation at ~ 400nm results in a 100x
increase in fluorescence when excited at
488nm (Patterson et al.,2002, Science, 297:
1873-77).
•KAEDE:
irradiation with UV or violet light converts
the protein to a red emitting protein (Ando
et al., 2002, PNAS, 99:12651-56).
•KFP1:
after irradiation with green light the Kindling
fluorescent Protein 1 alters
from non-fluorescent to a red fluorescent
state.
Excitation maximum ~ 580nm
//
emission maximum ~ 600 nm.
Intensity dependant, the conversion can be
reversible or irreversible. (Chudakov et
al., 2003, Nature biotechnology, 21: 191-94)
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| Caged
compound ( Un-caging ) |
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| Wide-Field
Microscopy 技術
-
Caged compound |
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Caged
Compounds - 德國 RAPP
公司的技術與系統設計
Examples: Ca2+
required in many cellular processes
ATP (Adenosine 5'-tri-phosphate)
energy
supplier of the cell
GTP (Guanosine 5'-triphosphate)
active
part in signal transduction processes
Solution:
Caged Compounds (Ca2+, ATP,
etc.)
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樣品的視野局部照明激發
(
Spot
illumination ) 應用:
螢光激發照明同時執行
:
整個樣品的全部視野照明
及 視野局部用不同光源或波長的雷射作螢光激發照明.
可應用於
flash photolysis of caged
compounds.
目前應用範例包括
:
Studies of cell
to cell communication - evaluation of
gap junctions. Release of
neurotransmitters or hormones.
Triggering of inter cellular calcium
waves.
選讀相關應用文獻
:
Presynaptic
capacitance measurements and Ca2+
uncaging reveal submillisecond
exocytosis kinetics and characterize
the Ca2+ sensitivity of vesicle pool
depletion at a fast CNS synapse.
Wolfel M, Schneggenburger R. J
Neurosci. 2003 Aug
6;23(18):7059-68.
Probing
the intracellular calcium sensitivity
of transmitter release during synaptic
facilitation. Felmy F, Neher E,
Schneggenburger R. Neuron. 2003 Mar
6;37(5):801-11.
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脈衝雷射(
RAPP pulsed UV Laser ) 雷射波長:
355 nm (Nd:YAG), 光纖傳導.
雷射脈衝功率高於
5
mJ, 脈衝時間
20 ns.
可應用於 Flash
Photolysis.
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| Leica
共軛焦系統提供最佳解決方法
:
FRAP, FLIP,
PA. Caged Compound ( Uncaging ). |
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