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了解 Leica 在
Super-Resolution 的解決方案 與 領導趨勢 .. |
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多一點了解 Leica
TCS SP5 的卓越, 與其他產品的差異性在哪裡 ? |
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Leica TCS SMD FCS
Precise analysis of particle transport and molecular
binding
引領"單分子偵測"的螢光顯微鏡技術進入細胞分子動力學研究與定量探索分析.
極具靈敏與精度的空間解析與時間解析, 成就了在極為微小的分子內外, 執行動態的單分子計數,
探索細胞內的粒子傳送與鍵結關係, 分子間的互動機制關係.
有助於分子擴散機制及細胞膜之流動性的探討.
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FCS
的技術原理
( Fluorescence Correlation Spectroscopy ) |
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Principle of FCS data acquisition and
analysis:
1. Laser illumination at a fixed
point of interest (beam-parking) excites fluorescent
particles in the excitation volume. The particle
movement in and out of the confocal volume causes
intensity fluctuations.
2. Registration of intensity
fluctuations using detectors in single photon counting
mode.
3. Calculation of the correlation
function.
4. Fitting of the corresponding
biophysical model to the correlation curve. Obtain
parameters of interest:
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particle concentration
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diffusion coefficient
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viscosity
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molecular mass
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binding constant
- photo-physical properties
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螢光分子間的相干光譜定量分析技術(
Fluorescence Correlation Spectroscopy, FCS )
隸屬非線性光學高解析三維影像技術,
是在多光子雷射掃描共軛焦顯微鏡下, 利用多光子飛秒雷射
( Ti-sapphire ) 在焦平面內產生極為微小的雷射焦點 (beam-park).
當螢光分子通過此雷射焦點時,
因為被激發產生釋放螢光光譜, 然後藉由極為靈敏的光子計數技術,
定量分析此焦點體積下的螢光分子的螢光強度變化,
因為螢光分子間的互動關係而產生的光譜變化量測,
即是所謂螢光相干分析 ( FCS )
技術。我們可用以探討分子間的動力學過程.
例如, 可以探討活細胞中不同區域內
EGFP 的流體運動.
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具有螢光染計標定的移動粒子,
當通過雷射焦點, 因為被激發而釋放出螢光光譜, 單分子螢光計數的強度變化及通過雷射焦點時間,
即是所謂的螢光相干量測, 可得到分子的擴散速率.
我們可通過雷射焦點的單位體積含有多少分子數目，以及利用通過雷射焦點的時間,
來求得分子的擴散速率.
因為螢光光譜變化的量測, 也可得到分子的擴散係數,
濃度, 以及分子間的相互作用關係.。利用此技術,
我們可以探討分子間的結合或鍵解機制,
了解分子間（蛋白質—蛋白質、蛋白質—核酸…等）的相互作用,
有助於了解細胞重大生命活動的發生和調節.也能深入瞭解疾病發生的分子機制，進而為尋找更有效的藥物分子、提高藥物篩選和藥物設計的效率提供新的思路。 |
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autocorrelation curve.
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Timescales of various processes
monitored by autocorrelation analysis
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FCCS
的技術原理
( Fluorescence
Cross-Correlation
Spectroscopy ) |
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FCCS 即是 FCS 技術的延伸. FCS
隸屬單一通道的量測技術, FCCS 則是雙通道量測技術, 言下之意, 即是可以利用兩個不同波長的雷射作為激發光源,
兩組偵測器測得兩組以上標定不同螢光染劑的分子的訊號. 藉以探索兩種分子的交互關係. ( 例如,
可同時標定 GFP 及 DsRed ) |
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FCS,
FCCS
的應用領域 |
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Analysis of
molecular binding in living cells with
FCCS
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FCCS facilitates the analysis of molecular binding.
Interactions are directly evident by the presence of a
cross-correlation between two spectrally distinct
channels. Furthermore, the ratio of free versus bound
fraction in equilibrium reactions can be quantified.
FCCS measurements are conducted with the
FCS wizard.
Example
Caspase6, a downstream effector of the TNF-dependent
apoptosis pathway, dimerizes upon activation (shown in the
graph). A GPF-labeled construct and an mCherry-labeled
construct were expressed in HeLa cells. 50% of the
resulting double-labeled dimers (grey box) are detected in
the cross-correlation signal.
FCCS thus proves the active form of Caspase.
We thank Prof. Dr. Roland Eils and Dr. Joel Beaudouin,
Bioquant, University of Heidelberg, Germany.
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Concentration mapping of TiF1a-GFP
in HeLa cells. The calibration of
APD image was performed with
FCS point measurement. ROIs for
FCS measurements are shown as black crosses. The
concentration was calculated from autocorrelation curves
(red and blue graphs). A spatial map of intracellular
concentrations is obtained. The correlation curves yield
information about the particle motility at the measured
points.
We thank Dr. Matthias Weiss, Dr. Jedrzej Szymanski, and
Nina Malchus,
DKFZ, Heidelberg, Gemany
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其他相關應用 |
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Concentration and aggregation measurements
Consideration of residence times: determining mobility and
molecular interactions
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Diffusion analysis
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Confined
and anomalous diffusion
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Active
transport phenomena in tubular structures
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Determination of molecular interactions
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Conformational changes
Consideration of cross-correlation amplitudes: a direct
way to monitor association/dissociation and enzyme
kinetics
Consideration of fast flickering: intramolecular dynamics
and probing of the microenvironment
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簡潔的使用流程,
讓工作簡化到任何人都可輕易的使用最精密的技術與應用. |
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FCS advantages
In vitro:
In vivo:
Complementary technologies available in
LAS AF
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FRET SE wizard
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FRET AB wizard
- FRAP wizard
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參考文獻 |
-
Wouters, F.S., Verveer, P.J., Bastiaens,
P.I.H. (2001) Imaging biochemistry inside cells. TiCB,
11:203
-
Kudryavtsev, V., Felekyan, S., Wozniak,
A.K., Konig, M., Sandhagen, C., Kuhnemuth, R., Seidel,
C.A.M., Oesterhelt, F. (2007) Monitoring dynamic systems
with multi parameter fluorescence imaging. Anal. Bioanal.
Chem. 387:1-82
Aragon, S. R.; Pecora, R.;
Biopolymers 14,
119 (1975)
Madge,
D., Elson, E., Webb, W. W. “Thermodynamic fl uctuations
in a reacting system – Measurement by fl uorescence
correlation spectroscopy” Phys. Rev.
Lett. 29:705-708 (1972)
Rigler, R., Widengren J.,
“Ultrasensitive detection of single molecules by
fluorescence correlation spectroscopy.”
Bioscience 3:180-183 (1990) http://www.biophysics.org/Portals/1/PDFs/Education/schwille.pdf
Gregor, I., Patra, D., Enderlein, J.
“Optical saturation in fluorescence correlation
spectroscopy under continuous-wave
and pulsed excitation” Chem. Phys. Chem.
6:164-170 (2005)
Ruttinger, S., Buschmann, V., Kramer,
B., Erdmann, R., Macdonald, R., Koberling, F.
“Comparison and accuracy of methods to determine the
confocal volume for quantitative fl uorescence
correlation spectroscopy”
J. Microsc. 232:343 (2008) http://www.picoquant.de/_scientific.htm
Tsien, R. Y. “ Constructing and
exploiting the fluorescent protein paintbox (Nobel
lecture) ”
Angew. Chem. Int. Ed. 48:5612-5626
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全世界唯一全光譜可調式的共軛焦顯微鏡系統,
結合 超高光學解析 與 超高速掃描 於一體. 結合最先進創新的光學解析提升技術, 創建出
Super-Resolution 的基礎平台. |
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相關資料 ( Information ) |
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雷射掃描共軛焦顯微鏡技術概論與應用介紹 ( CLSM
Technology & Application ) |
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Leica TCS 光譜式共軛焦顯微鏡的核心技術介紹
( AOTF, AOBS, Spectral Detector ) |
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Leica TCS 光譜式共軛焦顯微鏡的雷射光源 (
Laser system ) 介紹 |
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Leica TCS 光譜式共軛焦顯微鏡的物鏡 (
Objectives ) 介紹 |
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Leica TCS 光譜式共軛焦顯微鏡的應用軟體 (
Application Software ) 介紹 |
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使用於共軛焦顯微鏡上的活細胞培養設備 (
Micro-incubation ) |
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