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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 STED CW
The Fast Track to Superresolution
- 獨家設計, 創新領先
引領"超解析"螢光顯微鏡技術的領航者
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Observe
what’s inside – with confocal superresolution
Nanoscale
imaging – devoid of mathematical artifacts
Upgrading,
to STED – quick and affordable
Watch
live cell dynamics – at the nanoscale!
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甚麼是傳統光學解晰的繞射理論 ? 解晰的極限在那裡 ? |
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顯微鏡 ( Far-field microscopy )
的點光源會在焦平面 (focal plane ) 上產生一個模糊的繞射光斑(airy
disc)，此光斑的大小可以用其半波寬
(Full-Width at
Half-Maximum，FWHM)
來表示. |
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根據瑞利/阿貝
(Abbe) 原理,
光學顯微鏡的解析度 (resolution )定義為用顯微鏡可以分辨出來的兩個同等亮度的點光源之間的最小距離.
由於光的波動特性會造成干涉與繞射效應。所以在傳統光學顯微鏡中，僅能獲得約二分之一個量測波長的空間解析度，這稱為光學繞射極限 |
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而此解析度的極限是由光的繞射特性所決定的 ( diffraction
limitation ). 依照Rayleigh
定義,
兩物體的距離必須大於或等於（1.22λ／2n
sinθ）才能清楚地分辨出來，其中 λ
是所使用的光波長，n
是所在的光學介質折射率，θ
是用來收集或聚光至感測器 (物鏡)所用的物鏡光孔穴的半角。( 物鏡 NA = n x
sinθ , 光學解晰
Resolution = λ / 2
x NA ) )傳統遠場光學顯微鏡術的解析度受限於光的阿貝/
瑞利極限，理論上, 不能分辨出200
nm
以下的結構.
所以,
當我們要研究細胞內精確定位特定的蛋白質 (或, 微小胞器 ) 以研究其位置與功能的關係時,
就面臨分辨力不足的困擾了 .... |
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為了提高光學解析, 我們可以提高光學物鏡的
NA, 改變使用較短波長的光源, 提高介質的折射率.. 等, 或許,
可以在遠場光學中得到分辨力的改善.
然而, 通過改造光源的點擴散函數 (PSF) 來提高成像解析度的方法，其中之一的技術,
受激發射損耗顯微技術 ( STED
) 已被開發突破成為一種極具效益的光學成像技術與成像系統., 不僅已可使用於於固定的細胞樣品,
也可使用於活細胞成像. |
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甚麼是 STED 技術
? ( 受激放射耗乏顯微鏡技術 ) 為何 STED 是突破阿貝繞射理論的極限 ? |
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Resolution enhancement in STED microscopy
requires two different lasers. One for fl uorophore
excitation (CW STED: Argon-gas laser with 488 & 514 nm)
and one red shifted laser (CW: 592 nm fi ber laser) to
annihilate excitation by stimulated emission. This applies
for pulsed STED (red and green lines in the drawing) but
also for STED with continuous wave lasers (red and green
faint solid areas). Both laser beams are focused through
the objective onto the sample and moved, perfectly
aligned, by scanning mirrors (beam scanning). The
intensity distribution of the STED beam features a ring
shape with zero intensity in the center. Thus, no
excitation annihilation occurs in the inside of the STED
doughnut. This ring shape is generated by a highly effi
cient helical vortex phase fi lter so that fl uorescence
spot is minimized. |
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一般光學顯微系統的空間解析度 ( 光學顯微鏡的鑑別率極限 ) 最多只能達到約 50%
的 (FWHM) 波福的寬 (
約 0.61λ
/ NA 來估計 ). 所以, 要提高解析,
就是要突破光學繞射極限.. 在近場光學顯微技術 ( Near-Field microscopy ), 即是通過縮小光點的直徑
( < 50 nm ), 然後在樣本表面上, 距離約數奈米的情況下, 做移動掃瞄 ( scanning
) ，即可利用每一光點產生的訊號, 通過數學運算轉換拼成一張完整的焦平面顯微影像。此即是近場光學掃瞄顯微術的概念,
例如 near-field scanning microscope, AFM. 此種影像解析,
超過了阿貝光學繞射極限.
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突破光學繞射極限的途徑, 在 遠場光學顯微技術
( Far-Field Microscopy ), 是由德國
Stefan Hell
教授所發展的
受激放射耗乏顯微鏡技術
STED (stimulated emission depletion) 最有利於螢光成像.
我們已知道, 當螢光分子的間距, 近到小於繞射極限時，我們就會無法分辨個別分子位置.
即使在目前的共軛焦顯微鏡上, 也無法有效分辨兼具極微小的螢光分子. 所以, 為了提高分辨力, 必須把聚焦光點 (
Excitation - focus point ) 縮小，同時, 也必須讓發出螢光 ( Emission ) 的範圍縮小. 如何做 ???
簡單的說法, 即是,
靠受激放光的機制來抑制周邊螢光分子的發光能力. 當有兩個螢光分子 A
和
B
靠的非常近，一般的聚焦光點因為直徑太大, 會同時激發這兩個分子的螢光，造成難以分辨的模糊影像. 然而,
利用將鄰近焦點的螢光分子的發光能力暫時抑制掉, 讓其產生激發時卻又強迫旁邊的螢光 (B)不發光，只有焦點下的螢光分子
(A) 可以發光 (
產生釋放光譜 Emission )。利用此法交互運用, 通過掃描, 即可獲得突破阿貝解析極限,
依照使用激發雷射光源的不同, 分辨力可為infrared
STED: < 90nm FWHM, visible STED < 70nm FWHM,
TREX-STED < 25nm FWHM.
實驗顯示解析已達
XZ < 25 nm, XY < 15 nm.
(
視過飽和 STED 光場強度大小而定 )
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The involved photophysical processes are
confi ned to different areas of the STED scanning spot.
The conventional excitation of the fl uorophores that is
followed by spontaneous emission of photons with different
energies (= wavelength) dominates inside the ring, where
the STED intensity is close to zero. The STED laser
depopulates the excited electronic state S1 by inducing
stimulated emission in the periphery. The released photons
are indistinguishable from the STED laser photons and
spectrally fi ltered out. The process is not related to
bleaching and can be repeated many thousand times. |
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STED 螢光顯微鏡技術圖示 |
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STED
利用受激放光的機制來抑制周邊螢光分子的發光能力, 藉由調整
STED
光場強度，便可以任意地調整空間上發出螢光的範圍，因而使光學影像解析度不再受繞射極限所限制 |
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Leica 兩種技術 ( STED depletion
laser ) |
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All electronics and optics for operating
the 592 nm STED depletion laser and maximizing the
incoupling effi ciency are integrated into a stable and
compact rack. |
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The effective fluorescent area (green)
decreases with increasing depletion laser power (red). |
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Normalized excitation and emission
spectrum of ATTO 647N. For STED excitation and depletion
wavelength are at 640 and 750 nm respectively. The
depletion wavelength can be tuned. |
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The Abbe equation decribes the
achievable optical resolution. Stefan Hell extended this
equation by a –superresolution – term, breaking Abbe’s
diffraction barrier. |
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Extended form of Abbes law of
diffraction. (d = distance; λ = wavelength,
NA = numerical aperture, Is
=saturation intensity of depletion laser,
i.e. the power that is necessary to halve the
population of the excited state (this value depends on
dye molecules and wavelength, I = maximum intensity of
depletion laser) The term implies a theoretical
infinite resolution for I → ∞. |
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共軛焦成像 與 STED 成像的對應比較 ( 亦是 光學解晰
的比較 ) |
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3-fold reduction of the scanning spot size
in x and y yields 9-fold more accurate sampling.
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STED 的成像範例
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Structural studies of the nervous system
have been identifi ed as one of the most promising fi elds
for superresolution microscopy. With the new Leica TCS
STED CW
investigation of the neuromuscular
junction with subdiffraction resolution has become
possible – not only in fi xed specimens (as shown above)
but also in 3D, 10 μm inside the living larvae, in
time lapse recordings. Immunofl uorescent staining of
neuromuscular junctions of a drosophila larvae.
Labels: Bruchpilot (Chromeo 488, red), Res (green, Cy3).
Courtesy of Stephan Sigrist and
Wernher Fouquet, Freie Universitaet Berlin, Germany.
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Time lapse
experiment: movement of large dense core vesicles
labeled with the fluorescent protein Venus inside of
living PC12 cells |
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 Actin
Fibers |
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Sample Courtesy of Elise
Stanley, Division of Genetics & Development, Toronto
Western Research Institute (TWRI), Canada |
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 Nuclear
Protein |
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Nuclear protein in HeLa cells.
Marker: Alexa 488 |
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 Vimentin |
Vimentin in HeLa cells.
Marker: Chromeo 488.
Sample courtesy of Max Planck Institute for Biophysical
Chemistry, Dept. Nanobiophotonics, Goettingen, Germany (
Left : confocal, right : STED ) |
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Fluorescent Dyes for CW STED:
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Chromeo 488
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Alexa 488
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FITC
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Oregon Green
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ATTO 488
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and many more
Usable Fluorescent Proteins for the New
TCS STED CW:
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STED
可帶動那些重大的科研應用 ? |
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從 Leica 與 Hell 教授團隊的合作, 將 STED
技術演進在 TCS SP5 II 的平台上, 通過簡易的操作介面, 加上螢光染劑的演進, 科研人員已可輕易的使用
STED 技術, 探索微小的螢光分子的動態反應, 也可進行活細胞的實驗. 此一突破傳統解析的光學技術,
勢必引領風騷, 帶動許多突破性的重大實驗結果. 也可起超解析顯微鏡技術的新的一頁. |
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Leica
TCS STED 的系統, 是包括那些設備 ? |
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STED Module 基本組配
(
請洽本公司共軛焦影像團隊 ) |
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Mechanics |
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ultra stable
and compact device, fi rmly fi xed to scanner |
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STED Lasers |
excitation |
internal
Argon gas lasers (continuous wave), variable
excitation wavelength |
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depletion |
592 nm
visible fi ber laser (continuous wave) |
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Optomechanics |
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used imaging port UV-port (no UV
available) modulation of depletion PSF, automated beam
adjustment for perfect alignment of excitation and
depletion laser, average duration: < 1 min., alignment
inside the scanhead, no illumination by lasers of the
sample during alignment |
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Microscope |
inverted |
Leica DMI6000
CS Trino/Bino |
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STED
depletion |
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VFL depletion
1.5 W |
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Optics |
number of
laser ports for imaging |
3 (STED, VIS,
IR) |
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Confocal scanner |
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Leica TCS SP5 II |
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 Resolution
depends on:
intensity of depletion light
quality of central depletion minimum
NO FUNDAMENTAL RESOLUTION LIMIT!!!
infrared
STED: 90nm FWHM
visible
STED: 70nm FWHM
TREX- STED: 25nm FWHM
 High
energy depletion pulses needed
 Special
(pulsed) excitation and depletion lasers needed
 Fluorescence
dyes must perform efficient depletion at high
photostability => selected „STED dyes“
 Low
signal + high sampling => rel. slow image aquisition
 STED
is fully compatible to standard fluorescence technics
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Leica TCS STED 簡潔操作介面 |
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The Confocal and the STED tab: In scanning
mode two different settings of acquisition parameters can
be easily accessed by toggling between the STED and the
Confocal tab. |
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STED CW Features
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Flexible
STED-excitation: Ar Laser (488 & 514 nm) STED: Fiber
laser 592 nm; intensity modulated by AOTF
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XY-resolution
(FWHM) < 80 nm (measured on Chromeo 488 nano-beads),
depending on sample, embedding and staining
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Integrated
linear deconvolution
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Z-resolution: confocal
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Auto beam
alignment of excitation and STED beam for long term
stability
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Vortex phase
fi lter for maximum resolution
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Available in
combination with AOBS and dichroic systems
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Simultaneous
line sequential recording of STED and confocal possible
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Recording
speeds of > 20 frames per seconds with < 80 nm lateral
resolution
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Full range
of SP5 features supported, exclusive 405/UV
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STED
技術相關參考文獻 |
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1. Abbe E. (1873) Beitraege zur Theorie
des Mikroskops und der mikroskopischen Wahrnehmung. Arch.
f. Mikr. Anat., 9:413–420.
2. Hell S.W. and J. Wichmann. (1994)
Breaking the diffraction resolution limit by stimulated
emission depletion microscopy. Opt. Lett., 19(11):780–782.
3. Hell S.W. (2003) Toward fl uorescence
nanoscopy. Nature Biotechnol., 21(11):1347–1355.
4. Westphal V. and S.W. Hell.(2005)
Nanoscale resolution in the focal plane of an optical
microscope. Phys. Rev. Lett., 94:143903.
5. Sieber JJ et al. (2006) The snare-motif
is essential for syntaxin-clustering in the plasmamembrane.
Biophys J. Apr 15;90(8):2843-51.
6. Klar T.A. and S.W. Hell. (1999)
Subdiffraction resolution in far-fi eld fl uorescence
microscopy.Opt. Lett., 24(14):954–956.
7. Dyba M, S. Jakobs, and S.W. Hell.
(2003) Immunofl uorescence stimulated emission depletion
microscopy. Nature Biotechnol., 21(11):1303 – 1304.
8. Westphal V et al. (2008) Video-rate
far-fi eld optical nanoscopy dissects synaptic vesicle
movement. Science. Apr 11;320(5873):246-9.
9. Kittel T. et al. (2006) Bruchpilot
promotes active zone assembly, Ca2+ channel clustering,
and vesicle release. Science. May 19;312(5776):1051-4.
10. Willig KI et al. (2006) STED
microscopy reveals that synaptotagmin remains clustered
after synaptic vesicle exocytosis. Nature. Apr
13;440(7086):935-9.
11. Geumann U. et. al. (2008 ) SNARE
Function Is Not Involved in Early Endosome Docking. Mol
Biol Cell. Oct 8.[Epub ahead of printing]
12. Sieber JJ Science 2007 Anatomy and
dynamics of a supramolecular membrane protein cluster.
Science. 2007 Aug 24;317(5841):1072-6
13. Hell, S.W., M. Dyba, and S. Jakobs,
Concepts for nanoscale resolution in fluorescence
microscopy. Curr Opin Neurobiol, 2004. 14(5):p. 599-609.
14. Fouquet, W., et al., Maturation of
active zone assembly by Drosophila Bruchpilot. J Cell Biol,
2009. 186(1): p. 129-45.
15. Kittel, R.J., et al., Bruchpilot
promotes active zone assembly, Ca2+ channel clustering,
and vesicle release. Science, 2006. 312(5776): p. 1051-4.
16. Ding, J.B., K.T. Takasaki, and B.L.
Sabatini, Supraresolution imaging in brain slices using
stimulated-emission depletion two-photon laser scanning
microscopy. Neuron, 2009. 63(4): p. 429-37.
17. Nagerl, U.V., et al., Live-cell
imaging of dendritic spines by STED microscopy. Proc Natl
Acad Sci U S A, 2008. 105(48): p. 18982-7.
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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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