引領"超解析(Superresolution ) "螢光顯微鏡技術的領航者

在全世界, LEICA ( 德國 )  是獨家將超高光學解析顯微技術 ( STED, STED CW, 4Pi, CARS ... ) 導入商品系統化, 並推動一系列超過光學繞射理論極限的現代超高光學解晰顯微鏡技術 ( Superresolution Microscopy ).

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.

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, 皆可超過了阿貝光學繞射極限.

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.

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.

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.

3-fold reduction of the scanning spot size in x and y yields 9-fold more accurate sampling.

Presynaptic T-bar structure depicted by different imaging techniques. (A) NMJ labeled with two antibodies for BRP, either for the C-term (Nc82) or N-term. Because of its spherical structure of single subdivisions of the Drosophila NMJ (boutons), single synapses (see inlet) show a segregation of the BRPN-Term label towards the outside of the bouton. As in these regions the synaptic membrane lies perpendicular to the focal plane, a polarized orientation of BRP from the membrane to cytoplasm may be implicated. (B) Same structure now imaged with STED microscopy (green) and a confocal reference (red). In these images an architectural arrangement was described for BRP that depicts striking similarities to the T-bar observed in electron micrographs (C). Images taken with a Leica TCS STED.

Immunohistological co-staining of two antibodies which bind at different regions of the synaptic protein Bruchpilot (BRP). The increased resolution resulting from the STED technology (green, BRPC-Term) allows us to probe the spatial organisation of BRP at synapses. The overlay of the sequentially acquired confocal images (red, BRPN-Term) with the STED images clearly shows the higher resolution obtained by STED microscopy. Courtesy of S. Sigrist, Institute of Biology Freie Universitat Berlin, Germany

For the first time, STED brings light into darkness in the field of synaptic proteins. We recognise sub-structures of synapses and are able to localise proteins such as bruchpilot. Bruchpilot plays a key role in synaptic signal transmission in the nerve cells of the Drosophila fly by building up a specific Neuroscience 5 structure there for supporting signal transmission. If the Drosophila fly does not have much bruchpilot, it cannot sustain flight, if it has none at all, it dies. The protein is found in similar form in humans, too, and could be connected with diseases of the nervous system. Studying animals helps to understand  “Why are we interested in flies that turn into crash pilots?” the functions of the protein in humans. Understanding biological signal transmission is not only important for science in general. It is probable that synaptic defects trigger a large number of neurodegenerative diseases. In addition, it is almost certain that memory and learning processes are organised at synapses.

The synaptic protein Synaptotagmin 1 was labelled with a primary monoclonal mouse antibody and then with a secondary antibody carrying the fluorophore (Atto 647N). In STED mode, the synaptic vesicles can be clearly distinguished as individual points, which is not the case in confocal mode. Courtesy of S. Rizzoli, European Neuroscience Institute (ENI), Gottingen, Germany

Confocal (left ) and STED images (right ) of dentritic protrusions in cultured neurons. The inserts show the improved resolution with STED microscopy at the level of individual synapses. Green: Atto647-labelled GFP, red: synaptophysin. Courtesy of G. Wilczynski, Nencki Institute of Experimental Biology, Warsaw, Poland

 best choices for Leica TCS STED: ATTO 647N and ATTO 655

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

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.

CARS allows rapid and non-perturbative imaging of biological specimen with chemical selectivity. No specific staining is required. Visualization in CARS microscopy arises from the intrinsic vibrations of molecules only.

The result is an axial resolution of around 100 nanometers and an almost isotropic focal spot, ideal for 3D reconstructions with resolution beyond Abbe's limit. Key applications of the Leica TCS 4PI exist in a wide range of structural cell biology investigations and in the research of infectious diseases.

Based on the saturation of reversible single photon optical transitions in molecules, RESOLFT microscopy has the potential to provide resolution of tens of nanometres non-invasively in 3D structures