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Leica Intelligent
Structured Illumination Microscopy ( SIM )
智慧型 結構照明成像顯微鏡系統 |
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一個價格實惠低廉及設計簡易的壓電光柵格子 (Opti-Grid),
可簡單的安裝於顯微鏡螢光光路上, 可以提高光學解析及對比,
獲得類似共軛焦三維成像效果, 並作 3D 及光學切片成效. |
3D reconstruction of
a glomerulus (mouse kidney).
Maximum projection of a
stack containing 139 planes. With the software
package Leica MM AF a 3D reconstruction was
performed. The better contrast as
well as the better resolving power of small
structures is clearly visible after the use of
the OptiGrid. |
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| 圖示
: 安裝於 Leica DMI 6000 B 倒立顯微鏡 |
圖示 : 安裝於 Leica DM
6000 B 正立顯微鏡 |
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Eye anlage of E10 mouse embryo.
Maximum projection of 84 z-sections, 0.2 μm
z-distance, 40x/1.25. DNA: Hoechst 33342, beta-Tubulin:
AlexaFluorR488, AlexaFluorR633
Sample: Courtesy of Dr. Yves Lutz, Imaging Center
IGBMC, Strasbourg, France |
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Rat Brain; different neuronal cell types; 20x
air objective |
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Aspergillus nidulans; spore carriers; Courtesy
of Prof. Dr. Reinhard Fischer and Nicole
Zekert; KIT; Karlsruhe (63x Glycerol
Objective) |
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Leica 針對 SIM
螢光影像的光學解析 (resolution)及成像對比 (contrast),
提出了創新的光學設計. 利用特殊的 SIM-螢光照明光圈來提高解析,
利用結構照明光圈來提高對比. 是唯一的獨家設計. 同時採用特殊的 LED 405 濾鏡,
以避免紫外光光柵摺線的產生. ( 因為一般使用 365 nm 激發 - DAPI, 100x
OIL - 經常會看到影像的百葉型摺線) |
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Leica 智慧型 結構照明成像顯微鏡系統 核心技術 |
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SIM 是利用光柵格子（Opti-Grid）投射在樣品上的成像,
產生的位移運算組合, 得到高清析影像.
光柵格子會因為壓電效應產生精度極高的位移控制,
此類似百葉窗線條式的光柵格子會投射到標本上，以全幅影像的
1/3
作垂直於光柵格子的移動。一全幅光學影像是由瞬間獲取的三幅不同光柵格子影像組成的。第一幅光柵格子影像可在任意位置獲取，然後光柵格子線性地移動相當於1/3光柵格子條紋間隔的距離以獲取第二幅影像，最後再重複一次1/3的移動以獲取第三幅影像。以上這些運作都在瞬間完成，最後,
經由運算組合, 產生成一幅具有完整結構的光學影像或單層光學切片。
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Structured Illumination Diaphragm (SID)
- 獨家 SIM 專用光圈設計 |
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Leica SIM 系統, 採用 MetaMorph 驅動.,
應用功能包括 :
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– Multidimensional imaging
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– Image overlay
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– Colocalization
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– Morphometric measurements
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– Intensity quantifi cations
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– Time-lapse imaging
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– Morphological fi lters
- – Manual tracking
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Leica SIM
完整的系統 ( 架設於正立顯微鏡上 ) |
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Leica Intelligent Structured
Illumination – the Smart Solution
Resolution and contrast are
crucially important for fluorescence applications
in widefi eld microscopy. However, haze can be
aproblem when imaging thick specimens. Fluorescent
light from different planes in the specimen
reaches the focal plane, blurring the image and
reducing contrast. There are various options for
haze reduction, either confocal microscopy,
time-consuming deconvolution
– or the smart principle of
structured illumination. The result: excellent
contrast, superb axial resolution and ultra-sharp
2D sections of the specimen.
The principle
A grid structure inthe fi eld
diaphragm plane of the fluorescence axis is
sharply imaged in the object plane. At least three
raw images are captured, moving the grid structure
each time, and used by the patented OptiGrid
algorithm to calculate a final image.
The result
The clear image, haze-free and
with enhanced contrast, can be immediately viewed
on the PC monitor. Also, a sharply defined 3D
reconstruction of the object can be generated by
recording several z planes
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使用 SIM 顯微鏡必須注意事項 : |
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必須防振, 避免顯微鏡的震動. 可採用光學防振桌來克服此干擾. |
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避免樣品的移動. |
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穩定的激發光源, 可採直流式高壓燈源 或 LED 光源. 以維持螢光訊號的穩定. |
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樣品應避免快速螢光漂白. |
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當使用 UV 激發光時, 建議使用 405 nm 微激發光譜. 避免光柵格線的產生. |
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如有任何技術問題, 請洽本公司技術專員
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Leica
SIM 目錄下載 ................ |
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Deconvolution
影像處理軟體 |
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Deconvolution 影像處理軟體, 也可以得到令人激賞的清析影像. 本公司代理荷蘭 SVI 公司的
Huygens software 是廣為科研領域所使用. |
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A metaphase human cell
stained for DNA (red), centromeres (blue) and the
anaphase promoting complex/cyclosome (green).
Upper part: original data,
Lower part: deconvolved with Huygens Professional.
Recorded by Dr. Claire Acquaviva, Dr. Pines Lab.
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SFP Renderer.
This renderer is based on taking the 3D microscopy
image as a distribution of fluorescent material,
simulating what happens if the material is excited and
how the subsequently emitted light travels to the
observer. The computational work is done by the
Simulated Fluorescence Process (SFP)
algorithm. The unique properties of this algorithm
enable it to create depth cue rich images from
unprocessed data.
Because it does not rely on boundaries or sharp
gradients, it is eminently suited to render 3D
microscopic data sets. Since the SFP algorithm is
bases on ray-tracing it does not require a special
graphical board as the polygon based techniques do. |
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Measuring a line
profile in the Twin Slicer.
The
Twin Slicer allows you to synchronize views of two
images, measure distances, plot line profiles, etc. In
Basic Mode, image comparison is intuitive and easy,
while the Advanced Mode gives the user the freedom to
rotate the cutting plane to any arbitrary orientation,
link (synchronize) or unlink viewing parameters
between the two images, and more. |
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Surface Renderer.
The
Surface Renderer enables you to represent your
microscopy data in a convenient way to clearly see
separated volumes. It is not only capable of iso-surface
rendering; it is also able to show MIP projections
together with the surfaces to be used as a reference
to the original microscopic voxel data.
Because the Surface Renderer is based on rendering
continuous surfaces with fast ray tracing algorithms,
there is no need for any special graphic card. The
fast ray tracers can utilize 64 bit multiprocessor
systems, and are therefore able to render very large
microscopic volume data to high resolution output
images. |
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The Colocalization
Analyzer.
The
Colocalization Analyzer tool provides information
about the amount of spatial overlap between different
data channels, in 3D stacks or 3D time series. |
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The
Object Analyzer is a great tool to label
and analyze 3D and 4D single and multi-channel single
objects and their statistics. With the 3D region of
interest (ROI) selector tool you can limit the
analysis to a certain volume only, but also crop
your original data precisely like you want it for
further analysis. Next to analyzing single objects or
groups you can also analys the whole dataset in all
its aspects by clicking on one single button.
The Object
Analyzer.
The
Object Analyzer tool provides information about
objects in different channels and time points: it
reports physical properties, how objects relate
spatially to each other or to reference objects, and
how they overlap. |
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The
way Huygens works
The
Huygens Software
of
Scientific Volume Imaging
enables you to obtain a PSF in two ways:
In the
second case, given a model of the bead shape, the PSF is
computed 'distilled' which its convolution with the bead
model matches the measured bead image. That can be
understood looking back at figure 1 and equation 1. Now we
know how the object f is (the exact size of the
spherical bead must be known) and we have acquired its
image g, thus we can distill the remaining
unknown term h in the equation.
Once a
PSF is provided Huygens can use different mathematical
algorithms to effectively solve the convolution equation 4
and do deconvolution:
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Classic Maximum Likelihood Estimation
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Quick Maximum Likelihood Estimation
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Iterative Constrained Tikhonov-Miller
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Quick Tikhonov-Miller
The
Classic Maximum Likelihood
Estimation (CMLE) is the
most general
Restoration Method
available, valid for almost any kind of images. It is
based on the idea of iteratively optimizing the likelihood
of an estimate of the object given the measured image and
the PSF. The object estimate is in the form of a regular
3D image. The likelihood in this procedure is computed by
a
Quality Criterion
under the assumption that the
Photon Noise
is governed by Poisson statistics. (Photoelectrons
collected by a detector exhibit a
Poisson Distribution
and have a square root relationship between signal and
noise). For this reason it is optimally suited for
low-signal images. In addition, it is well suited for
restoring images of point- line- or plane like objects.
See
Maximum Likelihood Estimation
for more details.
There
are however situations in which other algorithms come to
front, for example when deconvolving 3D-time series, which
is very compute-intensive. In this case you may consider
to use Quick Maximum Likelihood Estimation-time (QMLE)
which is much faster than the CMLE-time and will give
excellent results as well.
An
advantage of using measured PSF as in Huygens is that in
essence it requires you to calibrate your microscope, and
stimulates the use of standard protocols for imaging.
Together, these will ensure correct functioning of the
microscope and vastly increase the quality and reliability
of the microscopic data itself, and with that of the
deconvolution results.
Lastly, an advantage of theoretical or measured PSFs is
that they facilitate construction of very fast algorithms
like the QMLE in
Huygens Professional
or the
New Batch Processor Tutorial*.
Iterations in QMLE are about five times more effective
than CMLE iterations and require less time per iteration.
Images affected by
Spherical Aberration
due to a
Refractive Index Mismatch
are better restored with
Huygens Software
through the use of depth-dependent PSF's (see
Parameter Variation).
Huygens algorithms generally do
Intensity Preservation.
See the Huygens
restoration applied to some accessible images in
Convolving Trains
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