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Leica
TCS SP2 AOBS
雷射掃描共軛焦光譜顯微鏡系統 |
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Leica TCS SP2
AOBS
是最新一代的
"
完全光譜式
"
雷射掃描共軛焦顯微鏡系統,
其設計上,
有創新與重大的突破設計 -
突破光學元件的障礙 ( Breaking all the barriers !
) : 1).
激發光設計 ( Excitation )
完全採用可程式光波調控設計
( AOTF, Acousto Optical Tunable Filter, programmable
), 包括紫外光雷射,
可見光雷射,
紅外光雷射,
皆採用 AOTF
調控雷射光波選取與強度控制.
2).
分光設計
(
Beam splitter )
完全採用可程式光波調控設計
( AOBS, Acousto Optical Beam Splitter, programmable )
3).
釋放接收光譜設計
( Emission )
完全採用可程式光波調控設計
(
SP, Spectrophotometer, programmable )
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Leica TCS
SP2 AOBS
提供了許多創新與實用的使用效益
:
The only
completely programmable fluorescence imaging system
for Confocal Microscopy
Excitation, Beam
Splitter and Emission modules are all electronically
tunable - any specification for excitation, beam
splitter and emission " filter " characteristics can
be programmed.
The
best efficiency ever achieved in a confocal microscope
Collect 30 - 50%
more emission light ( fluorescence photon ) than with
any other confocal. Enables the use of the confocal
microscope for analytical experiments like FRET, FRAP,
FLIM and FCS.
Simultaneous multi-color fluorescence imaging
Obtain the best
primary image data, image 4 fluorescence proteins
simultaneously ( CFP, YFP, GFP, DsRed ), enables the
use of more & new lasers and lines combinations,
use more dyes simultaneously, optimize dye separation,
Get stronger FRET signals. No pixel shift between
detection channel.
Simultaneous multi-spectral scanning
Emission
spectrum recording, No conventional beam splitter
modulation : Measure clean real emission spectra. Up
to 2000 spectral recording points ( vs up to 32 on
array detectors ). Better in-situ spectra due to
unmatched collection efficiency.
Protect live cell
use less
illumination power than with any other confocal, have
less cell damage than with any other confocal,
minimize bleaching, maximize cell viability, optimize
time-course measurements, improve signal-to-noise
ratio.
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| The
Leica TCS SP2 AOBS light path |
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詳細
TCS SP2 AOBS
光路圖示, 請點選連結. Leica AOBS Scanhead |
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AOBS ( Acousto Optical Beam
Splitter ) 技術原理 |
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Single or multiple excitation laser lines are directed
to the specimen by acousto-optical deflection. The
returning fluorescence light is passing the device
undisturbed and is directed to the confocal pinhole
and the prism spectrophotometer detector. ( Left :
AOBS ). Conventional beam splitters are not very
efficient for fluorescence ( Right : Blue curve ) They
have fixed characteristics and wide bands. The AOBS
has higher transparency, sharp bands and neutral
transmission across the visible spectrum. The neutral
characteristics of the AOBS enables recording of real
fluorescence spectra, un-modulated by beam splitter
transmission. No transmission " holes " exists. |
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| Leica
AOBS : Perfect
selectivity ( 0.6 - 2 nm typical )
programmable
multi-chroic up to 8 channels, 8 sharp bands can be
selected sequentially or simultaneously. The rest of
spectrum is free for collecting fluorescence light.
Conventional
beam splitters are not very efficient for
fluorescence. They have fixed characteristics and
wide bands. The AOBS has higher transparency, sharp
bands and neutral transmission across the visible
spectrum. |
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| Dichroic
beam splitter ( optical filter ) |
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Leica
AOBS |
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FITC,
TRITC, excitation with 488 nm and 543 nm laser
lines. The Leica TCS SP2 AOBS adapts beam splitter
characteristics dynamically to each excitation line
( Blue ). The result is optimal fluorescence
collection efficiency for each dye. The Fluorescence
light collection efficiency of the AOBS is far
higher than with conventional beam splitter. A
comparison of dual channel acquisition of FITC and
TRITC shows that the AOBS-enabled system collects
30% more FITC emission and 20% more TRITC emission
light. |
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| Dichroic
Beam Splitter ( optical filter ) |
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Leica
AOBS |
| Mouse
cerebellum, Cy3-Cy5 : Identical instrument
conditions. ( Laser power in focal plane, parameter
settings, specimen volume ) : Leica AOBS is more
sensitive, needs less laser power, less gain, allows
faster imaging speed. |
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| Dichroic
Beam Splitter ( optical filter ) |
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Leica
AOBS |
| Parasagittal
section of mouse cerebellum - Hoechest ( Nuclei ),
anti-GFAP/Cy2 ( Glia cells, fibers ),
calbindin-D28K/Cy3 ( Purkinje cells ) |
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c.
Elegans nervous
system, four fluorescent proteins separated in the
Leica TCS SP2 AOBS without image processing : CFP, YFP, GFP, DS RED,
courtesy Dr. Harald Hutter,
Max-Planck Institute for Medical Research,
Heidelberg, Germany
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| Leica
TCS SP2 AOBS 的性能比較例證
下圖樣品為果蠅 (
Drosophila ), GFP 螢光訊號非常的弱,
YFP 螢光訊號非常的強.
在 Leica
TCS SP2 AOBS, 用 HCX
PL-APO 63x/1.40 OIL 鏡頭,
以 476
/ 514 nm 雷射直接激發擷取影像,
得到左下圖的清析影像,
GFP 及 YFP
分辨的非常清楚,
極少 Cross-Talk
現象.
然而,
以同樣的樣品,
用相同的條件,
在另外德系廠牌的共軛焦顯微鏡做,
並再輔以 Un-Mixing
影像處理,
縱使用所謂的光譜偵測器
( 32 ch.
Spectral PMT ), 仍然無法有效分出
GFP 及
YFP. ( 如右下圖
) |
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| Leica
TCS SP2 AOBS |
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其他廠牌
(
Spectral Detector + Un-Mixing ), 仍然無法有效分辨
GFP 及
YFP. |
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Orange
594 nm Laser ( 橘色光雷射
) |
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| Leica
Orange 594 nm 橘色光雷射,
補足了 He-Ne
543 及 He-Ne
633 兩波長雷射間的間隔.
594 nm 固態雷射能對
543
nm 與 633
nm 間的螢光染劑,
做最有效率的激發效果,
可大幅提高解析,
更能保樣細胞及樣品.
最適宜長時間影像擷取應用.
當使用 TCS
SP2 AOBS 時,
能有效同時 (
選用 )
使用 457,
476, 488,
496, 514,
543, 594,
633 nm 雷射. |
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| FITC
/ Texas Red : 以 488
/ 594
nm 雷射激發,
能得到更高解析的螢光影像.
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FITC
/ Texas Red : 以 488
/ 543
nm 雷射激發 |
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| Dyes
excited with 594 nm |
Excitation
( nm ) |
Emission
( nm ) |
| Alexa
568 |
577 |
603 |
| 5-Rox |
578 |
604 |
| HcRed |
590 |
614 |
| SNARF
( carboxy ), PH 9 |
576 |
639 |
| Alexa
594 |
594 |
618 |
| Bodipy
TR-X SE |
588 |
616 |
| Calcium
Crimson |
588 |
611 |
| CTC
5-cyano-2,
3-ditolyl-Tetrazolium-Chloride |
602 |
630 |
| Red
FluoSpheres |
580 |
603 |
| Texas
Red |
595 |
620 |
| YO-PRO-3 |
613 |
629 |
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Leica
405
nm Blue Laser
( 紫外藍光雷射
)
固態雷射,
AOTF 連續調控雷射強度.
最適宜應用
UV-dyes
( DAPI, Hoechst ), CFP, FRET-CFP/YFP,
GFP2, FRET-GFP2/YFP, Photoactivation GFP. |
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夢寐以求的
405
nm 雷射
專用鏡頭
Leica
HCX 20x/0.70 Lambda-Blue
Immersion / Corr.
Leica
HCX 63x/1.40 Lambda-Blue
OIL.
Leica
HCX 63x/1.20 Lambda-Blue
Water / Corr. |
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| 全世界唯一的
Leica
Lambda-Blue 系列物鏡,
能確保 Co-localization
( Excitation spot and Emission in
focal plane ) , 能確定
Z-registration,
使得 FRET,
FRAP, Photoactivation 研究具有正確的意義,
此類物鏡最適宜應用於
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UV-Dyes |
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CFP,
GFP2 |
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FRET,
FRAP |
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Photoactivation |
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Lambda-Blue
物鏡 vs
一般無特別修正的物鏡 |
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| 一般物鏡,
無特殊 UV
( 405 nm, Lambda-Blue ) 修正 |
Leica
Lambda-Blue 系列物鏡,
有特別修正設計. |
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405
nm 及 543
nm 雷射同時激發螢光粒子
(
XZ sections of Fluorescence micro-beads
) |
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Plane, 紫外光及綠色光產生位移,
無法聚集於同一焦點. |
在
Focal
Plane, 紫外光及綠色光完全修正在同一焦點. |
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左圖 :
Fibroblasts,
DAPI/FITC, 405/488 nm excitation.
右圖 :
Smooth
Muscle Cell, DAPI counterstain |
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Leica TCS SP2
AOBS 相關技術資料與規格
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Microscope,
Upright, HC |
Leica DM R, RE,
RXE, RXA 2 |
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Leica DM 6000 |
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Leica DM LFSA (
Fixed Stage Microscope ) |
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Microscope,
Inverted, HC |
Leica DM IRB,
DM IRBE, DM IRE 2. |
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Z drive |
High precision
Galvo-Z-drive : 170 micrometer travel, XYZ : 40 nm
resolution, XZ< 1 nm resolution. |
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Internal
motorization of Leica RE, RXE, RXA2, IRBE, IRE 2
and DM 6000 ( Z-wdie ) |
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Lasers &
Attenuation switching modules |
Ar UV
351, 364 nm |
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He-Cd 442
nm |
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Ar 457 nm, 476
nm, 488 nm, 514 nm |
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Ar-Kr 488 nm,
568 nm, 647 nm |
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Kr 568 nm |
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CW Solid-State
561 nm ( NEW ) |
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He-Ne 543 nm |
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He-Ne 594 nm (
NEW orange ) |
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He-Ne 633 nm |
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DPSS 430 nm |
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Diode Laser 405
nm ( 20 mW ) |
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Ti:Sapphire
Lasers ( for Multi-Photon Imaging ) |
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AOTF, 4 or 8
channels ( for Visible range ) |
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AOTF, UV range |
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EOM, IR range (
Electro-optical modulator ) |
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Laser Merge
Module for 4 lasers. |
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Confocal Unit |
Optics : |
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User-switchable
between Leica microscope ( Upright and Inverted ) |
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Alignment-Free
for lifetime |
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3 individual
ports for external lasers for up to 6 lasers
connected simultaneously |
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Programmable
8-channel AOBS Beam Splitter |
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Spectral range
of Detector optics : 400 - 850 nm, programmable. |
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UV and MP
possible in one system ( UV-VIS-MP ) |
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Fiber coupling
( MP : either fiber coupling or direct mirror
coupling ) |
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Field-upgradable
to MP microscopy |
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UV system with
individual objective correction lens |
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Adjustable
PUPIL illumination |
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Single pinhole,
variable diameter size. |
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Scanner : |
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K-scanner with
two independent galvanometers. |
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Line frequency
: up to 2000 lines/sec. |
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Frame rate : 3
fps ( 512 x 512 pixels ), 20 fps ( 512 x 32 pixels
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Scan resolution
: up to 4096 x 4096 pixels |
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Scan Zoom 1 -
32x |
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Scan Rotation :
-2 to +92 degrees |
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Scan field : 22
mm diagonal in intermediate image plane |
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Control Unit |
Detectors |
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Highly
sensitivity prism spectrophotometer detector, 1 -
4 simultaneous channels. |
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Continuously
adjustable bandwidth and center wavelength |
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Spectral
steepness factor less than 1 |
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Low noise
photomultipliers |
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Up to 12 bits
digitization per channel |
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Transmitted
light / DIC Detector |
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Non-Descanned (
NDD ) Dual channel transmission detector ( MP ) |
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Non-Descanned (
NDD ) Dual channel Reflection & Fluorescence
detector ( MP ) |
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FLIM detector (
for MP-FLIM or D-FLIM pulsed diode 405 nm ) |
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FCS detector (
for Fluorescence Correlation Spectroscopy ) |
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Optional
External Detector |
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Programmable
Panel-Box, user-configurable |
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Electronics |
Scanner control
system with FRGA ( Field-programmable gate arrays
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Trigger-IN and
Trigger-OUT ports |
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8-Detector
channels, 12-bits digitization, Simultaneous. |
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Ultra-Wide SCSI
interface to PC |
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High
performance PC Workstation ( Windows XP
environment ) |
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One or Two
monitor configuration |
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Software |
Leica LCS
software package |
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Fully
operator-configurable user interface |
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Intuitive and
guided |
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Context-sensitive online help system |
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Multi-dimensional series acquisition ( up to 7
parameters simultaneously ) |
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Programmable
manual control panel |
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Region of
Interest scan |
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Excitation line
/ Frame multiplexing |
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Emission
spectrum recording |
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Multi-color
restoration, spectral un-mixing |
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Advanced Time
lapse recording |
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Surface
reconstruction |
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Multiple
measurement functions |
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Physiology
application software package |
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Multi-Color
application software package |
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FRET
application software |
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FLIM
application software |
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FCS application
software |
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FRAP
application software |
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3D
reconstruction with multiple reconstruction &
rendering functions |
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Macro developer
software |
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De-convolution
software |
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Material
software |
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The 2nd
workstation package ( off-line ) |
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Software new
function |
Leica LCS
MicroLab ( New ) |
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Dye separation |
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Pixel intensity
readout in documentation-viewer |
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Offline phase
correction |
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Zoom-IN
function combined with corp function ( AOTF
control, ROI-scan ) |
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Panning
function during scanning available with Panel Box |
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Objectives |
Leica PL-Fluotar,
PL-APO objectives ( CS ) |
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Live-Cell
Chamber |
Temperature &
CO2 controlled chamber system |
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Wide-Field
Imaging |
Cooled CCD
Camera with software, workstation. |
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Motorized
microscope stage |
Programmable
motorized XY-scanning stage |
 請參考
Leica LCS software 詳細說明 |
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共軛焦顯微鏡適用
- 活細胞培養系統 ( 溫控及二氧化碳濃度控制
)
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詳細規格,
請洽本公司共軛焦小組 |
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其他相關資料下載及連結 |
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