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Leica TCS MP2 

Multi-Photon Imaging System

雷射掃描共軛焦 - 多光子顯微鏡系統

如果同時使用 2 個以上的雙倍波長光子的能量, 以能量相加的交互共同方式, 來激發螢光染劑, 則此共同的能量, 就如同單光子的能量, 足以改變能階, 並促使螢光染劑釋出與單光子激發後所產生的相同波長的螢光. 此即表示雙光子技術.  例如, DAPI 的 Ex : 359 nm, Em : 461 nm  / 我們可使用單光子雷射 Ar-UV laser ( 約351 nm ) 來激發, 也可使用雙光子雷射 Ti-Sapphire ( 約702 nm ) 來激發, 同樣可得到相同波長 461 nm 的螢光.雙光子螢光影像已經是可謂是 “完全聚焦點 “ , 不是大面積樣品的影光影像. 故此聚焦點的反射螢光光路無須經過共軛焦掃描器內的感測光針孔 ( Detection Pinhole ), 可 100% 的直接於安裝在顯微鏡內的 Non-Descanned Fluorescence Detector 所感測.然而, 兩個長波長的光子以上的共同交互合作激發機制, 必須在極短的次微秒時間內完成 ( 也有人稱為飛秒雷射 或 超快雷射 ), 所以, 才有 Pico-second ( 10-12 sec ) 及 femto second ( 10-15 sec ) 技術.

     為何要使用 雙光子雷射掃描影像技術 ? 

► 可依應用做適度的取代紫外光雷射 ( UV )

因光傷害問題, 無法使用於活體細胞

許多螢光染劑都使用較短波長的紫外光來當作激發光源, 因其激發能量較高. 然而, 紫外光極易散射 ( high UV-scattering ), 傳輸與穿透不易, 所以, 必須使用特別製作的光學元件 ( 造價極為昂貴, 品質不易控制 ).

紫外光極易散射, 會沉降在樣品的表面層, 就如同晒傷, 會致使樣品的熱傷害. 紫外光的輻射, 會致使毒害, 致使 DNA 傷害. 

► 增加取樣的深度, 能更深入樣品裡層. 

► 不會產生光毒傷害 

► 減低螢光漂白的速率, 增加螢光觀察與掃描時間.

► 使用多種染劑時, 呈像無色差現象. 

► 較容易將激發光與釋出光的波長分開, 螢光影像比較不會有重疊現像 ( cross-talk ). 

► 無須感測光針孔, 所以反射螢光可 100% 的被感測. 

► 可使用絕大部份的螢光染劑, 尤其, 是須使用紫外光光源所激發的雷射 

► 可使用於活體細胞, 而不會造成傷害, 可做活細胞長時間影像紀錄.

► 有效的應用於胞外或胞內離子流定量分析及螢光影像, Fluorescence Life Time 研究 ( FLIM ), Fluorescence Correlation System ( FCS ).

　

Image : Single photon

       Image : Two-photon

雷射選配安裝
Laser Mai-Tai Tsunami
Coupling Direct-mirror coupling Fiber-coupling Direct-mirror coupling Fiber-coupling
Femto-second V 　 V 　
Pico-second V V V V
注意事項 如果使用人員, 未來有考慮要在 正力與倒立顯微鏡 間, 經常切換掃描器, 則建議採用 Fiber Coupling.

Mai-Tai 

多光子顯微鏡系統 - 超快飛秒雷射系統 ( Ultrafast Ti:Sapphire IR-Laser )
　 　
Mai Tai®: the first tunable, one-box Ti:sapphire femtosecond laser source. The field proven standard and mid tuning range Mai Tai® is also available using a single diode-pumped Millennia. It tunes over 140 nm with up to 1 W average power. With the largest installation base of all tunable one-box ultrafast lasers combined, you can’t go wrong.

Features:
  • Turnkey, single-box design for hands-off operation
    Fully automated and computer-controlled
    Tuning range from 750 nm to 850 nm, or 780 nm to 920 nm
    New StabiLok™ technology ensures <25 µrad pointing stability across the entire tuning range.
    Customized tuning ranges on request
    Compact laser head (23 x 14 x 5 inches, 59 x 35 x 12 cm)
    Uses regeneratively initiated, mode-locking
    Uses Millennia i Series single diode-pumped solid state laser.

New Wideband Mai Tai®Features:

  • More than 1.5 watts average power at 800 nm
  • More than 700 mW over entire tuning range
  • Hands off operation
  • Fully computer controlled from user friendly Graphical User Interface (GUI)
  • Tuning range 710 nm to 920 nm
  • StabiLok™ provides better than 70 µrad of pointing stability over the entire tuning range
  • Continuous tunability accomplished in a matter of seconds with no loss of mode locked operation
  • Maintains features of field proven standard Mai Tai®;

　

Tsunami ( Millennia )

 多光子顯微鏡系統 - 超快飛秒雷射系統 ( Ultrafast Ti:Sapphire IR-Laser )
　 　 　
Tsunami®
The first choice in mode-locked Ti:sapphire lasers
The Tsunami® mode-locked Ti:sapphire laser provides the widest pulse width range, the broadest wavelength coverage and the highest power levels of any ultrafast oscillator on the market. It also offers the best long-term stability and reliability in both femtosecond and picosecond domains.

Features:

  • Broadest pulse width coverage (< 35 fs to 100 ps) of any commercial mode-locked Ti:sapphire laser
  • Power output > 2 W for high-power harmonic generation and OPO pumping
  • New proprietary broadband optics
  • Uses Millennia s series all-solid state pump lasers (5 to 10 watts)
  • Regeneratively stabilized mode-locking for reliable starting mechanism, long-term stability, prevention of pulse dropouts and broadest wavelength coverage
  • Invar resonator with gimbaled mirror mounts
  • Three-point ball-bearing registry of all optics
  • Smallest footprint of any commercial, broadly tunable, ultrafast source
  • Accessories include Lok-to-Clock synchronization, harmonic generators, OPOs, high-energy regenerative amplifiers, kHz regenerative amplifiers, OPAs and more

　

Millennia

Pump source for Ti:sapphire Tsunami laser
High-Power, diode-pumped solid state lasers
The Spectra-Physics Millennia® s Series and the industrial Millennia i Series are the next generation in CW 532 nm, high-power, diode-pumped solid state laser technology.

The industrial Millennia i Series uses a monolithic architecture and advanced thermal design to provide unprecedented power stability and beam-pointing performance with a minimum footprint.

The Millennia s Series combines the industrial features of the i Series in a compact, CDRH-compliant package appropriate for a wide range of applications.

Features:
  • Enhanced, patented intracavity doubling process (QMAD) provides ultra-low optical noise, less than 0.04% rms for all models from 2 W to 10 W
  • Permanently aligned and sealed
  • Rugged, reliable X-cavity design requires a maximum of two high- power diodes
  • T-tune control software assure the pump diodes operate at their optimum current and pump wavelength
  • Ultra-compact size readily integrated into any application, the Millennia s Series is 60% smaller than the competitive design
  • Beam position mechanically indexed for reproducible installations
  • FCbar™ fiber-coupled source diodes are field replaceable
Applications:
  • Pump source for Ti:sapphire and dye lasers

　

Leica TCS SP2 AOBS

Confocal Spectral Microscope
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 )
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.

　
The Leica TCS SP2 AOBS light path

　
詳細 TCS SP2 AOBS 光路圖示, 請點選連結. Leica AOBS Scanhead

AOBS ( Acousto Optical Beam Splitter ) 技術原理
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.

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. 
　 　 　
　

Dichroic beam splitter ( optical filter )

 　

Leica AOBS
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.
Dichroic Beam Splitter ( optical filter ) 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.
Dichroic Beam Splitter ( optical filter ) Leica AOBS
Parasagittal section of mouse cerebellum - Hoechest ( Nuclei ), anti-GFAP/Cy2 ( Glia cells, fibers ), calbindin-D28K/Cy3 ( Purkinje cells )

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

　
　

Leica TCS SP2 AOBS 相關技術資料與規格

　
Microscope, Upright, HC Leica DM R, RE, RXE, RXA 2
Leica DM 6000
Leica DM LFSA ( Fixed Stage Microscope )
Microscope, Inverted, HC Leica DM IRB, DM IRBE, DM IRE 2.
　
Z drive High precision Galvo-Z-drive : 170 micrometer travel, XYZ : 40 nm resolution, XZ< 1 nm resolution.
Internal motorization of Leica RE, RXE, RXA2, IRBE, IRE 2 and DM 6000 ( Z-wdie )
　
Lasers & Attenuation switching modules Ar UV  351, 364 nm
He-Cd  442 nm
Ar 457 nm, 476 nm, 488 nm, 514 nm
Ar-Kr 488 nm, 568 nm, 647 nm
Kr 568 nm
CW Solid-State 561 nm ( NEW )
He-Ne 543 nm
He-Ne 594 nm ( NEW orange ) 
He-Ne 633 nm
DPSS 430 nm
Diode Laser 405 nm ( 20 mW )
Ti:Sapphire Lasers ( for Multi-Photon Imaging )
AOTF, 4 or 8 channels ( for Visible range )
AOTF, UV range
EOM, IR range ( Electro-optical modulator )
Laser Merge Module for 4 lasers.
　
Confocal Unit Optics :
User-switchable between Leica microscope ( Upright and Inverted )
Alignment-Free for lifetime
3 individual ports for external lasers for up to 6 lasers connected simultaneously
Programmable 8-channel AOBS Beam Splitter
Spectral range of Detector optics : 400 - 850 nm, programmable.
UV and MP possible in one system ( UV-VIS-MP )
Fiber coupling ( MP : either fiber coupling or direct mirror coupling )
Field-upgradable to MP microscopy
UV system with individual objective correction lens
Adjustable PUPIL illumination
Single pinhole, variable diameter size.
　
Scanner :
K-scanner with two independent galvanometers.
Line frequency : up to 2000 lines/sec.
Frame rate : 3 fps ( 512 x 512 pixels ), 20 fps ( 512 x 32 pixels )
Scan resolution : up to 4096 x 4096 pixels
Scan Zoom 1 - 32x
Scan Rotation : -2 to +92 degrees
Scan field : 22 mm diagonal in intermediate image plane
　
Control Unit Detectors
Highly sensitivity prism spectrophotometer detector, 1 - 4 simultaneous channels.
Continuously adjustable bandwidth and center wavelength
Spectral steepness factor less than 1
Low noise photomultipliers
Up to 12 bits digitization per channel
Transmitted light / DIC Detector
Non-Descanned ( NDD ) Dual channel transmission detector ( MP )
Non-Descanned ( NDD ) Dual channel Reflection & Fluorescence detector ( MP )
FLIM detector ( for MP-FLIM or D-FLIM pulsed diode 405 nm )
FCS detector ( for Fluorescence Correlation Spectroscopy )
Optional External Detector
Programmable Panel-Box, user-configurable
　
Electronics Scanner control system with FRGA ( Field-programmable gate arrays )
Trigger-IN and Trigger-OUT ports
8-Detector channels, 12-bits digitization, Simultaneous.
Ultra-Wide SCSI interface to PC
High performance PC Workstation ( Windows XP environment )
One or Two monitor configuration
　
Software Leica LCS software package
Fully operator-configurable user interface
Intuitive and guided
Context-sensitive online help system
Multi-dimensional series acquisition ( up to 7 parameters simultaneously )
Programmable manual control panel
Region of Interest scan
Excitation line / Frame multiplexing
Emission spectrum recording
Multi-color restoration, spectral un-mixing
Advanced Time lapse recording
Surface reconstruction
Multiple measurement functions
Physiology application software package
Multi-Color application software package
FRET application software
FLIM application software
FCS application software