Transcript
Leica TCS SP Spectral Confocal Microscope
Principle Benefits of Confocal Microscopy Horizontal (xy) image of kidney tissure, non-confocal
Horizontal (xy) image of kidney tissue, confocal
Vertical (xz) images of kidney tissue confocal (above), nonconfocal (below)
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Multi-dimensional imaging
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Resolution improvement
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Contrast improvement
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Optical sectioning
Resolving Power of Confocal Microscope (Oil Immersion)
Resolution (FWHM) 488 nm
Numerical aperture of objective
Spectral excitation and emission bands of common fluorophores Application
488mn
351+ 364 mn UVAr
520
490
FITC
647mn Ar/Kr
568mn
557
TRITC
576
350
2
670
648
CY5 400
450
500
550
600
650
700
Design FITC image
I C
B
H
F G
A A I
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TRITC image
D VIS AOT F
1998
VIS L
aser
VIS L
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VIS L
IR La UV AOT F
E
ser
aser
UV L
aser
CY5 image
Superior System Resolution Lateral resolution Axial resolution Temporal resolution Spectral resolution
Transmitted light differential interference contrast image
by design: • Alignment stability • Illumination pinholes • Fiber coupling • Leica optics • High optical transfer efficiency • Spectrophotometer • Perfect registration • AOTF line selection • Quick set-up of acquisition parameters • Quick set-up of time lapse interface
A B C D E F G H I
Illumination pinholes Main beamsplitter Scan mirror Microscope objective Photomultiplier (transmitted light) Confocal pinhole Prism Variable slit Photomultiplier (fluorescence)
Human chromosomes, composite image FITC/TRITC/CY5/ Transmitted light differential interference contrast
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SP Advantages
Two spectral GFP variants in stalk cells in Dyctostelium discoideum fruiting body
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“Design-a-filter“: Ultimate flexibility for new dyes and difficult dye combinations
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Easy wavelength selection. Pre-programmed spectra of all popular dyes
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Can be set to real emission spectra of dyes
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Better spectral steepness and transparency than filter combinations mean clearer images
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Less bleed through in multi-color applications
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Less averaging in live cell imaging
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Multi-channel recording of emission spectra
Drosophila embryo development, Label: FITC, TRITC
Mouse fibroblasts; Label: nucleus: Hoechst, vimentin: TRITC, actin: FITC
Mouse plasmocytoma; Label: chromosomes: red, tubulin: green, vimentin: FITC, blue
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SP Interface
System Highlights •
Superior image quality
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Alignment-free scanner
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Fiber coupled UV, VIS and IR lasers
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Shift-free multiline-illumination for perfect co-localization
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Illumination pinhole to assure perfect beam geometry
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Scanner is user-switchable from upright to inverted microscopes
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Cooled scanner for low dark noise and high sensitivity
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Live xz scanning for sample cross-sectioning
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Intuitive interface permits easy control of the imaging system
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Comprehensive on-line help functions
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Control panel with digital potentiometers for instant parameter access
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Superior objectives for Confocal Microscopy
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Multi-Photon upgradable (pico-second lasers)
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Network compatible PC with Windows NT
Mouse fibroblasts; Label: actin: FITC, tubulin: TRITC, vimentin CY5
Mouse fibroblasts; Label: vimentin FITC, actin TRITC, tubulin CY5
®
You will find further information on the Internet: http://www.llt.de
Nerium Oleander, three different spectral ranges
PAP smears, triple label
Radiolaria, reflection
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System Components
Confocal scanner head
Fiber coupling of UV, visible range and infrared lasers
Microscope – Leica DM RBE
High-precision focusing stage
Electronic focus
Anti-vibration table
Leica TCS SP with inverted microscope Leica DM IRB/E and single monitor
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Leica Design by Ernest Igl/ Christophe Apothéloz
Prionium serratum shoot, Safranin – Fastgreen stained, 3-channel overlay
Monitors
Computer desk
Control panel
Laser box
PC workstation
System electronics 7
Software Main Toolbar
Designed for the multi-user laboratory: Intuitive workflow Quick parameter access Control panel Online tutorial
Time-course toolbar
Powerful functionality: • Scanning sequences • Animation • Surface rendering • 3D reconstruction • Measurements • Time-course studies • Physiology software • Spectral scanning • Annotation sheet • Customized control with Leica QWINTM
Image window
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Orthoslicing
3d viewer
Physiology software
Large field Biomapping
Annotation window
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System Requirements Dimension of desks Electronics box Dimension of laser box Number of laser boxes Weight base system Human hair, reflection
Supply voltage Mains voltage fluctuations Mains frequencies Mains frequency fluctuations Mains fuses
Heat load max. TRITC – phalloidin labeld F-actin of XTH-2 cells.
Heat load average Standby mode Room temperature Separate cooling Coolant Room requirements Height of room (minimum) Opening (minimum) Magnetic protection shield Room darkening Electric supply mains Room environment Waste heat ArKr-laser Cooling air flow rate Heat load
1400 inverted stand 1420 upright stand
190 cm x 90 cm x 142 cm (without chair) integrated in desk 75 cm x 27 cm x 60 cm 2 for 3 laser system 1 laser box placed unter desk HeNe laser inside electonic boy max. 320 kg (without UV system), max. 428 kg (with complete UV system) 230 V ± 10 % 50/60 Hz ± 10 % 10 A microscope + workstation 16 A ArKr or Kr Laser 16 A Ar Laser 32 A UV laser 5 kW (without UV system) 10 kW (with complete UV system) 3 kW 2 kW 18˚C to 25˚C UV-laser air 1,8 m 1 m x 1,5 m only in neighborhood of strong magnetic fields recommended 2 x (or 3 x for 3 laser system) 230 V/16 A (separate supply) avoid close proximity to air conditioning equipment, protect from dust 240-320 m3/h ≤ 3 kW
885
750
600 730
270 Distance from wall standard system: 200 UV system: 300 All measurements in mm
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1890
Configurations DANGER LASER RADIATION – AVOID DIRECT EXPOSURE TO BEAM. < 400 mw ARGON 457–514 nm CLASS IIIb LASER PRODUCT
DANGER LASER RADIATION – AVOID DIRECT EXPOSURE TO BEAM. < 400 mw AR-KR 457–675 nm CLASS IIIb LASER PRODUCT
DANGER VISIBLE AND INVISIBLE LASER RADIATION AVOID EYE OR SKIN EXPOSURE TO DIRECT OR SCATTERED RADIATION.
< 400 mw AR-ION 351, 364 nm CLASS IIIb LASER PRODUCT
DANGER VISIBLE AND INVISIBLE LASER RADIATION AVOID EYE OR SKIN EXPOSURE TO DIRECT OR SCATTERED RADIATION.
< 400 mw HeNe 633 nm CLASS IIIb LASER PRODUCT
DANGER VISIBLE AND INVISIBLE LASER RADIATION AVOID EYE OR SKIN EXPOSURE TO DIRECT OR SCATTERED RADIATION.
< 400 mw KR 568 nm CLASS IIIb LASER PRODUCT
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