Leica TCS SP5 MP STED

 

Leica TCS SP5 equipped with a Spectra-Physics Mai Tai Ti:Sapphire laser is an inverted confocal laser scanning microscope. It is designed for high-resolution fluorescence and transmitted light imaging with single or multi-photon excitation, in 2D, 3D or 4D with live or fixed cells. It can also be used for Fluorescence Recovery After Photo bleaching (FRAP), pulsed Stimulated Emission Depletion (pulsed STED) and Fluorescence Correlation Spectroscopy (FCS). In combination with the Picoquant Microtime200, it can be used for Fluorescence Correlation Spectroscopy (FCS) coupled to Fluorescence Lifetime Imaging (FLIM).

Microscope body:
Inverted
Motroised Stage:
Yes
Temp Control:
Heating Stage
CO2:
No
Software:
LAS AF

Common Applications

Confocal: Confocal imaging is a fluorescence microscopy technique that optically sections the specimen preventing out of focus light from reaching the detector. This yields clear high contrast images, and together with the ability to acquire images sequentially at multiple positions the entire sample can be reconstructed in 3D
Colocalisation: Light microscopy lends itself very well to labelling multiple structures within a sample due to the ability to separate these spatially overlapping signals by the wavelength of light they emit. Colocalisation is the study of how the distribution of one probe relates to that of another within the same sample.
Multi-Photon: This technique is using a pulsed laser to send, at the exact same time, 2 photons of half the energy required to excite a fluorophore. This event is so rare, it is only likely to happen where the light of the laser converges, at the focal plan. This avoid bleaching the rest of the sample while acquiring data. Also, because it requires to use far-red wavelength to excite fluorophores usually excited by visible light, a deeper penetration trough the sample is possible as in these range, organic tissues show good transparency.
STED: Stimulated emission depletion microscopy is a technique using tow laser beams to illuminate the specimen. An excitation laser pulse created by a multi-photon laser is closely followed by a doughnut-shaped red-shift laser pulse (STED beam). The STED beam is designed to make excited fluorophores intravenously returning to non-excited states. When the two lasers are superimposed, only the molecule in the center of the STED beam are able to emit fluorescence. The point spread function is then drastically reduced and the resolution increased beyond the diffraction limit (around 60nm).
Second harmonic generation: Only non-centrosymmetric structures are capable of emitting Second Harmonic Generation light. One such structure is collagen. Using a short-pulse laser and a set of appropriate filters the excitation light can be easily separated from the emitted, frequency-doubled Second Harmonic Generation signal. This allows for very high axial and lateral resolution comparable to that of confocal microscopy without having to use pinholes.
FCS: Fluorescence Correlating Spectroscopy allows for determining diffusion coefficients and concentrations of fluorescently labelled molecules at nanomolar concentrations both in vitro and in live cells. It is mostly useful for indirect studies of molecular activity in plasma membrane, in cytosol and in nucleus via following relative changes in diffusive behavior and/or concentration.
FCS-FLIM: Each fluorescent dye displays a characteristic fluorescence lifetime, namely the length of time following excitation that the dye will remain in its excited state emitting fluorescence.  This fluorescence lifetime is a quantitative signature that can be used to distinguish between different fluorescent dyes, even those emitting the same fluorescent color. The FCS-FLIM mode operation allows for FLIM (Fluorescence Lifetime Imaging).
FRAP: Fluorescence Recovery After Photobleaching is used to gain insight into the dynamics or both diffusion and binding rates within a sample. Photobleaching is an irreversible process by which a fluorophore loses the ability to absorb and emit light. By deliberately inducing photobleaching within a restricted region of the sample observing the recovery within this region over time is affected by the rate at which unbleached molecules entre this region and bleached molecules are moved. The light sheet mode available on this instrument allows to study thicker samples than (<2mm) what an ordinary confocal microscope could image.

 

Objective Lenses

MAG N.A CORRECTIONS IMMERSION W.D MISC.
10x 0.3 HCX PL Fluotar AIR 11 mm  
20x 0.7 HCX PL APO CS GLY/OIL 0.26-0.17 mm  
40x 1.25-0.75 HCX PL APO OIL 0.1 mm  
63x 1.2 HCX PL APO CS WATER 0.22 mm Coverslip 0.14-0.18
63x 1.3 HCX PL APO GLY (37 C) 0.25 mm  
63x 1.4 HCX PL APO CS OIL 0.1 mm  
100x 1.4-0.7 HCX PL APO OIL 0.09 mm  

Light Sources

Source Wavelengths Nominal Power
Tungsten light Transmitted white light (Eye piece only)  
Mercury arc lamp Incident Visible light (Eye piece only)  
Argon Laser 458 nm, 476 nm, 488 nm, 496 nm, 514 nm 100 mW
DPSS Laser 561 nm 10 mW
HeNe Laser 633 nm 10 mW
STED excitation Diode 532 nm 9 mW (80 MHz)
STED excitation Diode 635 nm 9 mW (80 MHz)
STED depletion TitanSapphire From 725 nm to 850 nm

 

Variable
Mai Tai DeepSee MP laser 700 nm to 1040 nm 2.5 W (70 fs pulses)

Filters

name Excitation Dichroic Emission Description
Acousto Oprical Beam Splitter     400 - 800 nm Wavelength collection by acoustic waves
DAPI        
GFP   500 nm    
YFP   515 nm    

Detectors

name filter Description
2 x Photomultiplier (PMT)

(AOBS)

400 nm to 800 nm

high sensitivity low noise PMTs (R 9624). Tunable for emission bands (1nm)
1 x Photomultiplier (PMT) BP 680/60nm Used for STED detection
2 x Hybrid Detectors (HyD)

(AOBS)

400 nm to 800 nm

Tunable for emission bands (1nm)
4 x Non-Descanned detectors (NDD)   Used for transmitted (Briegtfield, DIC, Phase), 2-Photon (FITC and DAPI) and Incident Second Harmonic Generation detection 
2 x Photomultiplier Tube Avalanche Photo Diodes (PMT-APD)    
DFC360 FX Camera   high speed CCD Camera fully integrated into LAS AF

Scanning & Stage

NAME Range Description
Conventional scanner 10 to 1400Hz Commonly used scanner allowing a maximal image speed of 87 frame/sec (~1 fps typical).
Resonant Scanner 8000Hz Constant frequency for fast acquisition with a maximum of 500 frames/sec (~16 fps typical).
Z-drive  1500 μm travel range/3 nm step size Motorized stage or 3D imaging (maximum 250g)
Stage Heater   For live cell imaging