Leica TCS SP5 WLL STED

This Leica TCS SP5 confocal system is fitted for a broad range of experimental requirements. Along with a large range of fixed wavelength laser lines it now has a White-Light Laser (WLL) tunable within the range 470nm - 670nm for precise control over fluorophore excitation. Fitted with Hybrid Dectors (HyD) for enhanced sensitivity, and interchangeable Galvano and resonant scanners, this system is suitable for applications requiring both speed and/or improved signal-to-noise. Its Gated-STED module also enables colleciton of super-resolution images allowing investigators to distinguish objects and structures beyond the conventional resolution limit of light.
Microscope body:
Inverted
Motroised Stage:
Yes
Temp Control:
Full Enclosure
CO2:
Stage Insert
Software:
Las AF

 

Common Applications

Confocal: Confocal imaging is a fluorescence microscopy technique that optically sections the speciment 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.
Live Cell Imaging: Many biological processes are dynamic events and as such observing living cells is crucial to unravelling and understanding the mechanisms behind them. This requires special consideration when imaging. Firstly, the environmental conditions must be controllable and stable to ensure the cell remain healthy. Secondly, care needs to be taken that the act of imaging has as little impact as possible on the event being observed.
STED: Stimulated Emission Depletion (STED) is a super resolution fluorescent microscope technique designed to enhance image resolution by bypassing the diffraction limit faced by tranditional microscopy. 
FRET

FRET (Fluorescence Resonance Energy Transfer) is a technique used largely to investigate molecular interactions, or with specially designed reports it can show if molecules are in an active or inactive state. It works by using two carefully selected fluorophores that when in close enough proximity and suitable orientation energy from the donor fluorophore can be transferred to the accepter. This transfer can be observed by monitoring the acceptors emission or the donors fluorescence lifetime.

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    

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.

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.

 

Objective Lenses

MAG N.A CORRECTIONS IMMERSION W.D MISC.
20x 0.5 HC Plan Fluotar AIR 1.15 mm  
20x 0.7 HCX Plan Apo CS IMM 0.26 mm UV
40x 0.75 HCX Plan Fluotar AIR 0.4 mm  
63x 1.2 HCX Plan Apo CS WATER 0.22 mm UV
63x 1.4 HCX Plan Apo CS OIL 0.1 mm UV
100x 1.4 HCX Plan Apo OIL 0.1 mm STED

 

Light Sources

Source Wavelengths Nominal Power
Tungsten light Transmitted white light (Eye piece only)  
Mercury arc lamp Incident visible light (Eye piece only)  
STED excitation Diode 532 nm 9 mW (80 MHz)
STED excitation Diode 635nm 9 mW (80 MHz)
White Light Laser 470 nm  to 670nm  Tunable laser in visible range

 

Filters

Name Excitation dichroic Emission Description
AOBS     400nm - 800nm Emission splitting by acoustic waves
GFP   500nm   Incident light filter for eye piece only
YFP   515nm   Incident light filter for eye piece only

 

Detectors

Name Descirption
3x Photomultiplier (PMT) high sensitivity low noise PMTs (R 9624)
2x Hybrid Detectors (HyD)  
3x Photomultiplier (PMT) Tans Used for transmitted light
2x Photomultiplier Tube Avalance Phot Diodes (PMT_APD)  
DFC360 FX Camera high speed CCD Camera fully integrated into LAS AF

 

Scanning And 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 um Tranvel range/ 3nm step size Motorized stage or 3D imaging (maximum 250g)