Leica SP8 DLS

The Leica TCS SP8 DLS (Digital LightSheet) is an inverted laser scanning microscope capable to be used for confocal imaging and light sheet imaging. This microscope is able to image multi-colour, live, fixed or aqueous-based cleared specimens utilising a combinations of 405, 458, 488, 514, 561 and 635 nm excitation lasers through the employment of the AOBS with spectral high quantum efficiency detectors. The advantage of the two combined features (confocal/Lightsheet) on a single equipment allow to perform, with the confocal module, local photo activation or photo bleaching where the resulting dynamics can be capture via lightsheet imaging on thick sample (up to 2 mm thick and 1 cm long).
Microscope body:
Inverted
Motroised Stage:
Yes
Temp Control:
Full Enclosure
CO2:
Stage Insert
Software:
Las X

 

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.
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     
Photo-activation

Photoactivation is a technique of photomanipulation used to investigate intracellular protein movement. A laser is used to cause a structural change to a population of molecules like fluorophores. These photoactivated molecules can then be tracked to assess their movements.

Tissue Clearing

Tissue clearing is a preparation technique with the aim of reducing the inherently scattering nature of tissue. There are several means by which this is achieved but all enable clearer and deeper penetration into the sample using light microscopy.

Digital Lightsheet Microscopy

The Leica TCS SP8 DLS is based on an ordinary confocal microscope. To convert such a system into a digital light sheet microscope, small mirrors are positioned just outside the observed field, at the position of the focal plane and on each side of the specimen, to make the illumination beam pass through the sample perpendicular to the optical axis. The lateral scanning modes is used to scan the illumination line in the perpendicular direction, creating the required two-dimensional light sheet. The deflecting mirrors are mechanically connected to the observation lens to ensure the illuminated z-position is always in the focus of the image-generating optics. The image is then projected onto a video camera that converts the intensities into a digital image.

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

Lightsheet Objectives

Illumination

Mag NA Corrections Immersion Working Distance Misc
2.5x 0.07 HC PL Dry    
5x 0.15 HC PL Fluotar Dry    

 

Collection

MAG NA CORRECTIONS IMMERSION WORKING DISTANCE MISC
1.6x 0.05 HC PL Dry    
5x 0.15 HC PL Fluotar Imm 13.7mm  
10x 0.3 HC PL Apo L Water 3.6mm  
25xx 0.95 HC PL Fluotar Water 2.5mm  

 

Collection Mirrors

Description Immersion
DLS TwinFlect 7.8 mm Glycerol
DLS TwinFlect 7.8 mm Water
DLS TwinFlect 5 mm  Water
DLS TwinFlect 2.5 mm Water

 

 

Confocal Objectives

MAG NA CORRECTIONS IMMERSION WORKING DISTANCE MISC
10x 0.4 HC PL APO Dry 2.2mm 506285
20x 0.75 HC PL APO CS2 Dry 0.62mm 506517
40x 1.3 HC PL APO CS2 Oil 0.24mm 506358
63xx 1.4 HC PL APO CS2 Oil 1.4mm 506350

 

Light Sources

Source Wavelengths Nominal Power
Tungsten light Transmitted white light (Eye piece only)  
Mercury arc lamp Incident visible light (Eye piece only)  
UV Laser DMOD 405 nm 50 mW
Argon Laser 458 nm, 476 nm, 488 nm, 496 nm, 514 nm 65 mW
DPSS 561 Laser 561 nm 20 mW
HeNe 633 Laser 633 nm 10 mW

 

Filters

High Transmission AOBS® (Acousto Optical Beam Splitter)

Emission splitting by acoustic waves allows cusomized spectral collection between 400nm to 80nm.

 

Name Exication Dichroic Emission Description
DAPI       Incident light filter for eye piece visualisation of sample (Blue)
GFP       Incident light filter for eye piece visualisation of sample (Green)
DsRed       Incident light filter for eye piece visualisation of sample (Red)
DLS 575615     BP 575 - 615 nm  
DLS 575635     BP 575 - 635 nm  
DLS     NF 405, 488, 561, 633 nm  

 

Detectors

Name Descirption
3x Photomultiplier (PMT) high sensitivity low noise PMTs (R 9624)
2x Hybrid Detectors (HyD)  
Hamamatsu Orca Flash 4.0 V2 camera

CMOS image sensor for scientific measurement.

Maximum pixel format: 2048 x 2048

Pixel size 6.5µm (H) x 6.5µm (V)

Bit Depth:16 bit

Sensitivity and speed: 100 fps (2048 x 2048 format)

 

Scanning and Stage

Name Range Description
FOV (Field of View) scanner 1800Hz Large field (22 mm) of view 3 mirror scanner
Tandem Scanner Up to 12 kHz Line scan frequencies Combination of FOV and 8 kHz or 12 kHz resonant scanning system with switchable galvanometric mirrors. 40 fps at a scan format of 512x512 pixels or 428 fps at 512x16 pixels
Super Z Galvo flow (Stage)   For fast and clear 4D time series were the Tandem Scanner in xy direction and the SuperZ act self-synergistically

 

Miscellaneous

 

 

Inserts and specimens:

  • Stage inserts accommodating for slides, 35 mm dishes, and 6-well to 96-well plate
  • Specimen size for DLS imaging: Diameter of sample (short axis) ≤ 3.5 mm, diameter long axis determined by the mounting dish
  • Lightsheet thickness 1.7-15 um