Leica TCS SP5


Location: HWB room 3/51
Contact: Dr Kees Straatman (krs5/7085, 2263)


A guide to start and shut down the microscope can be downloaded from here
Safety Data Sheet and Technical Information Leica immersion oil
A free viewer for images from the Leica TCS SP5 can be downloaded from here.

View Equipment Booking Statistics here.

Specifications of the Leica TCS SP5 confocal laser scanning microscope

TCS stands for True Confocal Scanner and SP5 for the 5 SpectroPhotometer channels this system is equipped with. The microscope is a Leica DMI 6000 CS inverted microscope with a motorized XY-stage and Z-drive motor focus on the microscope stand (100 nm step size) as well as a SuperZ Galvo stage (1500 µm travel range) for higher speed and accuracy. An incubator is fitted on the microscope stage and CO2 can be piped into this chamber for live cell imaging.

Lasers

50 mW diode - 405 nm
100 mW Argon laser - 458, 476, 488, 496 and 514 nm
10 mW DPSS - 561 nm
2 mW HeNe - 594 nm
10 mW HeNe - 633 nm

The SP5 uses an acousto-optical tunable filter (AOTF) to control the excitation laser light and an acousto-optical beam splitter (AOBS) instead of filter blocks to send the excitation laser light to the sample and to reflect the emitted light from the sample into the scan head. This is a more light efficient way than using glass di- or multichroic mirrors and filters.

Objectives

TypeMagnification/NA/imm
Working distance (mm)
Coverslip

HC PL APO CS

10x/0.4 dry

2.2

0.17

HC PL APO Lbd. Bl

20x/0.70 immersion

0.26 (water) - 0.17 (oil)

-

HCX PL APO CS

40x/1.25-0.75 oil

0.1

0.17

HCX PL APO Lbd. Bl

63x/1.4-0.6 oil

0.1

0.17

HCX PL APO CS

>63x/1.3 Glyc 21°C

0.28

0.14-0.18

 

Detectors

In addition to standard PMTs at position 1, 3 and 5 this system has very sensitive HyD SP GaAsP-detectors at position 2 and 4 which combine high-sensitivity with low noise.

HyD-Efficiency plot compared to PMT

Figure 1; Quantum efficiency of the HyD detector compared to the "standard" PMT detector.

When you select a HyD detector you can choose out of three modes:
1) Standard mode - most commonly used. The photon count of the HyD detector directly correlates with the concentration of your fluorescent marker. A scaling factor is introduced using the gain to map the absolute photon count to a look-up table to give the final intensities.
2) BrightR  - rarely used, non-linear gain amplification, non-quantitative as dimmer signal is more enhanced than brighter signal.
3) Photon Counting Mode - most accurate for image quantification. The gain is disabled and the raw photon numbers are returned with no further signal amplification as in the Standard mode. You can improve the signal by Line Accumulation or Frame Accumulation.

Don't use the HyD detectors for channels with bright signal, like the DAPI/Hoechst signal. Check first with PMTs how bright the signal is. Overload will result in a shot down of the detector, a beep and a warning on screen. Overloads are also counted by software.

HyD detectors also always stay ON, even if they are not in use. So a very bright DAPI signal in PMT 1 can overload HyD 2, even when you have not selected anything for HyD 2. If not in use please make the slider for the HyD detectors as small as possible and never park them directly in the wavelength of a laser beam.

Besides the 5 detectors for detection of fluorescence light there is one detector for the detection of transmitted light. Keep in mind that light going to the transmitted light detector does not pass the pinhole and therefore is not an optical z-section!

More reading about HyD detectors:
Leica info
Detectors for sensitive detection: HyD

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