Experiments were conducted at the Central Imaging and Flow Cytometry Facility (CIFF), National Center for Biological Sciences (NCBS), Bangalore, India.]
We have a Becker and Hickl (B&H) Simple Tau 152 DX TCSPC system. The system uses the two sets of detectors:
The detectors are attached to the Direct Coupling (DC) port of an LSM 780 Zeiss confocal microscope. The detectors are connected to the DC port using a A-Z-DC-710-PMZ beam splitter adapter from B&H. Having two detectors in each set is useful for techniques like Time Resolved Anisotropy (TRA), Fluorescence Lifetime Gated FCCS, or simply two color FLIM. A MaiTai Ti:Sa femtosecond laser at 80MHz is used for multiphoton excitation.
The MCPs offer a shorter IRF but have a lower sensitivity and more after pulsing compared to the Hybrid GaAsP detectors. Depending on the application users prefer different sets of detectors. Hence we end-up swapping the detectors every once in a while. We thought it would be nice to describe the hardware configuration involved in connecting up the two sets of detectors, which is what we plan to do in this technical note.
Our B&H Simple Tau 152 DX TCSPC is a table top system that comes with a Laptop connected to a fast bus extension interface. The following cards are housed in the fast bus extension interface:
Configuration 1: Setup with R3809U Multi Channel Plate (MCP) detectors
Figure 1527/1 shows two MCP detectors connected to the DC port of the Zeiss LSM 780 confocal microscope using the B&H beam splitter. Note that mechanical shutter and the photodiode associated with each individual detector detectors were removed due to a small malfunction. The photodiode and shutter combination detect overload conditions and protect the MCP. If you are using the MCPs without this you need to be very careful about overload conditions.
The connection diagram for connecting two MCPs to the Sample Tau 152 system is described in the B&H TCSPC handbook and is shown in Figure 1527/2.
The R3809U MCPs donot have internal high voltage generation circuits. Hence an external high voltage supply needs to be used. Our system uses a FUG HCN 14-3500 high voltage power supply as shown in Figure 1527/3.
The power supply system can supply upto 3.5kV. It has however only one output. Hence a voltage splitter is used to create two outputs to power the two MCP detectors. Figure 1527/4 shows the high voltage splitter.
This is a passive splitter. This is fine since high voltage supplies operate at very low currents. So splitting the voltage passively does to load the power supply.
The DCC 100 card has three connectors con1, con2 and con3. The con1 and con2 connectors are connected to the DCC1 and DCC2 connectors of the on the P Box. The P Box is powered by a 12 volts AC to DC wall mounted adapter. This is shown in Figure 1527/5.
The P Box has there outputs Shut1, Shut2 and Detect.
Both Shut1 and Shut2 are connected to a connector with two output cables. These two cables are connected to the shutter and photodiode inputs respectively on the assembly of each of the MCP detectors. In our case since we had removed the shutter-photodiode assembly, we did not connect these.
The Detect output of the P Box is connected to a connector with 3 cables. One cable is for the high voltage control of the power supply. This helps the power supply to be controlled and the voltage varied using the TCSPC software. In our case, we usually donot control the power supply using the software. We manually set the high voltage to a constant value of 3.4kV. The other 2 cables are for overload (OVLD) triggers and they are connected to the OVLD inputs of two widebad amplifiers.
The signal output of each MCP is connected to the IN pin of an individual wideband signal amplifier (B&H HFAC-26dB) as shown in Figure 1527/6.
The amplifiers are powered by a 12V AC to DC wall mounted adapter. The signal output of each of the two wideband amplifiers is connected to the Constant Fraction Discriminator (CFD) input on the two individual SPC-150 TCSPC boards housed in the fast bus extension interface.
A breakout box BOB-4 is used to split the scan-clock from the LSM-780 confocal microscopes onto the two SPc-15 cards. Figure 1527/7 shows the breakout box.
The scan input of the breakout box is connected to a connector with 2 cables. The two cables are connected to the scan clock outputs at the back of the Zeiss LSM 780 real time controller. This is shown in Figure 1527/8. If the breakout box is used only for splitting the scan clocks, it need not be externally powered.
The SPC-1 and SPC-2 outputs are connected to the scan clock inputs of each of the SPC-150 cards.
The Sync inputs for each of the two SPC-150 cards are obtained by bleeding a small amount of the femotsecond laser light at 80MHz using a thin glass slide onto a B&H PHD-400 high-speed photodetector. This is shown in Figure 1527/9.
The output of the photodetector is split using an SMA Y splitter as shown in Figure 1527/10 and fed to the SYNC inputs of the two SPC-150 cards.
It should be made sure that length of the two cables and effective delay in them of the two coaxial cables connecting the SYNC inputs of the two SPC-150 cards as similar as possible. Also, the length should be adjusted to get the optimal timing. Else additional patch cords should be added.
Configuration 2: Setup with HPM-100-40 hybrid GaAsP detectors
Figure 1527/11 shows the connection diagram for connecting two HPMs to the Sample Tau 152 system as described in the B&H TCSPC Handbook.
The setup is very similar to the one described above for MCP detectors. The main differences are:
- HPMs have internal high voltage generators. Hence an external high voltage source is not required.
- The Con1 and Con3 connectors on the DCC card are used.
- P Box is not used
- Con1 and Con3 connectors on the DCC are directly connected to the power supply and control inputs on HPM1 and HPM2 respectively.
- The HPMs have internal broadband amplifiers hence the HPM signal outputs can be directly connected to the CFD inputs on the respective SPC-150 cards.