Important phone numbers:
Manny Benish 0546708136 |
This is an internal page where we keep a record of everything that is relevant for use of lab microscopes. This can be general protocol information, common errors or troubleshooting, as well as up to date information about microscope parts that are being repaired, who is taking care of this, where a specific objective is, etc.
To Do ListNew Green/Red Wide band pass filter set for TIS. Make sure to check which parts of a filter set we already have left over in lab.
Computer controller for decreasing power of the new Obis Solid 488 laser NIS software upgrade |
Important Updates
August 13th'14 - Rotem
How to photo convert on the A1R with very short pulses
NIS Elements has a few bugs when it's used for photo conversion pulses. Many times it is hard to set the desired duration of the photo conversion pulse. If you want to give a very short pulse, you can follow this procedure. Please notice that you currently cannot choose a frame and stimulation point and immediately give a pulse shorter than 30ms. However, if you don't find giving a few pulses at the same location, then using this procedure you can shorten the pulses to even single miliseconds.
1) Set the pulse duration to 30ms, then click the apply button (or if it's disabled, click the enable lasers button and then click apply). Usually what will happen is that the value that you wrote will change to 30.68ms, which is what NIS Elements thinks is the shortest pulse duration possible.
2) If you did not manage to set the pulse duration as 30.68ms, it probably means that you already applied a long photo conversion pulse at a previous experiment in the same session. A bug in NIS Elements enables you to decrease the pulse duration by no more than 2 fold each time. For example, if you used an 800ms pulse and you now try to set the new duration to less than 400ms and click the apply button, the duration will jump back to 400ms. The way to overcome this is to give a pulse at 400ms, then set the value to 200ms. It will work. Give another pulse, and set the value to 100ms, and so on until you reach the desired value. Alternatively, you can close and relaunch NIS Elements, and then you should be able to set the pulse duration to 30.68ms.
3) If you managed to set the pulse duration to 30.68ms and you want an even shorter pulse, it is possible only under certain conditions. You must first give a 30.68ms pulse and click the finish button. Then, *without* changing the frame, the zoom, or the location of the stimulation ROI, set a new value to the pulse duration. NIS Elements will enable you to decrease the duration by 2 fold, down to ~15ms. Give another pulse, and click finish. Again without changing the frame etc. set a new value for the duration. It will decrease down to ~7ms and so on.
Apr 17th'14 - Amen
A1R:
- after starting NIS software moving Transmitted Detector IN/OUT helps restore green and red channel detection
- sometimes in galvano scanning mode Z stack acquisition starts from the middle, relaunching software and interchange btw resonant/galvano modes helps
Jan 20-21th'13 - Amen
A1R - usually when starting the system one needs to change eyeport<--> L100 manually first time, only then use "live" in the software.
SIM - sim-tirf considerations:
1. Laser alignment in TIRF mode(=> single mode) easier to do first w/o grating block inside, then with.
2. Alignment is done after focusing on the sample, removing the plate and cleaning the objective
3. No need in beads and PSF in 2D-SIM and TIRF
4. To make image look better you can close filter diaphragm (on the left side of the mic. - lower one)
5. In order not to bleach the sample use sine wave mode while looking for the right place
6. After playing with angle do realignment.. or just don't play
7. Sometimes autoscale button is on
September 4th, 2013 - Dan
This could be a really useful plugin for SIM from ImageJ:
http://www2.bioch.ox.ac.uk/microngroup/software/SIMCheck.shtml
Oct 16th, 2013 - Ayeletchen
The laser power changes is not liniar under 2% power. This meens we cant know if we work with the same laser power as previeus experiments unless we use more than 2% power. To overcome this problem we can insert a filter to the laser box and work with higher laser power or connect the laser to the computer and change the power from there.
July 24th, 2013 - Meytal
Some SIM Updates:
The Piezo stage wasn't working efficiency, due to uncalibrated voltage. To align the piazo's voltage, one should go to the box under the microscope, and open the case attached to it. Inside there is a volt-meter, a BNC wire and a thin screwdriver.
Now follow these steps:
-Turn on the volt-meter.
-Attach the BNC wire to the left exit on the box itself, and the other ends to the volt-meter (Black to black, red to red).
-Disconnect both BNC wires that are attached to the piezo box.
(The voltage should be -0.1v to -0.5v)
-To change the voltage, use the thin screwdriver in the small adjustment hole that is located near the right BNC exit, (that is now connected to the voltmeter), and turn it (gently!) until the voltage is correct.
-Reattach the BNC wires that were previously connected and move the piezo stage up and down. The voltage should be correlated with the height of the stage. (at a height of 50nm the voltage should be 5v).
Good luck!:)
July 24th, 2013 - Ayeletchen
We ordered new fluorescent lamps for the TiS and for the A1R. The first can be ordered from few companies but the second can be ordered just from Agentek.
June 19th, 2013 - Dan
I have decided to move the 60X water na1.2 objective to the TIS scope in the TC room. I've noticed that given the availability of the 60x na 1.27 objective, no one wants to use the 1.2 for the A1R. I want to get maximal utility out of all of the equipment that we have - hence I think that the W60x 1.2 will be useful in the TC room for looking at cells. What this means is that we have to be extra careful with the TIS, since it now has an expensive objective on it. It is probably good policy to take the W60x off and store it above the scope, if its not being used.
July 1st, 2013 - Rotem
I am working with Kees at Nikon and with Lior at Agentek to enable the theoretically possible but practically problematic feature of the A1R to do photoactivation experiments in single line scanning mode. Trying to do such an experiment causes NIS-Elements to somehow lose connection to the hardware. Current status: I sent Kees a log file and I'm waiting for his diagnosis. The log file is on the A1R computer and is called SysInfo_20130630_002843.zip
July 22nd, 2013 - Rotem
I set up the workstation. It has Matlab and NIS Elements AR on it. Right now it's connected to the internet using a wifi card. That will change when Claire will be persuaded to connect the computer to the network. Meanwhile, that means that in order for Matlab to work, the computer has to be connected to HUJI VPN. To do that, activate Cisco AnyConnect application from the start menu and log in with your %ra account. Another thing that the lab might consider is to buy a Matlab license; the current instance of Matlab runs with a student license I got from the Aguda. As for NIS Elements, it does not need an internet connection, but it needs a dongle; Manny is supposed to bring an extra dongle soon and meanwhile you can use the dongle from the SIM if it's not in use.
July 24th, 2013 - Rotem
The workstation is now connected to the HUJI network. The wifi card is back in the a1r room. Manny says we need to buy a new dongle. I asked Lior for a hatza'at mechir.
A1R - usually when starting the system one needs to change eyeport<--> L100 manually first time, only then use "live" in the software.
SIM - sim-tirf considerations:
1. Laser alignment in TIRF mode(=> single mode) easier to do first w/o grating block inside, then with.
2. Alignment is done after focusing on the sample, removing the plate and cleaning the objective
3. No need in beads and PSF in 2D-SIM and TIRF
4. To make image look better you can close filter diaphragm (on the left side of the mic. - lower one)
5. In order not to bleach the sample use sine wave mode while looking for the right place
6. After playing with angle do realignment.. or just don't play
7. Sometimes autoscale button is on
September 4th, 2013 - Dan
This could be a really useful plugin for SIM from ImageJ:
http://www2.bioch.ox.ac.uk/microngroup/software/SIMCheck.shtml
Oct 16th, 2013 - Ayeletchen
The laser power changes is not liniar under 2% power. This meens we cant know if we work with the same laser power as previeus experiments unless we use more than 2% power. To overcome this problem we can insert a filter to the laser box and work with higher laser power or connect the laser to the computer and change the power from there.
July 24th, 2013 - Meytal
Some SIM Updates:
The Piezo stage wasn't working efficiency, due to uncalibrated voltage. To align the piazo's voltage, one should go to the box under the microscope, and open the case attached to it. Inside there is a volt-meter, a BNC wire and a thin screwdriver.
Now follow these steps:
-Turn on the volt-meter.
-Attach the BNC wire to the left exit on the box itself, and the other ends to the volt-meter (Black to black, red to red).
-Disconnect both BNC wires that are attached to the piezo box.
(The voltage should be -0.1v to -0.5v)
-To change the voltage, use the thin screwdriver in the small adjustment hole that is located near the right BNC exit, (that is now connected to the voltmeter), and turn it (gently!) until the voltage is correct.
-Reattach the BNC wires that were previously connected and move the piezo stage up and down. The voltage should be correlated with the height of the stage. (at a height of 50nm the voltage should be 5v).
Good luck!:)
July 24th, 2013 - Ayeletchen
We ordered new fluorescent lamps for the TiS and for the A1R. The first can be ordered from few companies but the second can be ordered just from Agentek.
June 19th, 2013 - Dan
I have decided to move the 60X water na1.2 objective to the TIS scope in the TC room. I've noticed that given the availability of the 60x na 1.27 objective, no one wants to use the 1.2 for the A1R. I want to get maximal utility out of all of the equipment that we have - hence I think that the W60x 1.2 will be useful in the TC room for looking at cells. What this means is that we have to be extra careful with the TIS, since it now has an expensive objective on it. It is probably good policy to take the W60x off and store it above the scope, if its not being used.
July 1st, 2013 - Rotem
I am working with Kees at Nikon and with Lior at Agentek to enable the theoretically possible but practically problematic feature of the A1R to do photoactivation experiments in single line scanning mode. Trying to do such an experiment causes NIS-Elements to somehow lose connection to the hardware. Current status: I sent Kees a log file and I'm waiting for his diagnosis. The log file is on the A1R computer and is called SysInfo_20130630_002843.zip
July 22nd, 2013 - Rotem
I set up the workstation. It has Matlab and NIS Elements AR on it. Right now it's connected to the internet using a wifi card. That will change when Claire will be persuaded to connect the computer to the network. Meanwhile, that means that in order for Matlab to work, the computer has to be connected to HUJI VPN. To do that, activate Cisco AnyConnect application from the start menu and log in with your %ra account. Another thing that the lab might consider is to buy a Matlab license; the current instance of Matlab runs with a student license I got from the Aguda. As for NIS Elements, it does not need an internet connection, but it needs a dongle; Manny is supposed to bring an extra dongle soon and meanwhile you can use the dongle from the SIM if it's not in use.
July 24th, 2013 - Rotem
The workstation is now connected to the HUJI network. The wifi card is back in the a1r room. Manny says we need to buy a new dongle. I asked Lior for a hatza'at mechir.
Additional Basic Microscope Policies
Please do not put ANYTHING on the PMT detector box OR on the laser box. These things are not lab benches.
Keep the microscope rooms clean. This should be maintained on a daily basis, and once every two weeks there should be a concerted effort to clean the rooms extensively. They do not get cleaned by the cleaning staff, and trash does not get emptied. There should not be dust on the floor or on the equipment, and there should not be excessive piling up of materials in the rooms that do not pertain to the equipment (slides, plates, liquids).
Please be careful about overcrowding in the microscope rooms. There is a tendency to bring in additional chairs and people - in the A1R room this can lead to undesirable situations where equipment gets knocked around.
When operating the water pump to deliver water onto the water objective, please make sure that the adaptor is firmly in place, and that it won't move and deliver water to the rest of the microscope. Double and triple check this, and also make sure the pipetting volume is not too high.
There should never be any of your data on the C drive. The D drive is appropriate for temporary storage, until the data can be moved to an external drive. Keep in mind that when you click the "save to file" box, the data is being saved whether you need it or not.
If you move something, send something to be fixed, change something - tell us about it. That's why we have this page.
Keep the microscope rooms clean. This should be maintained on a daily basis, and once every two weeks there should be a concerted effort to clean the rooms extensively. They do not get cleaned by the cleaning staff, and trash does not get emptied. There should not be dust on the floor or on the equipment, and there should not be excessive piling up of materials in the rooms that do not pertain to the equipment (slides, plates, liquids).
Please be careful about overcrowding in the microscope rooms. There is a tendency to bring in additional chairs and people - in the A1R room this can lead to undesirable situations where equipment gets knocked around.
When operating the water pump to deliver water onto the water objective, please make sure that the adaptor is firmly in place, and that it won't move and deliver water to the rest of the microscope. Double and triple check this, and also make sure the pipetting volume is not too high.
There should never be any of your data on the C drive. The D drive is appropriate for temporary storage, until the data can be moved to an external drive. Keep in mind that when you click the "save to file" box, the data is being saved whether you need it or not.
If you move something, send something to be fixed, change something - tell us about it. That's why we have this page.
