Zeiss -- Attenuator is here

The attenuator for the new mercury illumination system arrived last week and is in place. It allows for a very fine level of control over the light level, although it doesn't dial down in 1% increments like the Atto Arc. It sits in the Aperture slot right behind the red suppression filter in the Zeiss fluorescent light path. It's reasonably straightforward and easy to figure out.

Survey is out -- please respond

I emailed all the CMN Core users on the main email list a link to the new CMN Core services and equipment survey. Responses from this will be critical in shaping how we make purchase decisions, what services we offer, what types of training and workshops should be offered, and how the website will be managed. Thank you to anyone who has already filled out the survey, and please fill one out right away if you haven't yet done so!

Zeiss -- Fluorescence is working now

Our rep, Sam Byerly, was able to bring by a loaner X-Cite illumination system, and it works great. There is no alignment to do, and the field is evenly illuminated due to the "liquid light" system, I believe. The bulbs last 2000 hours, instead of just 300, so that's an improvement too. The only downside is that instead of using the Atto Arc and dialing down by 1% increments to adjust intensity, there is a slider on the X-Cite unit that allows 0%, 12%, 25%, 50% or 100% of the light through. We have ordered an additional attenuator that should effectively allow us a similar level of control but with a less simple interface (coordinating the attenuation using both controls in tandem). Anyway, I took my first virtual slice today, using red fluorescence at 10X, spanning about 10 fields of view, and WOW! It looks great, and without me having to tweak the alignment. We can now do fluorescent montages without having to crop as many pixels and without having to account for the uneven illumination. Our X-Cite unit will be identical to the one we're borrowing, and should arrive next week.

Confocal -- Back up and running

Our rep, Colleen Lavin, came and installed a new DOM unit in the confocal. Apparently that's a small hard drive in the C1 controller that communicates with the computer. It seems to have done the trick; everything's working now.

Zeiss - New illuminator ordered, may get loaner

We have put in an Emergency Transaction to try to get a new fluorescence illumination system (X-Cite) right away. Our rep said that if there was any delay with the order processing, he would loan us a system to use until our new one comes in. It sounds like we could expect that later this week or early next week.

Confocal - looks like it'll be a while

I went through a brief troubleshooting session with a Nikon rep and confirmed that the problem was not with the BIOS of the controller. Thus, he said we need to ship the controller and the scan head to NY for service. I asked if we could have anything loaned to us for the time being, so experiments could continue, and he said he'd get back to me. (I wouldn't count on it) I spoke with Colleen, our local rep, and she said that she would try to get a Nikon confocal service engineer up to Madison if possible, to see if it could be fixed locally, and would also talk to Steve Fryer and/or anyone who might be able to help. I requested that as a minimum, could she send a Nikon person to come do the disassembly as it appears to be a bit complicated.

Zeiss and Confocal both down

As of today, both the confocal and the Zeiss mercury lamp were not working.

The Atto Arc for the Zeiss went down sometime between Tuesday afternoon and Wednesday morning. Sam Byerly, our new Zeiss rep, came promptly on Thursday and checked it out. He said that both fuses were blown, replaced them both, unplugged the power supply, then plugged it in and fired it up again. It blew the fuses again, so he said that it needs to be replaced. Zeiss no longer services Atto Arcs, so we need to buy either a FluoArc to go with our existing mercury lamp housing or upgrade to an X-Cite system that should provide homogeneous illumination without needing any alignment whatsoever. It looks like the X-Cite is the way to go, but it could be 1-4 weeks before it arrives and gets installed.

The confocal apparently went down sometime between Thursday early evening and Friday morning. The problem is the laser controller, which won't communicate properly with the computer. The power light comes on, but not the ready light, and if you try to boot the software anyway, it says it can't communicate with the IP address (of the controller, presumably). I had Heather from the IT dept look at it, and she felt that it wasn't just a cable problem. I called Nikon and they were baffled by the problem. They said they'd never heard of anything like this happening.

I grew concerned about the possibility of a power surge causing our problems, but spoke with an electrician who said that we would likely have noticed more damage to other pieces of equipment if there had been a surge. I picked up some surge protectors from the IT dept and also had Bruce take a look at the mercury power supply, since Zeiss won't service it in any way regardless.

Nikon eventually called me back and told me that there is some troubleshooting we can try on Monday, but that we probably need to pack things up and ship them to Nikon for repair.

New calendaring up and running

I've been working on the website trying to integrate the new Google calendars into it, and things seem to be working well. Anyone wishing to self-schedule on the new calendars will need to use a Google account, and all others can simply fill out a request form to be scheduled for equipment use. Haven't had any major problems as yet, and have received a fair amount of positive feedback. The website has been revamped, and all the instructions and forms are now available online.

FACSCalibur - Failed time delay again

I went to run FACSComp again today and again it failed time delay (signal out of range). I hit the reset buttons and did a hard prime without fixing the problem. The error changed to the "more than 5 channels difference" message. The sample voltage had been fine. So, I went to the Time Delay Update folder and got the previous settings back. Then, it was ok.

Confocal - "Line-iness" issue, in timelapse

I got a call from Austin a few days ago saying that there seemed to be some sort of "line-iness" problem with the confocal. I went up to investigate and saw that something seeemed a little weird with how their time series images looked, generally when you went from image to image. It seemed like maybe it could be due to a very subtle laser flicker, so that individual rows of pixels were slightly brighter or fainter than the ones in the same spot on the next image. Since they use pseudocolor to detect very subtle changes in fluorescence, it is more noticeable than it would be if they were not using pseudocolor.

I called Colleen who said she would make sure Steve Fryer stopped by when he was in town.

He stopped by on Thursday to check things out and he's not sure anything can be done. It was difficult to fully replicate the problem because Jason Weick didn't save the files in .ids format, but he got a sense of it. I think he thought that because we're talking pseudocolor, which is so sensitive, very subtle laser fluctuations can result in this effect. He mentioned another potential problem that is probably not the cause but would be of concern if it was. I can't recall what it was, but something like the laser stabilizer or something like that.

Meanwhile, we set up for him to come clean the system thoroughly once a year, piggy-backed onto some scopes at the vet school that are cleaned annually and will be serviced within the next few weeks. Steve Fryer emailed me to ask how the week of November 5 looked for coming out and I said it was wide open. Hopefully, we'll draw up an agreement that we'll be serviced annually and save money on travel expenses by doing it at the same time as other campus scopes.

FACSCalibur - O Ring resolved, failing FACSComp now, was bubble

So, I forgot to update the blog about the O Ring issue.
Basically, we could get it to work with the O ring that was replaced by Straub, but tubes kept falling off and it just didn't feel right. The rep called to follow up and I told him what we were experiencing and that because it was the one that Straub placed on the cytometer, I felt they should send us a new one. They sent it right away and I received it the next day. It still feels a little different but is working overall, so we're happy. The rep, Oscar I think, called to check on things, which was nice.

The latest problem was that it was failing FACSComp. Torey Browning came to use the scope on Wednesday and couldn't get it to pass. She left me a message and I checked things out again. It was failing time delay, and the status showed low voltage. I reset the small black buttons, because there was a red and orange light. It didn't help. I did a regular prime and it didn't help much. Then, I did a hard prime and that worked. It must have been a bubble. Passed FACSComp.

Then, I finally got a call back from BD, Ref # 889248. The rep, who I've never spoken with before, wanted to be sure that all was well and that the problem was more than just temporarily fixed. I told him what I had done and that the sample voltage to go from 8.30 to 10.23, and he said that sounded good. He had me created a folder on the desktop called Time Delay, with two files inside: a Time Delay Cell Quest Pro document and a Time Delay Settings file to keep the settings that worked. He said our red laser looked good because the voltage was 684 (up to 750 is good, above 850 means you need a new laser). He also explained that the colored LEDs inside the machine relate to specific requirements. The event rate needs to be >200/sec, the FSC and FL4 need to be over channel 400, and the threshold needs to be above 200. Otherwise, it turns yellow. There's a window into which the events need to fall for the red light to not be on, and when the red light comes on, you're "out of range" and fail FACSComp. He told me that the lower black button is the update button and updates the range to match the events (to a certain extent) and the upper button should rarely be used because it's the resent button and removes the window altogether.

He also said that it's normal to get bubbles, as we do, and that probably they are usually in the sheath filter. To check this next time, I can bypass the sheath filter and see the effect. I had thought our bubbles were in the flow cell. We'll have to see.

FACSCalibur - O ring not forming seal

Last night, I received an email from Jason Meyer indicating that he had problems getting the RUN light to turn green, and that the seal with the tube didn't seem tight. He also said that he changed the sheath fluid but couldn't stop it from leaking.

I came in this morning and fixed the new sheath fluid cap and checked the system. There were a few air bubbles in the line, but everything else looked fine. I pressed RUN and the light stayed amber. I changed the tube and could feel that the seal was not tight. I took apart the assembly and put it back together with no improvement. So, I called BD support. Reference number was 886802; the rep said someone would call me.

Background: Periodic Maintenance was done by Dave Straub on the 4th. Prior to the PM, the cytometer was failing FSC in FACSComp, due to high minimum scatter values. Straub's reports show it passing with flying colors after the PM. At the time of the PM, I realized that our unlabeled and PE beads had expired over a month ago. We used them anyway and I asked Eric to order some new sets of beads as well as some FACSRinse and FACSClean solutions.

10:30 am: finally received a call back. Initially, they said that consumables such as O-rings are the customer's responsibility. After telling the rep that PM was just done and that the problem occurred right after PM, he said that Straub would have replaced the O ring for us, and that it may have been tighter than expected, such that the next user might have been too forceful and bumped it out of position, causing the problem. I again took the assembly apart and reassembled it and moved the O ring around, and while I was on hold, it began working properly again. I told Jason Meyer about it, and he will be trying to run his samples soon.

Blog start

Here is the start of the CMN Core Blog. Inside will reside records of issues, concerns, problems, and anything else that comes up with any of the equipment or services.

Each entry will show the equipment name and a brief summary about the entry in the title, and the details in the body. IT issues will have IT next to the equipment name in the title.

Anything that came up before the creation of this blog may at some point be listed in an entry, to provide background information.