Farewell

Today was my last day at the Waisman Center; my vacation begins tomorrow, and when I return I'll be taking a new position in the department of Surgery. I have enjoyed the opportunity to work with everyone and hope my efforts have been appreciated. Best of luck to all.

Jolien

FACSCalibur -- pump died, will be replaced

Our service guy came and took a quick look at the FACSCalibur today. He confirmed that the pump is shot and ordered one to be shipped tomorrow. He'll be back to install it.

FACSCalibur - out of service and user accts created

Flow users,

I just finished trying to troubleshoot a pressurization problem with
the FACSCalibur and found that a fluid pump has failed (is presumably
dead). A service technician is being contacted right now and should
be here within 48 hours according to our service contract. I
apologize for the inconvenience.

Also, I finally finished setting up user accounts on the computer today.
Please sign in to your user account whenever you use the flow
cytometer and continue to sign the log book so we have your grant
information for billing. For now, we are still only charging for
cytometer usage, so please note FlowJo usage in the log book to help
us differentiate.

User accounts do not currently have passwords assigned, but you are
welcome to password-protect your account. The data is still under
User Data on the main hard drive, and you can copy your data over to
your own Documents folder. Unfortunately, if you'd like the
applications you use (CellQuestPro and possibly FACSComp and/or
FlowJo) to be on your dock, you need to drag them from the BD
Applications folder or Applications folder (for FlowJo) onto your
dock.

FlowJo users need to re-enter the serial number the first time they
use FlowJo from their new user account. I have printed this out and
put it in the drawer under the cytometer.

Thanks for your co-operation and sorry for any inconvenience.

Both Scopes Cleaned/Serviced Today

Sam Goodall, our old Nikon rep and now in business for himself servicing microscopes, came today and finished cleaning the confocal. Now he's working on the Zeiss and expects to finish today. We're setting up an arrangement with him to clean and service both scopes twice a year and change bulbs (mercury or halogen) as needed.

RT-PCR -- Still no software upgrade available

Our rep emailed me a few weeks ago that the software upgrade would be available today, but it wasn't on the site for me to download. I booked some time on Thursday afternoon in hopes that it will be available by then.

Confocal -- focus motor back, appears to be working

The focus motor arrived this morning and I promptly installed and tested it. It appears to be working alright now, but I didn't test it rigorously, i.e. I didn't attempt to replicate the subtle problems we were having. Please let me know right away if there are any issues or concerns with it.

Confocal - Focus motor repaired and on its way back

The focus motor is en route from Oregon after being repaired by the manufacturer. I should have it in my hands on Tuesday morning and will get it on the scope right away when I have it. I'll send out an email to confirm that it is on and working properly. Thanks for your patience.

Confocal -- Focus motor partially removed

I was able to partially remove the remote focus controller from the confocal, but am waiting for our rep to come finish removing it for shipping to Oregon. The manufacturer will repair it and ship it back as quickly as possible.

Z-stacks will not be possible until it's back, but capturing single images, including channel series (frame lambda) should be fine. I have a few sessions booked this week, and will let people know if there seem to be any problems. I would expect us to be up and running again for z-stacks by around the 21st or so (of May), but will update the blog as I hear back about it.

Calendars -- Event privacy considered

At the monthly CMN Core meeting, I raised the issue of event privacy on the CMN Core calendars. Thus far, I have been making sure all events are public, meaning anyone can see your name and timeslot. I've gotten the sense that some users would prefer not to have their names quite so public and would prefer privacy. From my end, the problem is that I definitely need to know who's signed up for which timeslot, and I think it benefits other users to be able to see who's before or after them in case of schedule changes at the last minute.

If we designate an event to be private, then all those authorized to self-schedule on the calendar can see the details, but not the general public. They can see the details when logged into Google, though when viewing the calendar via the CMN Core web pages, one often needs to click reload to get the details to appear. The general public and anyone not logged into their authorized Google account for viewing the calendars simply sees the word "busy" for each private event.

I've recommended that we increase privacy by having reservations be private on CMN Core calendars. So, I'll send out an email about this when I'm ready to implement it, then will consistently make sure events are made private, except for service events. Those who consistently use Google and are frequently logged in may not notice much, but others will see the word "busy" instead of names. Let me know if there are questions or concerns.

Confocal -- focus motor to be sent for repair

I got information from our Nikon rep and then the manufacturer of our focus motor as to the best way to get it repaired. We'll be sending it out next Friday, May 9, for repairs and should have it back by the end of the following week. As far as I know, we should still be able to capture images just fine, but not z-stacks.

GeneSifter - License to be purchased, cost $75/month or less

We have decided to purchase a license for GeneSifter software.

This software is used to analyze microarray data and is considered very user-friendly and extremely useful. The main benefits over other microarray software are that it is web-based (so your lab can use it anywhere), well-supported, and intuitive to use. It does statistical analyses using the R statistical language, picks out differentially expressed genes with various statistical algorithms, performs cluster and pathway analysis, corrects for multiple comparisons, has one-click gene summaries, identifies biological themes, allows searches by function, and provides ontology reports. The gene annotations combine information from GenBank, UniGene, LocusLink, The Gene Ontology Consortium, Affymetrix NetAffx, Homologene, and KEGG. More info can be found at www.genesifter.net.

Note that P30 Projects (those listed on the Waisman Center Core Grant) receive subsidies for CMN Core services and also for services (such as microarray runs) at the Biotechnology Center.

We decided to get a one year license and allow investigators to gain access to the software by committing to up to $100/month for 12 months. The more investigators commit, the lower the price is for everyone. We currently have four investigators ready to contribute to the license, so for now the cost is $75/month. One more will bring it down to $60/month, and two will bring it down to $50/month. If anyone is interested in trying out the software or in contributing to the license to gain full access, just contact me.

Confocal -- mercury bulb alignment and field of view

On Friday, I had our service technician come to help figure out what was going on with a concern about the field of view. Essentially, a user has been looking at cells in the dentate gyrus of mouse sections -- granule cells specifically -- and finds that the field of view looks much larger than on the widefield fluorescence scope in his lab. This makes the cells look very small and harder to count, and images captured with the entire field of view don't "look right" because they seem zoomed out.
Earlier in the week, I had taken our calibration grid slide that I use to calibrate the Zeiss and we put it on the scope to see if there was any calibration issue. We were able to get an image of the calibration grid in the red channel and a granule cell in the green channel, and indeed the cell we were looking at appeared to be only about 5 um in diameter. The PI checked the literature and found these cells to generally be described as at least 10 um in diameter.

After much discussion with the rep, we concluded that indeed our confocal tends to have a larger default field of view than many other scopes, which can be reduced if desired using the Zoom or Crop feature of the Navigation menu. Talk to me if this is of concern to you or you'd like to learn more about it.

Later that day, we found that the mercury bulb was again out of alignment. This is the second or third time this has happened, and we are very puzzled as to how this could occur. I called our service technician back and asked him to come check it out, both to get it back in optimal alignment and see if he could find any reason for it to be going out of alignment without anyone intentionally touching the knobs. This mystery remains unsolved at the moment.

Seminar -- What's New in the CMN Core -- went well and will be held again soon

On Wednesday at 11 am, I gave an hour long seminar titled, "What's New in the CMN Core". I gave an overview of the equipment and services that are available as well as updates on what we've upgraded, plan to upgrade, or are anticipating difficulties with. I'll be giving the talk again sometime soon, since many people weren't able to make it on such short notice (both the neuroscience program and Waisman Center didn't get announcements out as early as would be ideal). I'll also be putting the talk on the web site for download.

There will also be more talks in the CMN Core seminar series, with topics including Flow Cytometry analysis using FlowJo software, planning and carrying out a confocal or stereology project, and image analysis using ImageJ and MATLab software. Please let me know if there are other seminar or workshop topics you'd like to see in the series.

Confocal -- Mercury bulb went out of alignment -- fixed now

We started having a strange problem on Wednesday morning with the confocal. When looking through the eyepiece, only a blobby looking area in the center was illuminated (and very brightly). The rest of the field of view was relatively dark. As I was hurriedly preparing for the seminar, I didn't have time to check out the problem thoroughly. I had the user try the confocal, to see where in the light path the problem was, and it appeared that the problem was also in the laser light path in addition to the mercury light path. I called our scope service rep and he said he would try to get someone to come take a look.

I checked back several times later in the day, and the users that were scheduled used the confocal and reported that it worked fine (the confocal light path being fine, and the view in the eyepiece still abnormal). Then on Thursday, I was able to take a minute to check the mercury bulb alignment and it was way off. There are three knobs for adjusting the positioning of the bulb, and it was the one farthest forward that needed adjustment to get it back in alignment. Hopefully, no one is intentionally touching this knob, as it disrupts the mercury illumination for all users. Contact me if you have questions about this or would like for me to show you how to get the mercury bulb back in alignment (or what knobs NOT to touch).

Another strange phenomenon this user reports is that at 40X, often the field of view appears zoomed out, his cells of interest appearing very small. I'm looking into getting a fluorescent micrometer slide of some sort, to be able to check this type of thing out. In the meantime, let me know if you observe this same phenomenon.

FACSCalibur -- FlowJo license not to worry

FlowJo users: our license is set to expire on Friday, but not really. I just contact them each year and they give us a new serial number for the year. I should receive that soon and there should be no lapse in our license. Let me know if you have any problems, and just ignore the message that the license is about to expire. Thanks!

Confocal - access Viewer and Thumbnailer installers on MyWebSpace

University of Wisconsin users (with NetIDs) can now access the installers for the C1 Viewer and Thumbnailer from MyWebSpace. Just log into it (http://mywebspace.wisc.edu/xythoswfs/webui) then search for NetID jxconnor, folder named confocal. Open the folder, and the installers should be there. Let me know if you have any problems.

Newsletter sent out and on web site

The 4th issue of the CMN Core newsletter went out today, detailing the changes and news since the last issue about a year ago. It is also posted on the website on the News page.

Confocal -- Gear problem subtle but confirmed

Our Nikon service rep, Steve Fryer, took a look at the confocal yesterday and was able to replicate in a large z-stack the subtle abnormal behavior with the focus motor that has been observed by several users lately. He found that the steps in a z-stack occasionally would get very small for a few steps then "catch up" or come back to normal size. He cleaned and regreased the motor but felt that it may simply be a mechanical problem with no good solution. I'll be contacting the sales department to get a quote on a new focus motor, and at the same time will ask about getting a stage with XY motor control to allow some semblance of stereological sampling capability.

In the meantime, the recommendation is to watch your scans for this phenomenon, save your files as .ids format (for z-stacks), and report to me if and how often you observe problems.

Confocal -- Gear sticking problem?

A user reported to me that she noticed among many z-stacks in a row that a few times the z focus gears seemed to stick, such that 3 or 4 images were clearly in the same focal plane rather than different ones, but then the focus would change. She thinks it's a sporadic, unpredictable event, and didn't save the .ids files so that we could examine the images (she renders them and saves the resulting image).

I contacted our service rep, Steve Fryer, and he'll be coming to take a look on Wednesday afternoon next week. My recommendation for users is to watch your scans in case the focus motor behaves erratically, carefully engage the focus motor and check by trying to move the fine focus knob (gently), and SAVE .ids FILES FOR YOUR Z-STACKS. As I've probably mentioned to most users, even though they're large, they keep all of your data, so that you can judge if anything has gone awry, redo your projections, make new movies, make scale bars, and pick out the settings you used, just to name a few of the important reasons to save your .ids files.

If you are concerned about the amount of disk space this would take up, remember that DVDs are less than a dollar apiece and can store over 4 GB of data, which can be burning while you clean off the objectives at the end of your session. Talk to me if you have any questions or concerns about this.

Confocal and Zeiss -- Local Accounts deleted

I went in today and deleted all local accounts on the Zeiss computer and the Confocal computer. Thus, to access either of these computers, a Waisman logon is required. Please talk to me if you have any concerns or questions about this. All data from the Documents and Settings user folders was burned onto DVDs then deleted, but files that were elsewhere were left alone. If you need any of the data from the Documents and Settings folder (which would include anything on your desktop or in your My Documents folder), the dvds are in the small filing cabinet next to the confocal scope.

Since we are doing fine at the moment on disk space, I'll wait to clear off any other images or data from these computers until there is concern about this. The confocal computer currently has 75 GB free out of 250GB, and the Zeiss computer currently has 200 GB free out of 250GB, so I would say that the confocal computer will need clearing off in the next few months, but not the Zeiss for the forseeable future. An email will go out before I do this, and if possible, removed data will be compressed and burned to DVD, just in case.

Please make an effort to keep your hard disk usage to a minimum on these computers. Thanks.

RT PCR -- Beginning upgrade process

We are beginning the process of upgrading the ABI7500 real time thermocycler system from the computing standpoint. We would like to put the computer on the network, for several reasons, but the cost of getting the laptop imaged may not be worth it, especially since we would need to add RAM to the computer to be able to upgrade to the latest version of the software. Our ABI service rep said that the newest version of the software is very good, allowing for quick and easy export of data to Excel and PowerPoint, and with an improved interface. This upgrade is free if we download it, but we need to have enough RAM to run it.

So, Seprek (the CMN Core workstation computer) has been moved down to room T529 to be our experimental computer on which to try installing the upgrade and ensuring that the thermocycler software can complete runs without conflicts due to virus scanning. It has been reported that the software often has issues with Norton and other virus software, and with the computer on the network, we need to configure the virus scanning software (Symantec, in our case) to allow thermocycler software to run properly.

Ultimately, a different computer will run the software, either Quiberon, which we already have, a hand-me-down computer that has been de-commissioned from elsewhere in the building, the laptop (but upgraded and imaged), or perhaps a new computer. The laptop is still in the room, on top of the thermocycler (and connected to it), and we'll let users know how the upgrade process is going. Once the process is complete, all users will need to use Waisman logons to use the thermocycler. We will likely be changing to an hourly charge for thermocycler use at some point as well (probably July 1). Let me know if you have questions or concerns.

Real Time Thermocycler -- Seems ok

Our rep came today to check the machine due to our strange problem with the strip caps on the plate. He couldn't find anything wrong with the machine, especially the alignment. He did measure the block temperature and said it was a little hot (106 when it should be 105), nothing to worry about, and he adjusted it. He also found some contamination in four of the wells and cleaned that. He recommends we always use film with the plates and let him know if there are any further problems. He also noticed that many of the plates in the trash can by the machine have scratches all over the tops of the caps. If this was like this during the run, it would cause artifacts. We couldn't figure out where the scratches were coming from. Let me know if you know anything about this.

RT PCR -- strip cap problem

Last week, a user reported running some reaction plates on the Real Time PCR machine and having a problem with her strip caps popping off during the reaction. She noticed that the data from that column of reactions was messed up, so she's pretty sure it happened early in the reaction, probably during the initial 95 degree denaturation step. For her next reaction, she made sure all the caps were on tight, yet it happened again, with the caps in the very same column (#5).

It may have been a bad batch of caps, though it still would be weird that the problem happened twice in the same column. Please let me know if you've had a similar experience or if you have insight into this problem.

I spoke with technical support and our engineering rep at ABI, and they had the following advice: first they said to make sure we use the flat optical caps, not the domed (we already do). Next, they suggested using film instead of caps; we have some film and an application kit if anyone would like to use it. Lastly, the engineering technician said to go ahead and try running an empty plate using the RNAse P run to see if it happens again. He couldn't think of any explanation that involved a problem with the machine, but he's willing to come take a look at the machine on Monday.

FACSCalibur -- sheath fluid arrived

Our 10 boxes of sheath fluid arrived today, so we should be good on that for some time. They are all under the table by the confocal. Let me know if there are any questions or concerns about this.

Confocal -- locked up by security log

The confocal locked up last night due to the security log being full. I remotely cleared the log, so we should be fine for now. I'll put a ticket in to the IT staff to help develop a way for this to happen automatically or for there to be more space for the log so it doesn't lock up the computer again.

FACSCalibur -- No sheath fluid

We ran out of sheath fluid yesterday, and I hadn't realized that we were even low. I ordered 10 boxes right away, but they probably won't be in until Thursday at the earliest. I'll make sure to be more diligent about noticing when we're low in the future, and help from users on this would be greatly appreciated. In the meantime, if there's anything urgent you might need to do, you can use PBS; I have some in the Core room (T635B) if anyone needs some.

Confocal - focus motor gear slippage

I spoke with Steve Fryer, our scope rep recently about the problems some users have seen with the gears slipping when the focus motor is engaged. He stopped by and looked at it himself and said that the problem is that when you do manual focus, then engage the focus motor, it occasionally and randomly happens that the gears don't perfectly mesh right away. Then, when you start moving the focus, the gears slip into place, there's a clicking or popping sound, and from then on the gears are properly meshed. He said that the best way to handle it is to check the manual focus knob right after engaging the focus motor, and trying to move it a little should click it into place if it was not aligned. Then, it won't slip during the z-stack or when setting up the top and bottom. Let me know if you have any questions about this.

FACSCalibur -- sheath filter replaced

This morning when I came in, the FACSCalibur was on in Run-High mode, though the sheath fluid had long since run out. The user from yesterday had left it on like this all night. I tried to get it running again, but couldn't get anything to flow through the sheath filter. I called instrument support and they confirmed that the sheath filter was no longer useable and had to be replaced. Luckily, I had just ordered and received a spare sheath filter last week, so I used that one. I also changed the waste tank cap to the unbreakable type. With the new sheath filter, I was able to get the system running again, ran FACSComp, and it passed. We're up and running again. Hopefully, this won't happen again.

Zeiss -- Stereo Investigator 8 is installed

MicroBrightfield was willing to let us have version 8 of Stereo Investigator now because of all the problems we've been having with corrupted files. They also know that I've been frustrated with several bugs in version 7.5 that apparently have been fixed in 8. Sure enough, everything seems to be much better now. I'm very happy with the icon customization that we can do now, and my work has been going very smoothly.

Zeiss -- Our new X-Cite illuminator is here

Our X-Cite illuminator arrived and was installed by our rep yesterday. We let it sit on for two hours to "burn in" the new bulb or something like that (instructions from Sam, our rep). It works the same as ours, and we can expect to get about 2000 hours out of it before having to change the bulb.

One thing he mentioned is that we should try to close the iris any time we're not using the light, because it will allow the "liquid light cable" to last much longer. He explained that the head of the mercury vapor illumination gradually wears on the cable and that simple step can extend the life of the cable by years.

Zeiss (Stereo Investigator) -- Doing much better

We had been having greatly increased crashes and errors from around the first of the year til around the 8th of January. That evening, Christine was having such severe problems with Stereo Investigator that she couldn't continue any productive work. I came in that night for troubleshooting, and was unable to resolve the problems after many attempts. However, I did update the software from version 7.53.2 to 7.53.3. We read the release notes, and there was no indication that any of the errors Christine, Kevin, or I had been having would be resolved, but figured it probably couldn't hurt. I also showed Christine how to run the Optical Fractionator Probe without the workflow (the old-fashioned way, as I call it). This worked, while the workflow crashed every time she attempted to begin counting (and also lost her parameters every time).

Well, both Kevin and I noticed reduced crashes and errors since then (I haven't been having any at all, knock on wood). I called MBF the next day and they requested a copy of the file that was causing crashes. I sent it, and they "fixed" it and sent it back. However, the "fixed" version was an .asc file, and both Christine and I opened the .dat file, which was really the original, buggy file. Since then, Christine has completed that monkey using the old fashioned method and moved on to the rest of her work, so we haven't tested the "fixed" file.

They also told us that version 8 of Stereo Investigator will be released soon, and should fix all the bugs we've been having, and also should have many new useful features. They won't reveal the release date, however. Let me know if you have any questions about this.

Zeiss -- Stereo Investigator is crashing

Users have been having numerous Stereo Investigator crashes lately, and we don't yet know the reason. I personally have been experiencing frequent Ludl stage errors, but it has not really impeded my work much. Two users who are doing Optical Fractionators have been struggling to get anything done. Tonight, I came to check it out and found that using the Optical Fractionator without the workflow appeared to work, and that starting a new file from my own account (rather than the user's account) worked alright, but we couldn't get the workflow to not crash when she was logged in and using her data file. I'll call MBF tomorrow to see if they have any insight.