Bugs and current status

Below is a not-necessarily-complete record of pending and past technical problems with the LMCF microscopes.

11/3/09 - 510inv - Replaced the fast axis scanner, ran pinhole adjust.

11/3/09 - 510upright - Replaced the motorized FD in the microscope stand, this may be causing the reasonably frequent microscope hangups.

10/9/09 - SP5 - mirror in TL path was misaligned, fixed.

10/6/09 - SP5 - 405 fiber replaced, power improved to 6.2 mW. 405 and visible lines aligned.

7/28/09 - 510 upright - High voltage board replaced. Intermittent failure of PMT3. Changing the gain to max produced no noise. Offset produced uniform background. Sometimes these only fail when gain goes above a certain threshold.

7/20/09 - SP5 - 405 laser was by serviced by John Zhang of Leica. What he found was that the AOTF that controls the laser intensity gave two frequency peaks. The smaller and abnormal one absorbs and wastes the laser power. He minimized the abnormal peak and increased the major frequency peak. In addition he optimized the pinhole for the 405 laser. The output of laser at the objective increased from 0.15 mW to 2.3 mW.

The other problem: the 405 laser will be turned on whenever the laser power switch is on. This problem was examined, but not solved. John suspected that the laser control circuit board was at fault. He will examine other SP5 machines when he visits other institutes recently and determine the root of the problem.

Laser intensity:

405nm 2.3mW
458nm 1.8 mW
476nm 2.3 mW
488nm 6.7mW
496nm 1.7 mW
514nm 5.2 mW
594nm 0.44 mW
633nm 2.9 mW

7/18/09 - SP5 and TIRF - The bug (about 6-week old now) that the computer was trying to install a LAS file when logging on to the HOMER domain on SP5 and TIRF was fixed by Michelle of NetFriends. The problem was caused from an automatic inventory management software on the server.

7/9/09 - 510 upright - 510 upright - power supply on microscope blown, awaiting part 7/8/09 Zeiss parts ordering system crashed so part delayed until 7/9/09. . . Charlie installed the part and the problem was fixed.

June 2009 - Live cell station - stage had stability/accuracy problem, sent away for repair, returned from Marzhauser with issue with encoding in Y-axis completely unable to be controlled with rotary controller or by MM via MTB. Y-axis position didn't change with stage movement. Zeiss found replacement stage for now. Stage is made specially for Zeiss so MH unable to support directly.

5/11/09 - Live cell station - The alpha-Plan-Apochromat 100X/1.46 Oil DIC (UV) Vis-IR objective was found defective. The front element came off and was floating. Lens was sent back to Zeiss for repair (RMA 41050549). (6-19-09, quoted expense for repair; $1907 came back as $1348). Reinstalled on the scope 7/28/09.

4/20/09 - 510 upright - replaced x-scanner, sounds much better and no image defect visible with alignment grid.

4/20/09 - SP5 - Installed 405 nm laser, 1 mW at objective. Dana had difficulty aligning the laser (most of the power was to the uncontrolled spot after the AOTF). Needs improving.

4/13/09 - SP5 - 405 nm laser replaced but new part defective. Will be replaced 4/20/09. Software upgraded to LAS AF 2.0, remote diagnostic unit installed.

3/24/09 - Live cell station - Fuse went in the XL heater, and the light guide cracked and was letting through a few % of normal light transmission

3/2/09 - SP5 - 405 realigned but power only 10 mW at laser, should be 50 mW. Ok for dapi imaging at 100% AOTF, will be replaced 4/13/09. Fixed bug of changing to DAPI cube (Jim tightened the turret).

2/24/09 - 510 upright - Condenser replaced, condenser aperture has stoped moving so TL contrast was poor

11/4/08-1/29/09 - 510 inverted - 100x objective was damaged, front lens cracked let oil in. Sent to Germany for repair, cost $1300.

1/19/09 - SP5 - Tests of alignment and spectral calibration. 405 and 561 images were precisely aligned in both XY and XZ. Spectral accuracy checked in fluorescence and reflection with all PMTs with several lasers. All within 2 nm (ie background drops from 488 when lambda window moved to 490.

405 = 3.7 mW
458 = 1.5 mW
476 = 2.8 mW
488 = 7.6 mW
496 = 2.1 mW
514 = 6.6 mW
561 = 5.6 mW
594 = 0.5 mW
633 = 3.2 mW

1/19/09 - TIRF - image imperfection visible in TL only. Turned out to be speck of dust on the c-mount, not obvious why not visible in epi. Camera has very slight spot, will buy first contact for cleaning.

11/6/08 - 510 upright - Ar/Ar laser (458, 488, 514 nm) replaced. Both AOTF controller cooling fans also replaced. General system PM.

11/5/08 - Axio imager - The colour/fluorescence camera switch didn't always stay in position, Zeiss replaced the part.

11/3/08 - SP5 - filterwheel on DMI6000 became unusable. Small bearing wheel snaps the wheel into position but the metal holiding this into position had fractured. Jim replaced the part which required diassembly of the entire microscope. Dana realigned scan head and tweaked 405 nm laser to increase power from 3.0 mw to 4.0 mW.

10/9/08 - SD - new laser installed. Improved images. Excitation is still not completely even and a dark region is present at the top of the image. Seems on excitation side as TL is normal.

9/16/08 - 510 inverted - Offset problem with PMT3, replaced 8-channel PMT digitizer board.

9/16/08 - SP5 - replaced 405 laser. Laser powers measured . . .

405 = 4.4 mW
458 = 1.4 mW
476 = 2.4 mW
488 = 6.6 mW
496 = 1.7 mW
514 = 5.0 mW
561 = 6.0 mW
594 = 0.5 mW
633 = 3.3 mW

9/12/08 - Axio Imager - fine focus control had a lot of play in the gears, difficult to use. Charlie replaced focusing mechanism.

9/5/08 - Kiehart SDup - Non uniform field. Green=laser, black=scanhead end of fiber (laser end is angled at 8degrees to prevent reflections into the laser). Two adjustable mirrors, one at 45degree angle closest to scope port adjusts angle of light going to lens disk, if off 90 then the 30Hz oscillation in intensity measured with the BN meter and Yusuke's oscilloscope will be more pronounced. Adjust two smaller 1.5 mm allen screws to adjust to minimize AC component and max overall DC component = transmission.

9/03/08 - Live cell - CO2 wasn't working, line between gas mixer and scope incubator was full of water. Recalibrated zero point again 9/5/08 as seemed to high, media off-red, seems ok but swings around 5%.

8/29/08 - SP5 - 405 laser is weak. Should produce 50 mW, 7 mW max measurable. Leica will replace laser and do PM when this is installed.

8/12/08 - 510 up - Scanner sounds slightly noisy, laser power ratios checked. Both were ok for now.

8/12/08 - 510 inv - Scanners calibrated so non-multitime tiling gaps are eliminated.

6/30/08 - SP5 - Dana fixed the UV collimation optic wheel - replacement part proved unnecessary as adjusting the tension on the motor solved. System working much better, pinhole alignment good. Stage seems ok still. Laser powers -

405 = 4.3 mW
458 = 1.5 mW
476 = 2.7 mW
488 = 7.0 mW
496 = 1.75 mW
514 = 5.3 mW
561 = 6.0 mW
594 = 0.52 mW
633 = 3.4 mW

6/16/08 - SP5 - continued problems with the stage. Problem with UV beam expander, dana realigned all but still not great. Power at objectives reasonable but still down from install -

405 = 3.98 mW
488 = 7.0 mW
561 = 5.5 mW
594 = 0.45 mW
633 = 3.06 mW

Dana will send manual 3-plate stage until motorized stage problem is resolved. Other issues with scan head - AOTF vis sweeps were flat, pinhole alignment was bad. Swapping scope control box seems to be helping with the stage. UV wheel got worse.

4/16/08 - SP5 - installed insulation on power supply. Dana aligned lasers and measured power while there (to turn off aotf blanking - LAS diag/testcenter/scanner low level saotfm 0, zoom >x25)-

405 = 4.9 mW
458 = 1.75 mW
476 = 3.0 mW
488 = 7.8 mW
496 = 1.94 mW
514 = 5.8 mW
561 = 6.1 mW
594 = 0.52 mW
633 = 3.3 mW

1/18/08 - SD2 - measured laser power (out of laser, before fiber), run at full power after 5 min warmup, 488 line = 31 mW, 568 line = 33 mW. In comparison, kiehart wounding scope produced ~40 mW after fiber = all lambda.

11/28/07 - SP5 - replaced AOTF controller for visible lines, all visible lines then controlled properly. Measured laser powers and they seemed to be as install (the argon laser didn't warm up for very long): 12/3/07 installed AOTF controller for 405 and power measured as 5.8 mW. 561 measured as 6.3 mW. It then died, new one installed 12/10/07, powers measured as 405 = 5.2 mW, 561 = 6.3 mW.

488 = 8.1mW
514 =6.5 mW
561 = 6.5 mW
594 = 0.55 mW
633 = 3.6 mW

11/13/07 - SP5 - FRAP bleaching not that great on checking laser powers AOTF seem to be either on or off and offer no AT control. Diag gives okay frequency, powers about 2x over and install. Laser powers measured:

405 = 4.83 mW
488 = 6.81 mW
561 = 5.0 mW
594 = 0.16 mW
633 = 2.33 mW

9/18/07 - 510 inverted - On opening the software a stream of CAN_ error messages appeared (lack of communication with the laser modules). Appeared to be a problem where the PC was unable to open port7000 to communicate to the real time computer. Spent time looking for problems in firewall or TCP/IP settings. Problem was failure of the main data board downstream of the realtime computer which for some reason makes the RT computer unable to connect to the main PC.

9/5/07 - SP5 - Installation. All went well, except scope had additional port on side not compatible with the incubator, scratched shield on the condenser, no DAPI cube,

Laser powers through 10x objective, lasers at full power, aotf 100%, deactivate x blanking, 400 Hz, zoom 32

405 = 3.9 mW
458 = 1.6 mW
476 = 2.9 mW
488 = 8.4mW
496 = 2.2 mW
514 = 7.2 mW
561 = 6.0 mW
594 = 0.53 mW
633 = 3.6 mW

8/22/07 - SD1 - Laser power low. Measured power at 10x objective with BN meter and Kiehart lab meter before and after mirror and fiber alignment. Before alignment: BN (K)
488 = 130 microwatt (87 microwatt)
568 = 141 microwatt (62 microwatt)
647 not visible or detectable

After alignment:
488 = 342 microwatt (220 microwatt)
568 = 537 microwatt (230 microwatt)
647 = 390 microwatt (125 microwatt)

5/07/07 - Live cell station - Focus drift from faulty encoder in docking station, charlie disabled it until new part arrives. New one seems okay.

April 2007 - New Stereoscope - Broken dial, Middle TL dial doesn't stay forward and sometimes sticks depending on the position of the others. Need - HE red filters, dual light guide, 1x c-mount. Holding down RL and TL minus buttons = double tap. Custom buttons for all users and check reliability.

3/13/07 - 510 - Todd noticed when tiling, one edge of field consistently brighter than the other, possibly not completely flat also. Charlie fixed with scan field rotation macro.

3/7/07 - Axio - Metamorph crashes after DIC acquisition, shutter closes but camera fails? Check hard ware settings and see if fault is persistent.

11/22/06 - 410 - PMT 1 was replaced with a brand new Hamammatsu cooled PMT. Contrast values still register some 100 points higher than pre-PMT upgrade, but pictures & sensitivity look great. Scanners aligned and shading adjusted on PMTs. External lasers aligned. Microscope cleaned & cover relabeled. All this service was performed by the senior service technician (Brad Bobbitt).

11/17/06 - 510 - Alex measured laser power. In Admin mode 7.5 amp 80/20 10x = 1.2 to 1.4 mW. 3.67 mW with 488/543 dichroic. Seemed some problem in measure in normal mode. Alex recalibrated AOTF to improve linearity power, went up to about 1.3 mW. Not certain if this equates to increased bleaching or just measuring? Scanning must be in bidirectional mode to measure, uni = half. Need to remeasure with Dan's power meter to check is of comparable sensitivity.

10/31/06 - SD - Couple of users and have had problem with laser igniting.

10/19/06 - SD - Mike Smith installed new fiber and aligned laser scanhead etc. Replacing 568 filter removed image imperfections in that channel. 647 filter was beginning to show signs of burning so Mike is going to post me a replacement. Whole system working well. Measured laser power (warmed up for about 10 min) - 50 mW total: 488=8.3 mW, 568=8.5 mW, 647 13.1 mW. Guideline for when replaced under SC - <5mW per channel. Cleaning cost ¥300 000 ($2500).

10/12/06 - SD - Installed cleaned scanhead. Looks much better but still some imperfections visible on the red channel but much reduced. New fiber yet to be installed. Also thoroughly cleaned the microscope and surroundings, installed 63x/1.4 oil objective from BN scope. Returned scanhead had no details of what they had done.

10/5/06 - 510 up - Further improvement of laser power by Alex and Charlie. Laser power at objective 4 fold higher: New fiber (transmits 75%, old one 45%), improved alignment of fiber into scanhead, primary VIS mirror and routing mirror - better centered on back aperture of objective (moving collimator leads to movement of 3 pixels). No obvious damage to scanning mirrors but y mirror had a slight hum. Cleaned dust from primary mirror. Board controlling Ph3 broke, replacing at 3:00 10/6/06. Done.

Laser power measured at 10x objective, 80/20 objective (power at install) all 2-3 higher with optimal dichroic

* 351 nm (0.785 mW)
* 364 nm (2.02 mW)
* 458 nm 0.24 mW (0.391 mW)
* 477 nm 0.38 mW
* 488 nm 1.4 mW (2.19 mW) same as demo 1.2-1.4 mW "good"
* 514 nm 0.804 mW
* 543 nm 0.090 mW (0.126 mW)
* 633 nm 0.36 mW (0.451 mW)

9/25/06 - 510 - laser power too low for rapid bleaching. Back aperture of objectives dirty, primary mirror of Ar/2 adjusted = 25% improvement but the laser power measured at objective at least 5 fold too weak. Lasers themselves not adjusted but all working well -
543 - 1.12 mW; >1.00 after AOTF
633 - 4.92 mW out of laser; 4.67 mW after AOTF
Ar/2 @ 7.5 amps - total after first mirror 35 mW (all lines), after AOTF

* 458 3.20 mW
* 477 4.26 mW
* 488 16.0 mW
* 514 8.79 mW

(ie >32 of 35 mW reaches fiber optic to go to scope)

laser output delivered through 10x objective is pathetic - 488 0.4 mW, want more than 2.0 mW, red lasers similarly bad. Possibly the fiber optic - but illumination looked even when objective removed (couldn't take out fiber because of UV/Vis alignment). If not fiber, primary dichroic could be blemished = factory repair in germany. Alex and Charlie checking further next week.

9/12/06 - Axio - Upgrade: Uniblitz shutter fine, MAC 5000 controller for LUDL filterwheel can't be driven by metamorph through USB so we need to get extra I/O card for pc. Thread pitch of 25 mm analyzer different to filters/filterwheel, need to get unthreaded version, homemade one for now. Bill installed threadless analyzer 10/5/06. Uniblitz shutter i have doesn't fit on ex filterwheels, needs an adapted form from shutter - about $950 so keep using ND wheel for now. 10 for 6 position?

9/06 - 510 - Ar/2 laser getting weak, pinhole alignment fine, Alex checking laser at end of month.

8/7/06 - ? - Spinning disk - Away for cleaning, internal mirrors needed cleaning and perkin elmer had to send it back to yokogawa in japan. About 3 weeks has turned into about 8 weeks.

7/06 - Axio - Audrey noticed that DIC images differ between have illumination set to none and any setting and having RL shutter closed. Not sure why, possible software having to move filterwheel or not affects exposure time even though not in light path?

7/13/06 - Axio - Various problems with metamorph, required resetting of all electronic equipment, hot pixels remained from camera controller problem? Temporary problem.

7/11/06 - 510 - stage had become loose, Alex replaced missing screw and tightened. Fine now, problem was only noticeable if stage was far forward.

6/29/06 - 510 - Find function produced unusable gain values for the transmitted light detector. Maintain/setfind/AutoB+C controls. Select ChD Two sliders default values = 100, set amp gain to 1.0 checked.

6/29/06 - 410 - PMT2 high contrast/sensitivity problem proven to be in the detector itself (moved to detector position 1, and the high contrast values moved with it). Another PMT off of LSM Tech's demo unit was overnighted and installed in detector position 1, replacing the malfunctioning PMT. While this replacement PMT has contrast values some 150 points better than our old one, it isn't brand new and is ~50 contrast points less sensitive than our detector currently occupying position 2.

NEEDED: LSM Technologies needs to supply us with a *brand new* detector and to switch the PMT currently occupying position 2 back to position 1. Ultimately, we should have the more sensitive PMT in detector position 1.

6/23/2006 - 410 - failure of PMT3. No image, no noise. Dave Myers replaced failed socket for PMT3, now functional.

6/22/2006 - Axio - Halogen bulb not working, output switch on back had been changed.

2006 - Axio - DIC quality. Since changing microscope stands the DIC quality is sometimes poor. Installing new 100x seems to improve. Bill Faught will bring HC slider - currently testing.

5/24/2006 - Axio - Unevenness of Hg illumination. Despite realignment and bulb change seems to be a tendency for a bright central stripe. Aligning the bulb using the 100x objective improves as this is where the problem is worst.

5/15/2006 - Axio Imager - MetaMorph crashed upon opening, no obvious cause, changing to single user mode then back to multi user mode solved the problem.

5/13/2006 - 510 - Transmitted light appeared not to be functional, user had entered previous image mode so the scope was offline. Other issues with transmitted light - Alex replaced cable, errors still occur.

5/2006- 410 - Several problems with PMT2. Filters were incorrectly installed, after changing these contrast values still seem high. The pinhole was replaced for PMT2 in optic upgrade but is working normally and not the cause of the problem. Brad improved brightness of samples under Hg lamp - cleaned collecting lens of Hg arc lamp.

5/11/2006 - Spinning Disk - PM visit from Mike Smith, Laser alignment improved power increased to 50 mW at scan head. Fiber might be slightly imperfect - some haze around bright central spot. 60/1.4 seems ruined by oil, 60/water added. Dark spots around edge of image - contamination in scan head as rotation of camera see the contamination patterns rotate with the image; rotation of the entire scan head and camera sees the contamination pattern fixed and the image rotates. Green filter cube has red transmission.