Paulsson:Microscopes

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*02/09/11: Bulb replaced on DG4 lamp - 7967.4.
*02/09/11: Bulb replaced on DG4 lamp - 7967.4.
*June 2012: ORCA-R2 switched to 16-bit acquisition mode.  Much greater sensitivity!  May need to modify code for segmenting phase images.  To switch between 12-bit and 16-bit, go to SysBio/Paulsson Lab/Imaging_data/Zeiss_200M and extract "Orca R2 Patch.zip", then double click on the "Orca R2 default.reg" file and it will go back to 12-bit.  For 16-bit, use the "HDR mode" file (edited it so that it doesn't turn on the HDR mode but stays at 16-bit).
*June 2012: ORCA-R2 switched to 16-bit acquisition mode.  Much greater sensitivity!  May need to modify code for segmenting phase images.  To switch between 12-bit and 16-bit, go to SysBio/Paulsson Lab/Imaging_data/Zeiss_200M and extract "Orca R2 Patch.zip", then double click on the "Orca R2 default.reg" file and it will go back to 12-bit.  For 16-bit, use the "HDR mode" file (edited it so that it doesn't turn on the HDR mode but stays at 16-bit).
 +
*07/24/13: Replaced DG4 lamp (Excelitas Technologies, PE175BF). Hour meter read 10393.
=PALM  secret weapon=
=PALM  secret weapon=

Revision as of 15:25, 24 July 2013

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Contents

Zeiss 200M microscope

Sign-up list

User / Date / Duration

e.g. Shay / whenever he wants / as long as it takes
e.g. Rotons / when Shay lets them / not long

Jeanne 06/12/11: 10.00-16.00 (37°C)
Raul 06/16/11: 16:00-21:00 (37°C)

Configuration

Objectives

Filter cubes

  • 1. GFP: BP 470/40; FT 495; BP 525/50
  • 2. RFP: BP 565/30; FT 585; BP 620/60 (Zeiss Set 31)
  • 3. RFP: BP 560/40; FT 585; BP 630/75 (Zeiss Set 45)
  • 4. CFP: BP 436/25; FT 455; BP 480/40
  • 5. YFP: BP 500/25; FT 515; BP 535/30

spare filter cubes:

  • DAPI: BP 390/22; FT 420; BP 460/50 (Zeiss Set 34)
  • mystery red filters from Bill (loose filters)
  • Burak's Qdot filterset: QD605-A from Semrock

http://www.semrock.com/Catalog/SetDetails.aspx?SetBasePartID=2
ex: 435/40 Dichroic: 510LP em: 605/15

  • CFP : BP 436/25; FT 455; BP 480/40

Maintenance log

  • 03/01/07, new halogen bulb, replaced by Shay (old one blew out/up, due to power surge?)
  • 04/01/07, PROBLEMS: 1)CFP filter cube position is not able to perform z-stack.
  • 06/15/07, CFP filter located at CFP position, GFP filter is substituted by YFP filter cube.
  • while doing z-stack, the stage shakes - reflected light shutter in scope causes shaking

Solution: Adjust all settings so that shutter stays open and use shutter on DG4 light source (which is a lot faster anyways!)

  • 07/15/07, shift in register between images... Somehow (?!) settings changed back so reflected light shutter was set to open/close for fluorescence acquisition.

Solution: as before, Christine changed all settings back to using DG4 shutter instead

  • 070730, Doug installed new version of Axiovision (4.6.3)
  • 070808, Mason connected computer to MED domain. "Users Data" folder moved to FS-SYSTEMBIO\PAULSSON LAB\Acquisition Scope\Users Data - shortcut on desktop! Login to Administrator account locally, login to server at MED domain.
  • 070809, Christine installed a Hot-Fix8 to improve communication speed between "old" microscope and "new" Axiovision. Also added scaling settings for 63x objective used with 1.6x and 2.5x Optivar, and 100x phase objective with 2x2 and 4x4 binning. Copied Carl Zeiss\Data folder that has settings to C:\Documents and Settings\All Users\Shared Documents\Carl Zeiss\Data. Also at \\fs-sytembio\systembio$\PAULSSON LAB\Acquisition Scope\Users Data\Carl Zeiss\Data.
  • 080424, Per replaced Xenon bulb, hour meter read 3890h (wwoooww)
  • 0808??, Doug installed new Autofocus algorithm that allows user manipulation of autofocus search range and step-size. Accessed through toolbox/options(?) and is modifiable through a text file stored in the Zeiss preferences folder(?). Dirk has used it and knows the details. Might work with less fuss if we had AxioVision 4.7, but Doug says there may be hardware conflicts that make the system less stable.
  • 09/02/08: new autofocusing algorithm is fully installed and finally working (current version of the software is AxioVision 4.63 SP1). You can also run time lapse with decoupled channels (e.g. image phase every minute and GFP every 5 min) without the scripting module. Look up smart experiments in the help menu or talk to Dirk for more information. Smart experiments are smart but make the software more unstable. I had them crash a couple of times. It helps to leave the computer alone (don't even touch the mouse) when it performs a smart experiment. Tim Pratt from Zeiss recommended to update our software to AxioVision 4.7. Believe it or not: the update is for free ...
  • 02/04/09: Jim from Zeiss came by to discuss the update of our software to AxioVision 4.7.
  • 02/08/09: The fluorescent light doesn't seem to work. The microscope is down.
  • 02/10/09: The microscope is functional again. Now you must use the Lambda SC shutter Instead of the DG4. See Shay's email for details.
  • 02/24/09: The Lambda SC shutter broke (i.e. the shutter is not responding when called from AxioVision). As a short term solution we connected the fluorescent light source (XCite) of the PALM scope. You have to use new AxioVision configurations/settings for imaging. Talk to Shay, Dann, Dirk or Burak if you plan to use the microscope and need help to get started.
  • 03/09/09: The DG4 lamp is back and running.
  • 08/15/10: Upgrades to enable use of ORCA-R2:
    • Installed PCI card for FireWire 800 (PCI-express slot not available so can't take advantage of 800 speed anyway).
    • Mason backed up C:and D: (one physical drive), replaced it with a 250GB drive with two equal size partitions containing data from old C:and D: (we have old drive and Mason has a copy backed-up somewhere he can access)
    • Installed AV 4.8.2, including new Hamamatsu drivers
    • Left sideport configured for ORCA-R2; baseport configured for EM-CCD
    • Beware of:
      1. Recognizing ORCA-R2: If AV fails to recognize ORCA-R2, quit and reopen. (Problem with HDCAM looking for EM-CCD?)
      2. Using EM-CCD: Phoenix board disabled to help with above problem (does it??). Need to activate to use EM-CCD. Right click on MyComputer in Start menu, select Manage, select Device Manager, expand 'Imaging devices' menu, activate 'Silicon Phoenix (D24CL PCI32)'
      3. Autofocus preferences: In AV 4.6.3 the settings file was common to All Users, but in AV 4.8.2 each user has their own, stored in whatever directory you have used for storing User data (preferences), so I couldn't set up all users with the old modified settings file. To get yourself set-up to use the Autofocus, I suggest doing the following:
        • In AV 4.8.2, go to the 'Tools' pulldown and select 'Options'. On the left side of the dialog box, select 'Acquisition'. Under the sub-heading 'General' check the box for "use calibration-free autofocus". This means that AV will use a text file for the settings, rather than depending on (re)calibration. Leave this box checked!!! If you uncheck, and later re-check this box the settings file will default to the original, overwriting any changes you've made to the Autofocus parameters file (so I'm told - haven't tested if it's true).
        • Then select the 'Folder' tab on the left of the 'Options' dialog window. The 'User Data' field tells you where your preferences are being stored (not the data you collect while doing experiments). Navigate to this folder in the finder. There should be a Calibration folder in there, and inside it a file called "Autofocus2_Parameters". Replace it with the file of the same name that I put on your desktop (in the 'Autofocus parameter files' folder).
  • 02/09/11: Bulb replaced on DG4 lamp - 7967.4.
  • June 2012: ORCA-R2 switched to 16-bit acquisition mode. Much greater sensitivity! May need to modify code for segmenting phase images. To switch between 12-bit and 16-bit, go to SysBio/Paulsson Lab/Imaging_data/Zeiss_200M and extract "Orca R2 Patch.zip", then double click on the "Orca R2 default.reg" file and it will go back to 12-bit. For 16-bit, use the "HDR mode" file (edited it so that it doesn't turn on the HDR mode but stays at 16-bit).
  • 07/24/13: Replaced DG4 lamp (Excelitas Technologies, PE175BF). Hour meter read 10393.

PALM secret weapon

Configuration

Sign-up list

User / Date / Duration


Burak & Victor: 07/17/09: 10:00-18:00


Objectives

Position Objective
1 empty
2 10x A-Plan, 0.25 NA, Ph1
3 empty
4 40x LD Plan-NEOFLUAR, 0.6 NA, Korr, DIC(II)
5 63x, Plan-NEOFLUAR, 1.25 NA, oil immmersion, DIC(II)
6 100x FLUAR, 1.3 NA, oil immersion, DIC(III)
  • Sitting on the shelf, waiting for installation: 100x EC Plan-NEOFLUAR 1.3 NA, oil immersion.

Usable with excision laser - apparently 100x FLUAR isn't high enough quality to be used for excision.

Filters

Excitation wheel:

Position Filters
1 blocked
2 BP390-420
3 D485/20X (55748)
4 D535/50X (60742) (splotchy on inner surface of filters?)
5 blocked
6 open
7,8 blocked


Emission cubes:

Position Filters
1 HR355+1064; HT395+VIS/45° (15AZ106812)
2 HR355+1064; HR390HT450+VIS (15AZ106815)
3 GFP (424931)
4 RFP: BP 565/30; FT 585; BP 620/60 (from other Zeiss; need to move excitation to Wheel?!?)

Maintenance log

  • 07/15/07, Jeff in to install software for joystick control of PALM stage (including Axiovision, as a temporary use license thing)

Failed!!!! Jeff couldn't get PALM and AxioVision software to recognize the camera at the same time.

  • several intervening efforts
  • 09/06/07, Jeff tried again. PALM recognizes joystick, but AxioVision doesn't find stage
  • 11/07/07 PALM and Axiovision were loaded onto the same computer, but you can't use them at the same time. It seems that you can't open up Axiovision, close it, and have PALM work...
  • 02/19/09 The new PALM scope got delivered.




Nikon Ti microscope station #1 (aka "Mesut")

Sign-up list

User / Date / Duration


Dirk: 10/15/10: 100x TIRF objective will be on Nikon Ti #2; will not use Nikon Ti #1
Dirk: 10/19/10: 100x TIRF objective will be on Nikon Ti #2; will not use Nikon Ti #1
Dirk: 10/21/10: 100x TIRF objective will be on Nikon Ti #2; will not use Nikon Ti #1
Dirk and Burak: 11/5/10: 10:00 - 20:00
Dirk: 11/6/10: 100x TIRF objective will be on Nikon Ti #2; will not use Nikon Ti #1
Burak: 11/17/10: 10:00-14:00 - will use the 60x TIRF objective
Burak: 11/18/10: 10:00-17:00 - will use the 60x TIRF objective
Dirk/Nate: 11/23/10 10:00-14:00 (Using both TIs)
Burak: 11/23/10 14:00-19:00
Nate: 100x PA objective after Burak is done (overnight)

Burak: 11/24/10 10:00-18:00
Burak: 11/25/10 10:00-18:00 (60x TIRF objective)
Burak: 11/25/10 12:00-18:00 (60x TIRF objective)
nate/meriem: 11/30/10 10:00-O/N-- 100x PA Objective on TI-2
Burak: 12/02/10 12:00-18:00 (60x TIRF objective)
Burak: 12/03/10 12:00-18:00 (60x TIRF objective)
Dann: 12/07/10 15:00-17:00 (60x TIRF objective)
Burak: 12/08/10 13:00-18:00 (60x TIRF objective)
Dann: 12/09/10 15:00-18:00 (60x TIRF objective)
Burak: 12/10/10 14:00-18:00 (60x TIRF objective)
Burak: 12/14/10 17:00-20:00 (60x TIRF objective)
Burak: 01/25/11 16:00-22:00 (60x TIRF objective)
Meriem+Burak: 02/3/11 13:00-18:00 (100X PA objective)
Meriem+Burak: 02/8/11 13:00-18:00 (100X PA objective)
Meriem+Burak: 02/17/11 13:00-18:00 (100X PA objective)
Meriem+Burak: 06/28/11 13:00-18:00 (100X PA objective)


Configuration

General

Objectives

  • CFI Plan Fluor Dll 20x objective
  • CFI Apo 60x NA1.49 oil TIRF objective
  • CFI Apo 100x NA1.49 oil TIRF objective

Cameras

Optional:

Filter cubes

  • 1. CFP (excitation 438/24, dichroic 458, emission 483/32)
  • 2. GFP (excitation 472/30, dichroic 495, emission 520/35)
  • 3. YFP (excitation 500/24, dichroic 520, emission 542/27)
  • 4. RFP (excitation 562/40, dichroic 593, emission 641/75), this filter was designed for true RFPs like mCherry
  • 5. custom-made brass filter cube for laser
  • 6. Bf/Phase

spare filter cube:

  • DAPI (Semrock LF405-A: excitation 390/40, dichroic 405, emission 452/45)


filters for the lasers

dichroics sitting on the optical table, for diverting the beams:

ZT405RDC (chroma) - for 405 nm

ZT488RDC (chroma) - for 488 nm

ZT514RDC (chroma) - we have this for 514 nm but not the laser so this is NOT installed

LM01-552-25 (semrock) - for 532 nm

http://www.semrock.com/Catalog/Detail.aspx?FilterPartID=859&CategoryID=83

  • TagRFP:

dichroic: Di01-R532-25x36 (Semrock) http://www.semrock.com/Catalog/Detail.aspx?FilterPartID=870&CategoryID=81
emission:FF01-607/70-25 (Semrock) http://www.semrock.com/Catalog/Detail.aspx?FilterPartID=817&CategoryID=27

  • GFP:

filters are sitting on a custom made brass filter cube

dichroic: Di01-R488 (Semrock) http://www.semrock.com/Catalog/Detail.aspx?FilterPartID=775
emission 1: BLP01-488R-25 (Semrock) http://www.semrock.com/Catalog/Detail.aspx?FilterPartID=94&CategoryID=78
emission 2: FF01-525/45 (Semrock) http://www.semrock.com/Catalog/Detail.aspx?FilterPartID=787

  • misc:

Di01-R635-25x36

http://www.semrock.com/Catalog/Detail.aspx?FilterPartID=777

Lasers

  • 405nm, 50mW, Coherent Cube
  • 488nm, 75mW, Coherent Sapphire
  • 532nm, 300mW, Ultralasers.com (part#: G300N)

Maintenance log

  • Microscope got delivered.
  • 01/16/2009: Computer was setup by Pamela. ImageJ and Micro-Manager (version 1.2.39 beta) are installed. The computer is not connected to the microscope yet.
  • 01/27/2009: Thanks to Bruno's help we were able to setup and install Micro-Manager. The scope is up and running now. The second camera (for the phase image) is not installed yet because of the lack of an appropriate driver.
  • 02/06/2009: It is not clear if PixeLink will provide appropriate drivers to make their camera work with Micro-Manager.
  • 02/08/2009: Orca R2 camera is only operating in the 8- and 12-bit mode (but not 16-bit). The Micro-Manager Hamamatsu_DCAM driver causes some problems with the Orca R2. Sometimes Micro-Manager won't start because of an error with the camera driver. Nico from Micro-Manager is looking into this and will trouble shoot it.
  • 02/25/2009: The computer/Windows crashes when you turn on the camera controller. The problem seems to be a driver incompatibility between Windows and the Hamamatsu camera.
  • 02/27/2009: I called support and I was able to fix the problem by deinstalling the camera drivers and reinstalling a newer version of the Hamamatsu_DCAM driver.
  • 03/10/2009: The computer runs very stable and Micro-Manager starts without any problems (no camera driver error anymore).
  • 03/12/2009: Scion camera got delivered.
  • 03/13/2009: Scion camera is setup (E100 port). The camera seems to work fine with Micro-manager.
  • 03/16/3009: I measured the PSF of the 60x objective and the objective seems to have "serious" coma.
  • 04/09/2009: Second filter turret was installed.
  • 04/11/2009: I installed Micromanager 1.3 but I wasn't able to setup the X,Y stage under 1.3. Micromanager version 1.2 is still installed on the computer and fully functional (you can install different versions of MM on the same computer).
  • 04/11/2009: Phase (the phase ring of the condensor is not visible at the BFP) and PFS are not working (display is showing 'PFS OUT').
  • 04/13/2009: Barry's assistant deinstalled the second filter turret. A second filter turret is not compatible with the the external phase module. Alternatives?
  • 04/13/2009: I installed Micromanager 1.3.30. The Hamamatsu Orca R2 camera operates in 16-bit now but the Scion camera is not working under 1.3.30. Also, the PFS switches off if you change the x,y position (this is fixed in 1.3.37). Time-lapse on a single position with functional PFS is possible though. Micro-Manager 1.2 is fully functional.
  • 05/28/2009: Room temperature was changed to 30 C.
  • 05/29/2009: Barry exchanged the fluorescence illuminator and the 60x TIRF objective. The previous illuminator produced an uneven illumination over the field-of-view. This problem was known to Nikon.
  • 06/02/2009: 488nm and 405nm laser lines are fully aligned for three illumination modes (epi, TIRF and focused illumination). The focused illumination path is from the backport of the microscope, and the epi/TIRF is through the opening on the filter turret. Switching between focused illumination vs. epi/TIRF is achieved via the flip mirror.
  • 06/08/2009: Filter turret was fixed to give the proper positions of the filter cubes
  • 07/01/2009: Micro-Manager version 1.3.37 is working with Matlab now.
  • 08/05/2009: spill, light path is contaminated, RFP background is very high
  • 08/17/2009: Sometimes the computer does not boot correctly (unfortunately rather often). Switching the computer OFF and ON again helps. Moving the mouse around during the startup process seems to help too (-> don't forget it is windows ;-)
  • 09/04/2009: Microscope is up and running again. The RFP background looks good.
  • 09/10/2009: Uniblitz Vincent D3 shutter is installed and controls both laser lines (405 nm and 488 nm). We also cleaned up the Micro-manager .cfg file and this improved performance of the scope - it is much snappier now.
  • 11/19/2009: Micro-manager update. The current version is 1.3.41c beta. System is not running smooth yet (especially Matlab integration needs more work).
  • 12/11/2009: Matlab scripts are updated. Movement of fluorescence shutter causes blurring of images if the exposure time is very short (e.g. 50 ms).
  • 01/24/2010: Burak: Inserted the PCI card for Hamamatsu EMCCD C9100-02 on the computer - thanks to the flexibility of PC, would not have been possible with a Mac which is a fancy but unmodifiable pet computer. EMCCD can now be also used on Nikon.
  • 01/28/2010: Brightfield images are blurred if the exposure time is short. This is probably caused by the transmitted light shutter. 500 ms exposures work fine.
  • 03/03/2010: Room temperature is changed to 23 C.
  • 03/25/2010: Transmitted light shutter is moved further to the back to accommodate the incubator.
  • 04/06/2010: I upgraded to MM 1.3.45 a few days ago but I had to downgrade to version 1.3.41c because of Matlab incompatibility problems.
  • 07/14/2010: I inserted an IR absorbing filter (KG1) in the transmitted light path. This filter will cut out 870 nm light and should prevent PFS errors.
  • 08/27/2010: Our new member, the 532nm laser is here to be installed soon.
  • 08/31/2010: Dirk: installed Nikon SDK (version 4.2.3) and upgraded Nikon Ti drivers. I also installed Micro-manager version 1.3.46_20100830. The Micro-manager GUI worked well but Matlab threw errors (mainly Nikon TI Z-drive).


To-do list:

  • install Ph3 phase plate in the external phase ring (microscope body)
  • order and install Ph1 phase plate in condensor
  • cylindrical lens?
  • Dry thermometer for incubator.
  • Matlab problems with newest drivers and Micro-manager 1.3.46_20100830



Nikon Ti microscope station #2 (aka "Nuri")

What about Victoria -- crown princess of Sweden
Or Mameluk -- the famous turkish warriors (these guys were tough and could handle a large workload like the Nikon Ti ... hopefully)

Sign-up list

User / Date / Duration

Dirk: 5/02/10 8:00-8:01
Dann: 7/19/10 10:00-18:00 borrowing 100X objective
Dann: 7/27/10 19:00-22:00 borrowing 100X objective...Dirrrrrrrrrrkkkkkkkkk!!!!
Dirk: 8/16/10 12:00-20:0
Mason: 8/20/10 8:00-18:0 Change of hard drive
nate: 9/8/10: 15:00-O/N
Dirk: 9/9/10: 13:00-15:00
nate/Tom: 9/9/10: 15:00-O/N
Dirk: 9/10/10: 19:00-21:00
Nate/Tom: 9/13/10: 15:00-O/N
Dirk: 9/14/10: 12:00-20:00
Nate/Tom: 9/15/10: 12:00-O/N
Nate: 9/16/10: 16:00-O/N
Meriem/dirk: 9/17/10: 11:00-18:00
Dirk: 9/19/10: 14:00-20:00 change temperature to 30 C
Dirk: 9/20/10: 16:00-20:00
Meriem: 9/21/10: 11:00-17:00
Nate: 9/21/10: 17:00-O/N
Dirk: 9/23/10: 14:00-20:00 change temperature to 30 C, use 100x TIRF objective
Nate: 9/23/10: 20:00-O/N (37 C )
Meriem: 9/24/10: 11:00-17:00
Nate: 9/26/10: 14:00-20:00
Dirk: 9/27/10: 12:00-20:00
nate: 9/28/10: 17:00-O/N (Let me know if you want to use it in the morning!)
Dirk: 9/29/10: 13:00-20:00 change temperature to 30 C, use 100x TIRF objective
Dirk: 9/30/10: 12:00-20:00 change temperature to 37 C, use 100x TIRF objective
Meriem: 10/3/10: 10:00-14:00 (will probably finish earlier)
Nate: 10/5/10: 15:00-O/N Will set it to 37 around noon, if there are no objections
Dirk: 10/6/10: 12:00-20:00
Meriem: 10/7/10: 11:00-17:00
Dirk: 10/8/10: 12:00-20:00 change temperature to 37 C, use 100x TIRF objective
Dirk: 10/13/10: 12:00-20:00 change temperature to 37 C, use 100x TIRF objective
Nate: 10/13/10: 20:00-24:00
meriem:10/14/10: 11:00-15:00
Nate: 10/14/10: 17:00-O/N (will set to 37 C at ~13:00, if there are no objections)
Dirk: 10/15/10: 12:00-20:00 change temperature to 37 C, use 100x TIRF objective
Nate: 10/18/10: 15:00-20:00 (37 C at noon if possible)
Dirk: 10/19/10: 12:00-20:00 change temperature to 37 C, use 100x TIRF objective
Nate: 10/21/10: 20:00-O/N. Dirk: 10/21/10: 12:00-20:00 change temperature to 37 C, use 100x TIRF objective
Tom: 10/25/10: 3:00 - O/N (37 C at say 1 if possible)
Nate: 10/26/10: 17:00-O/N (37 a bit earlier, if no objections)
Tom: 10/27/10: 17:00-O/N (37 at say 2 if no one is using)
Meriem: 10/28/10: 11:00-20:00 (37 in the morning if possible)
Meriem: 10/31/10: 11:00-15:00
Nate: 10/31/10 : 16:00-O/N
Dirk: 11/01/10 : 16:00-20:00 (no heating, 100x PA objective)
Nate: 11/2/10: 17:00-O/N (37 C, 60X TIRF objective from TI-1 if possible)
Tom/Nate: 11/3/10: 17:00-O/N (37 C, 60x TIRF from TI-1)
meriem: 11/4/10 11:00-17:00(37C, 100x PA objective)
Dirk: 11/5/10: I will be using the 100x PA objective on Nikon Ti #1
Dirk: 11/6/10:15:00 - 20:00 (37 C, 100x TIRF objective)
meriem: 11/8/10 11:00-17:00(37C, 100x TIRF objective)
Tom/Nate: 11/10/10: 16:00-O/N (37 C, 60x TIRF from TI-1)
Nate: 11/11/10: 19:00-O/N (37 C)
Dirk: 11/12/10:12:00 - 20:00 (37 C, 100x TIRF objective)
Dirk: 11/15/10:12:00 - 20:00 (37 C, 100x TIRF objective)
Nate: 11/16/10: 17:00-O/N (37C, 100X PA objective)
Nate: 11/17/10: 17:00-O/N (37C, 100X PA objective)
Dirk: 11/18/10:12:00 - 20:00 (37 C, 100x TIRF objective)
Nate: 11/18/10: 20:00-O/N (37C, 100X PA objective)
Dirk: 11/19/10:12:00 - 20:00 (37 C, 100x TIRF objective)
Dirk/Nate: 11/23/10 10:00-14:00 (Using both TIs)
Nate: 11/23/10: 17:00-O/N 37 C, 100 PA objective
meriem: 11/24/10 10:00-18:00
Tom: 11/24/10: 18:00 - O/N 60X TIRF objective, 37 C
Dirk: 11/25/10: 12:00 - 20:00, 37 C, 100x TIRF
Dirk: 11/29/10: 17:00 - 20:00, RT, 100x TIRF
meriem: 11/30/10 11:00-17:00 100x PA 37C
Nate: 11/30/10: 17:00-O/N 100x PA 37 C
Nate: 12/1/10: 17:00 - O/N 100x PA 37 C
Dirk: 12/2/10: 12:00 - 20:00 100x TIRF
Nate: 12/3/10: 17:00-O/N 100x PA 37 C
Dirk: 12/5/10: 12:00 - 20:00 100x TIRF, 37 C
Dirk: 12/6/10: 12:00 - 16:00 100x TIRF, RT
Tom: 12/6/10: 16:00 - O/N 60X TIRF, 37 C
Nate: 12/7/10: 17:00 - O/N 100x PA 37 C
Nate/Jue: 12/8/10 10:00-14:00 100x PA RT
Dirk: 12/8/10: 17:00 - 19:00 100x PA, RT
Nate: 12/9/10: 17:00-O/N, 100x PA, 37 C
Dirk: 12/10/10: 12:00 - 20:00 100x TIRF, RT
Nate: 12/10/10: 20:00-O/N 100x PA 37 C
Tom: 12/11/10: 14:00 - O/N 60X TIRF, 37
Dirk: 12/12/10: 15:00 - 20:00 100x TIRF, RT
Dirk: 12/13/10: 15:00 - 20:00 100x TIRF, RT, if this is okay with Nate
Dirk: 12/14/10: 15:00 - 20:00 100x TIRF, RT, if this is okay with Nate
Nate: 12/14/10: 17:00-O/N 100x PA, 37 C
Dann: 12/15/10: 10:00 - 14:00 100x ?, RT
Tom: 12/15/10: 16:00-O/N 60X TIRF, 37 C
Nate: 12/16/10: 17:00-O/N 100x PA, 37 C
Dirk: 12/17/10: 12:00-22:00 100X TIRF, RT
Jue: 1/14/11: 14:00-18:00 100X PA, RT
Nate: 1/23/11: 16:00-O/N 100x PA, 37 C
Dirk: 1/24/11: 12:00-20:00 100X TIRF, RT
Nate: 1/24/11: 20:00-24:00 100X PA, RT
Nate: 1/25/11: 17:00-20:00 100x PA, RT
Nate: 1/27/11: 17:00-O/N 100x PA, RT
Nate: 1/29/11: 17:00-O/N 100x PA, RT
Dirk: 1/31/11: 16:00-18:00 if this is okay with Nate
Nate: 2/1/11: 17:00-O/N 100x PA, 37 C
Dann/Yoon: 4/19/11: 15:30-O/N 100x PA
Nate: 5/13/11-5/14/11: 17:00-O/N 100x PA
Nate: 5/16/11-5/17/11: 17:00-O/N 60x TIRF

Configuration

General

  • Spectra light engine form Lumencor, see http://www.lumencor.com/spectra_light_engine.html
  • LED system with 7 wavelengths:
    • 1. Violet (excitation filter = 379/34 nm, power = 152 mW)
    • 2. Blue (excitation filter = 434/17 nm, power = 271 mW)
    • 3. Cyan (excitation filter = 485/20 nm, power = 129 mW)
    • 4. Teal (excitation filter = 513/17 nm, power = 96 mW)
    • 5. Green (excitation filter = 549/15 nm, power = 874 mW)
    • 6. Yellow (excitation filter = 586/20 nm, power = 652 mW)
    • 7. Red (excitation filter = 650/13 nm, power = 119 mW)

See "Certificate of Conformance" for details (upload file)

Measured LED light intensities out of the 20x objective with optical power meter (Thorlabs PM100D).
The LED intensities are set to 100% in the software. Field diaphragm is fully open.
The violet LED has to be measured at 400 nm because of technical limitations.

20100819 Date 2
1. Violet (400 nm) 14.8 mW Cell C
2. Blue (434 nm) 49.3 mW Cell C
3. Cyan (485 nm) 23.6 mW Cell C
4. Teal (513 nm) 10.5 mW Cell C
5. Green (549 nm) 47.8 mW Cell C
6. Yellow (586 nm) 21.7 mW Cell C
7. Red (650 nm) 7.75 mW Cell C


Flat-filed is pretty good with Lumencor but could be better


Serial ports:

  • COM1 is Ludl XY stage
  • COM3 is Lumencor light engine
  • COM5 is Arduino Duemilanove I/O board (ON/OFF control of CoolLED for Bf/Phase)


Objectives

  • CFI Plan Fluor Dll 20x objective
  • Plan Apo 100x NA1.4 oil objective

Cameras

Filter cubes

The filter cubes have no excitation filter because the excitation filter is in the Lumencor light engine.


The dichroics for GFP, YFP and mCherry could be more red-shifted.
All filter cubes, except the YFP and mCherry cubes, have only the dichroic and the emission filter in place. The excitation filters are in the LED. We still need excitation filters in the YFP and mCherry cubes to prevent excitation light leaking through the dichroic. Should be fixed soon.

Incubator

Turn incubator on ~3 hours before imaging to equilibrate the microscope to the new temperature.
The LED display of the incubator does not correspond to the correct temperature.
For 37 C, set the incubator to 32 (Nate: according to the digital thermometer) C.
For 30 C, set the incubator to 26.5 C.

Maintenance log

  • Microscope got delivered.
  • Installed Micro-manager (1.3.45: http://valelab.ucsf.edu/~MM/nightlyBuilds/1.3/Windows/MMSetup_1.3.45_20100419.exe) and Matlab.
  • Downgraded the Nikon Ti driver to version 2.0.0.0
  • 05/02/2010: nothing done ...
  • 07/14/2010: I inserted an IR absorbing filter (KG1) in the transmitted light path. This filter will cut out 870 nm light and should prevent PFS errors.
  • 08/05/2010: Delivery and installation of Lumencor light engine
  • 08/13/2010: Installation of Micro-manager 1.3.46_20100809 and setup for Matlab
  • 08/17/2010: TIZdrive error with various versions of MM 1.3.46, MM 1.3.41 works fine though.
  • 08/24/2010: I installed the Nikon TI SDK version 4.2.3
  • 08/25/2010: Upgraded Micro-manager to version MM 1.3.46_20100824
  • 09/08/2010: Microfluidic chamber broke and some liquid spilled over the 100x PA objective and a few drops on PFS unit. Z-drive stalled after the spill (which might have been a coincidence). Further tests did not indicate of any damage to the scope.
  • 09/21/2010: Installed new Scion driver. The updated Scion driver should solve some of the synchronization problems. Scion driver works only with MM 1.3.46_20100919 (not with MM 1.3.46_20100824).



Work-around of known problems:

  • Live camera image looks weird
    • Right click -> properties -> Settings -> click Advanced -> Troubleshoot -> set the hardware acceleration to the 3rd lowest setting (this will disable all DirectDraw and Direct3D accelerations) -> restart computer



To-do list:

  • Change hard drive. Small HD for OS and large HD for images.
  • The dichroics for GFP, YFP and mCherry could be more red-shifted. YFP and RFP filter cubes have currently two excitation filters (one in the cube and one in the Lumencor). Exchange dichroics.




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