Paulsson:Microscopes: Difference between revisions

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Nate: 7/26/10: 20:00-o/n <br>  
Nate: 7/26/10: 20:00-o/n <br>  
Dirk: 7/27/10:  12:00-20:00 <br>
Dirk: 7/27/10:  12:00-20:00 <br>
Burak: 7/28/10: 15:00-20:00 <br>


==Configuration==
==Configuration==

Revision as of 15:09, 27 July 2010

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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

Burak: 5/5/10: 10:00-16:00
Burak: 5/6/10: 12:00-20:00
Per: 5/7/10: 10:00-22:00 (35°C)
Billy: 5/8/10: 17:00-22:00 ... probably 17:30
Billy: 5/9/10: 17:00-22:00
Billy: Tuesday 5/11/10: 14:00-18:00pm
Nate: 5/11/10: 18:00-22:00
Nate: 5/12/10: 10:00-15:00
Billy: 5/12/10: 15:00-18:00... Forgot about lab meeting, so actually reserving for a few hours after whenever it ends.
Mason/Shay: 5/13/10: 11:00-16:00 - Disk image (preparing for software upgrade!)
Billy: 5/13/10: 17:00-20:00, but this is pretty flexible.
Billy: 5/14/10: 17:00-20:00
Billy: 5/18/10: 15:00-23:59, perhaps a break at 6pm
Tom: 5/19/10: 13:00-16:00
Shay: 5/23/10: 10:00-20:00 (44°C)
Shay: 5/25/10: 12:00-22:00 (44°C)
Shay: 5/27/10: 10:00-20:00 (44°C)
Shay: 5/28/10: 10:00-20:00 (44°C)
Raul: 6/1/10: 15:00-20:00
Shay: 5/3/10: 10:00-20:00 (44°C)
Billy: 6/4/10: 2:00pm-4:00pm
Nate: 6/7/10: 18:30-24:00
Shay: 6/8/10: 10:00-20:00 (44°C)
Billy: 6/10/10: 2:00pm-4:00pm
Per: 6/10/10: 4:00pm-7:00pm
Shay: 6/17/10: 10:00-20:00 (44°C)
Meriem+Burak: 6/24/10: 11:00-22:00
Burak: 6/25/10: 10:00-20:00
Per: 6/28/10: 10:00-22:00 (35°C)
Burak: 6/29/10: 14:00-20:00
Meriem+Billy: 7/1/10: 15:00-18:00
Meriem+Burak: 7/2/10: 11:00-20:00
Per: 7/9/10: 10:00-22:00 (35°C)
Shay: 7/20/10: 9:00am - noon
Shay: 7/21/10: 10:00-20:00 (44°C)
Billy: 7/22/10: 4:30pm - 7:00pm
Billy: 7/23/10: 3:30pm - 6:00pm

Configuration

Objectives

Filter cubes

  • 1. GFP: BP 470/40; FT 495; BP 525/50
  • 2. mCherry: BP 560/40; FT 585; BP 630/75 (Zeiss Set 45)
  • 3. DAPI: BP 390/22; FT 420; BP 460/50
  • 4. empty
  • 5. YFP: BP 500/25; FT 515; BP 535/30

spare filter cubes:

  • mystery red filters from Bill (loose filters)
  • Burak's Qdot filter cube (QD605-A from Semrock)

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.




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

Sign-up list

User / Date / Duration

Nate/Dirk: 3/15/10 10:00-17:00
Burak: 3/15/10: 17:00-22:00
Burak: 3/16/10: 10:00-20:00
Burak: 3/18/10: 12:00-20:00
Burak: 3/19/10: 14:00-19:00
Burak: 3/22/10: 12:00-18:00
Burak and Dirk: 3/24/10: 12:00-17:00
Dirk: 3/26/10: 12:00-20:00
Burak: 3/30/10: 10:00-22:00
Burak: 3/31/10: 9:00-16:00
Dirk: 4/01/10: 15:00-20:00
Burak: 4/02/10: 10:00-22:00
Dirk: 4/06/10: 14:00-20:00
Burak: 4/09/10: 10:00-19:00
Dirk: 4/12/10: 14:00-20:00
Burak: 4/13/10: 12:00-18:00
Burak: 4/16/10: 10:00-18:00
Burak: 4/19/10: 10:00-20:00
Dirk: 4/20/10: 13:00-20:00
Dirk: 4/22/10: 14:00-20:00
Tom: 4/28/10: 10:00-20:00 (37 degrees, not actually using till ~1)
Dirk: 4/29/10: 10:00-22:00
Nate: 4/29/10: 22:00-10:00 (4/30)
Burak: 4/30/10: 10:00-22:00
Dirk: 5/03/10: 14:00-20:00
Burak + Dirk: 5/04/10: 12:00-20:00
Burak: 5/5/10: 10:00-16:00
Burak: 5/6/10: 12:00-20:00
Billy: 5/7/10: 15:30-20:00
Nate: 5/13/10: 16:00-9:00 (5/13)
Nate: 5/14/10: 10:00-17:00
Nate: 5/20/10: 17:00-10:00 (5/21)
Dirk: 6/12/10: 18:00-20:00
Burak: 6/15/10: 10:00-18:00
meriem+Burak: 6/24/10: 11:00-22:00
Dirk: 6/25/10: 16:00-21:00
Dirk: 6/27/10: 14:00-22:00
Dirk: 6/28/10: 17:00-20:00
Shay: 7/1/10: 9:00-20:00
Meriem+Burak: 7/2/10: 11:00-20:00
Meriem+Billy: 7/9/10: 15:00-17:00
Meriem+Billy: 14/9/10: 15:00-19:00
Burak: 7/15/10: 10:00-18:00
Dirk: 7/20/10: 16:00-20:00 (with EMCCD)
nate: 7/21/10: 16:00-20:00
Dirk: 7/26/10: 16:00-20:00
Nate: 7/26/10: 20:00-o/n
Dirk: 7/27/10: 12:00-20:00
Burak: 7/28/10: 15:00-20:00

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

  • Hamamatsu Orca R2 (Bf/Fluorescence camera, R100 port, 12- and 16-bit, dimension: 1344 x 1024, pixel size: 6.45 um x 6.45 um)
  • Scion CFW-1612M (Bf/Phase camera, E100 port, 12-bit, dimension: 1600 x 1200, pixel size: 4.40 um x 4.40 um)

Optional:

  • Hamamatsu EM-CCD C9100-02 (Bf/Fluorescence camera, L100 port, 14-bit, dimension: 1000 x 1000, pixel size: 8 um x 8 um)

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 (e.g. 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

  • GFP:

filters are sitting on a custom made brass filter cube

dichroic: Di01-R488 (Semrock) emission 1: BLP01-488R-25 (Semrock) emission 2: FF01-525/45 (Semrock)

Lasers

  • 488nm, 75mW Coherent Sapphire
  • 405nm, 50mW Coherent Cube

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.




Nikon Ti microscope station #2

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

Configuration

General

  • Lumencor?

Objectives

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

Cameras

  • Hamamatsu Orca R2 (Bf/Fluorescence camera, R100 port, 12- and 16-bit, dimension: 1344 x 1024, pixel size: 6.45 um x 6.45 um)
  • Scion CFW-1612M (Bf/Phase camera, E100 port, 12-bit, dimension: 1600 x 1200, pixel size: 4.40 um x 4.40 um)

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)
  • 5. empty
  • 6. empty (Bf/Phase)

Maintenance log

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