Paulsson:Microscopes

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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

Nate: 4/1/09 18:00-O/N
Nate: 4/2/09 14:00-20:00
Burak: 4/3/09 14:00-20:00
Burak: 4/6/09 14:00-20:00
Shay: 04/07/09 10:00-21:00 (43C. You may be able to use it before 14:00 if you don't mind the temperature)
Burak: 4/8/09 16:00-19:00
Billy: 4/9/09 18:00-20:00
Shay: 04/13/09 10:00-20:00 (43C. You may be able to use it before 14:00 if you don't mind the temperature)
Dann: 04/14/09 10:30-14:30

Configuration

Objectives

• 20x objective
• 40x objective
• 63x phase objective (is it phase?)
• 100x phase objective
• 100x objective
• find out more about the different Carl Zeiss objectives (http://www.zeiss.de/C1256D18002CC306/0/502076E7CBDC5264C12573530050F208/$file/Objectives_for_Microscopes_from_Carl_Zeiss.pdf)

Filter cubes

• 1. GFP: BP 470/40; FT 495; BP 525/50
• 2. RFP: BP 565/30; FT 585; BP 620/60
• 3. DAPI: BP 390/22; FT 420; BP 460/50
• 4. CFP : BP 436/25; FT 455; BP 480/40
• 5. YFP: BP 500/25; FT 515; BP 535/30

spare filter cubes:

• mCherry RFP (is ordered)
• Burak's Qdot filter cube (QD605-A from Semrock)

ex: 435/40 Dichroic: 510LP em: 605/15

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

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

Sign-up list

User / Date / Duration

Bruno: 01/27/09 o/n time-course
Dirk: 01/29/09 10:00-22:00 (Jim comes to take measurements of the table and I will run some tests)
Dirk: 02/02/09 - : setting up the scope, interfacing Micro-Manager with Matlab, solving some camera problems, etc
Burak: 02/09/09 18:00 - o/n - 02/11/09 11:00 (I will disassemble the upper part to take off the turret for Jim to take some measurements)
Dirk: 02/25/2009: 18:00 - 20:00
Paul: 02/27/2009: 18:00 - 21:00
Dirk: 02/28/2009: 15:00 - 20:00
Dirk: 03/09/2009: 13:00 - 20:00
Dirk: 03/11/2009: 12:00 - 20:00
Dirk: 03/12/2009: demonstration for John (Murray Lab)
Dirk: 03/13/2009: installation of Scion camera
Dirk: 03/19/2009: Barry will drop by
Dann: 04/04/2009: 17:00 - 18:00
Dirk: 04/11/2009:14:00 - 18:00 (Phase and PFS are not working)
Burak: 04/12/2009: 14:00 - 22:00 - optics alignment

Configuration

General

• find out more about the Nikon Ti on the official Nikon website (http://www.nikoninstruments.com/ti/)
• the microscope is controlled by Micro-Manager (http://micro-manager.org/)
• control via Matlab is also possible (https://valelab.ucsf.edu/~nico/MMwiki/index.php/Matlab_Configuration)

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)

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. Bf/Phase
• 6. Bf/Phase

filters for the lasers

Lasers

• 488nm, 75mW Coherent Sapphire
• 405nm, 50mW Coherent Cube
• 515nm, 50mW Cobolt Fandango

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. Also, the PFS switches off if you change the x,y position. Time-lapse on a single position with functional PFS is possible though.