Zeiss Axio Observer.A1 Inverted Microscope (S972) Use Google Calendar

Inverted fluorescent microscope, Olympus DP80 RGB color and NIR capable monochrome camera
Location: S972

Zeiss Axio Observer.A1 Inverted Microscope in Ross S972 has an Olympus DP80 RGB color + monochrome camera, capable of both RGB histology images and fluorescence. Phase contrast is available for several objective lenses.

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as of 20190709 (July 9, 2019) the online scheduler is on our Google Calendar,

https://calendar.google.com/calendar/embed?src=fsbhh2vstjd11foaems9muikk0%40group.calendar.google.com&ctz=America%2FNew_York

 

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Quck tip, Feb 20, 2019: Optimizing for brightfield image capture, i.e. H&E or IHC (DAB&H):

  • get training from George on how to do the steps below (I am deliberately not including pictures here - these steps are the brightfield microscope equivalent of 'learning to walk', or more mechanically, learning to ride a bicycle - should become automatic for you.
  1. turn on Zeiss.A1 microscope power, light path to eyes, transmitted light shutter open, condenser turret to "H" (German for Hole? ... not Ph# or DIC), filter cube turret to empty position, turn up the lamp voltage to be comfortable to your eyes (and white light, not orange). I recommend start with the 10x objective lens.
  2. Focus your specimen (i.e. H&E slide) with the microscope focus (left and right side of base).
  3. Shrink the condenser light path field aperture diaphragm all the way. If this is an in focus octagon, great ... if not, use the condenser focus knob to bring it to focus.
  4. Adjust the condenser numerical aperture (the 'dial' on the left side of the condenser) to that the illuminated area (conter of the octagon) is (almost) full brightness, while the aperture diaphragm is black (or nearly black).
  5. Open the field aperture diaphragm enough to get it out of the way ... I like to park it just inside the eyepiece field of view (which is way outside the camera field of view).
  6. In Olympus CellSens software, use manual exposure time (ex. 40 milliseconds), to get to 'reasonable brightness', while not saturating the empty (bright) areas. 
  7. If you change objective lenses: ideally re-do for each objective lens. Typically one objective lens images will be most critical for your experiment, so simplest to optimize the Numerical Aperture Diahragm for that lens, and not mess with it during your session (the other lenses will be not-quite-perfect, but the premise here is one lens is the priority).
  8. Color balancing: CellSens software has a control for this (icon).
  9. I note there is plenty of free (or almost free) software to stitch. For example, Microsoft ICE (Image Composite Editor). Fiji ImageJ and Adobe Photoshop can also stich.
  • if already acquired, see my 2005 article, color balancing with Adobe Photoshop, http://www.home.earthlink.net/~geomcnamara/McNamara2005JoH28n2pp81-88.pdf

 

Big News January 11, 2019: We replaced the DP72 RGB camera with Olympus DP80 RGB + monochrome camera. The two CCD sensors are controlled by the same two CellSens buttons for RGB and monochrome, but now moves a hardware component to switch between the two CCD sensors. This will enable much better imaging of Cy5 and Alexa Fluor 647 and other near infrared (NIR) fluorophores. The DP80 RGB color sensor is identical to the DP72. Please note that currently the eyepieces and DP80 are not parfocal - we recommend spending as much time as possible "live" with the DP80 and little time looking through the eyepieces. We are confident the benefits of having RGB + monochrome CCD sensor options outweight the minor inconvenience of not being parfocal. GM thanks John Gibas (Olympus) and Bill Griffith, (rgriff49@jhmi.edu), Sr. Instrument Designer, BME Machine shop, Traylor 4th floor, for big help.

  Jan 14, 2019 note: we will probably remove the Eppendorf micromanipulator from this microscope for lack of use. It will remain close by and can be set up again. Users interested in it will need to plan ahead and contact George to see about getting it set up on the "A1".

Suitable specimens include:

* microscope slides ... histology and/or fluorescence (DAPI, green, red, the microscope has a Cy5 filter set - please use the DP80's monochrome camera for single channel fluorescence.

* multiwell plates.

* 35 mm or 60 mm plastic (modest resolution and fluorescence sensitivity) and glass bottom (i.e. Mattek.com P35 series imaging dishes).

Our CellSens 2.02 license includes:
MIA = Multi Image Alignment Algorithm ... panorama
EFI = Extended Focal Imaging

for summary, see brochure at https://unicam.hu/files/olympus-cellsens/cellSens_Attekintes.pdf   (PDF also on our server and on acquisition PC).

Olympus CellSens tutorials (videos) online at https://www.olympus-lifescience.com/en/software/cellsens/?utm_content=sf92551011&utm_medium=social-media&utm_source=googleplus&utm_campaign=Olympus%20Life%20Science&sf92551011=1#!cms[tab]=%2Fsoftware%2Fcellsens%2Fresources 

 

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instrument info

 

McNamara 20190517Fri – Zeiss AxioObserver A1 filter sets

Zeiss ## filter set specifications are from https://www.micro-shop.zeiss.com/en/de/shop/filterAssistant/filtersets

Filter sets
  Zeiss Exciter Dichroic Emitter Zeiss catalog
B 49 G 365 FT 395 BP 445/50 488049-9901-000
G 38 BP 470/40 FT 495 BP 525/50 000000-1031-346
R 43 BP 545/25 FT 570 BP 605/70 000000-1114-101
RGB 25

TBP 400 + 495 + 570

TFT 410 + 505 + 585 TBP 460 + 530 + 625 488025-0000-000
Cy5         -

Our Olympus DP80 camera has dual RGB and monochrome CCD sensors (Cellsens acquisition has a toggle for RGB and monochrome). The monochrome sensor is much higher sensitivity, including for Cy5, so should be used for fluorescence. That makes the “RGB” filter set only useful for you to look by eye. The DP80 RGB sensor is ideal for H&E, H&DAB, and similar transmitted light applications (empty cube position between Cy5 and DAPI cubes, or RRGB cube – your choice).


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