Zeiss Imager.Z2 upright microscope
SB 4th floor
Contact Caterina Berti and Michael Cammer for access

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Instructions:  [Word doc]   [PDF]   [Full Zeiss manual]

This microscope has a monochrome camera and lenses with minimal focus shift due to color for high quality fluorescence imaging. It also has an automated stage for tiling large areas and a focus motor for Z series collection. It has DIC optics for transmitted light imaging. All lenses require #1.5 coverslip (170 to 175 um thick glass).

A few of the specifications:

• Zeiss Imager.Z2
• Zen 2.6 Blue
• HP Z240 32GB Windows 7 Ultimate
• 5X N.A. 0.16 Planapochromat 420630-9900
• 10X N.A. 0.45 Planapochromat 420640-9900
• 20X N.A. 0.80 Planapochromat 420650-9902
• (may be available by request 40X N.A. 0.95 Planapochromat 420660-9970 Adjustable collar)
• 63X N.A. 1.4 OIL DIC Planapochromat 420782-9900-1
• X-Cite-120LED Boost Wavelength Range: 370-700nm for reflected light, Power Consumption: 230W
• LED for transmitted light
• Axiocam 506 Mono Technical specifications and instruction manual
• DIC
• Fluorescent filter blocks:     [click here for photos of the filter blocks]
  1. near-IR; EX 625-655; split 660; EM 665-715; (Cy5, Alexa 647)
  2. Red; EX540-552; split 560; EM 575-640; (Rhodamine, Cy3, Alexa 568, RFP, mCherry...)
  3. Green; EX450-490; split 495; EM 500-550; (FITC, Alexa 488, eGFP, eYFP (inefficient but works)...
  4. Blue; EX335-383; split 395; EM 420-470; (dapi); n.b. that the lamp spectra make this effectively a 360-383 nm exciter.
• There is no filter set for Alexa Fluor 594; the Rhod set may be used but is inefficient.

People often ask, what about 100X? The 63X N.A. 1.40 lens has the same spatial resolution at the 100X lens when matched with the small pixel AxioCam on this microscope but with benefits such as a wider field of view.

Note about chromatic aberration. Different wavelengths focus at different focal planes. These lenses aim to correct this by being planapochromat. However, they are not perfect. For instance, we found a 2 um shift in Z from dapi to Cy5 with the 20X lens in a sample mounted in Prolong Diamond. To correct this, the Cy5 channel was set as the reference, precise focus is done in this channel, and a 2 um offset was set in the dapi channel. (Nomarski is imaged without an offset.)

Antivibration table needs to be floating properly for high resolution microscopy. Please turn off the gas after each use.

How to set the correct exposures for widefield fluorescence.

Typically images are too bright with too many saturated pixels. This undermines spatial resolution and the ability to quantify relative numbers of molecules. This video explains how to set good exposures.

It assumes excitation intensity fixed at 100% and imagers will only change exposure time. There is no discussion of camera gain, binning, or field size. This video exclusively addresses exposure time and its influence on dynamic range and LUT adjustments to change image appearance without altering the raw data.

Do you find that your sample bleaches while you are setting up imaging conditions? This video explains how to avoid this.