Zeiss Light Sheet Z1 notes

Lightsheet microscopy is excellent for transparent live organisms such as embryos and zebrafish and for small cleared organs. It complements confocal microscopy by imaging optical sections of larger tissues.

The lightsheet microscope here works with living samples in water or media and with fixed and cleared samples such as CLARITY. Aqueous media only. Refractive indexes officially 1.33 and 1.45.

With lasers at 405, 488, 568, and 638 nm, this system images most genetically encoded fluorescent proteins and other popular fluorescent probes. Magnification is at 5X N.A. 0.16 or 20X N.A. 1.0. Images are collected with two 1920 X 1920 pixels sCMOS cameras.

Schedule instrument at at http://nyusaphapp01.nyumc.org:8080/limsprod/logon.jsp

Sales info Brochure

2013 Zeiss PDF instruction manual

2014 Zeiss PDF instruction manual (other languages available in Zen/manuals folder on the microscope computer)

Zeiss Sample Prep manual

 

Instrument Startup

 

Lasers

405, 488, 561, and 638 nm

 

Lenses

When switching lenses, need to go to Maintain tab to set both illumination and detection.

Illumination for 20X Water, 10X/0.2 400900-9000-710

20X Water lens W Plan-Apochromat N.A. 1.0 421452-9700

Always:
5X illumination optics for 5X objective lens
10X illumination optics for 20X objective lens

When installing chamber for live work, if want to monitor temperature, must plug in temperature probe BEFORE starting software. Restart Zen if necessary.

When screwing in optics, do not tightened. Simply turn until they stop. Do not tighten. Did I say do not tighten?

 

Sample Prep

A live zebrafish is embedded in agarose inside a small bore pipette. After the agarose hardens, it is pushed out into the sample chamber of the microscope (teflon tip of plunger at far left). Imaging cannot be performed through the glass.

 

CLARITY sample embedded in agarose:

 

The syringe is a special type available in Europe. However, we have found at least one availablein the U.S. that works well too.

 

Alternative CLARITY sample method glued to side of capillary with Krazy Glue (only allows for 270 degrees of imaging):

We have imaged with slight refractive index mismatch in 80% glycerol/water and will post better media soon.

 

How to switch from 5X CLARITY to 20X CLARITY click here

 

Things you need:

 

 

Image Processing

We need to write macros for IJ to open a series of images, process, and save out for display. First try here.

One example would be to cross subtract channels, or percentages of channels from the other cahnnel.

Here is an example of how to do this in Zen. This takes each slice of a ZT series and subtracts the autofluoresence in channel1 from the signal detected in channel2. This highlights cells that are in channel2, but has the danger of removing channel2 real signal that is in the same XYZ position as autofluoresence.

This has to be done on the raw data because projections don't preserve data specific to Z locations.

 

If there are spillover problems and you can image a little slower, you can set up tracks such that different laser and filter combinations are imaged in sequence. In the following example where on the left the green signal spills over into the red channel when both the 488 and 561 nm lasers are illuminating, the images on the right show a track with 488 nm alone and camera 1 followed by 561 nm alone and camera 2. In this case, no filters were changed so mode "Frame Fast", but filter changescan be effected too although relatively slow. Also, two tracks have the benefit that the two cameras may have different exposure times.

 

Chromatic aberration?

 

Note to self:
Need to add:

  1. more instructions on how to change the filter block.  
  2. I’m not sure what the illumination optics are
  3. There isn’t a ring for the 20x oil clarity objective, right? 
  4. Add segment on shut off/clean up procedure?

 

To Clean Chambers

Replace glass windows, glass against metal and O ring on outside.

 

Early attempt to image lung.

 

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comments, questions, suggestions: Michael.Cammer@med.nyu.edu