Commercial post:Light sheet fluorescence microscopy

[John Zemek]

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Dear List,

What ASI has available for iSPIM & diSPIM systems is all of the necessary
hardware for generating the light sheet from a laser, and for scanning the
sample. We have systems for iSPIM where we focus the light sheet onto the
sample with one objective & imaged with the other, and dual iSPIM where we
can illuminate & image from both objectives. The objectives both can be moved
very rapidly & precisely with one of our piezo objective movers. The scan head
utilizes Micro-Mirrors for fast, reliable & economical positioning of the
illumination beam. We have developed this in collaboration with Hari Shroff at
the NIH/NIBIB; here are a couple of You Tube videos on his work:

http://www.youtube.com/watch?v=evBpK8OuKnE 
http://www.youtube.com/watch?v=KriozUQXpN4

The components that we have can be mounted onto the transmitted
illumination pillar of an inverted microscope, or onto our RAMM system. In this
configuration a third objective can be used to image the sample from below.
We currently do not provide the lasers, objectives, and cameras for these
systems.  The software that Hari’s group has developed is also available, and
you would need to talk with their group regarding this.

There are three basic configurations of SPIM systems that we have built for
various users.  
These are:

1)  Fixed sheet systems.  (single sided) The light sheet is stationary, with only
mechanical adjustment for the sheet position.  The two objectives are manually
adjusted to correctly focus on the sheet.  The specimen is scanned through
the sheet using the X and Z stages to generate volume images.  Advantages of
this system is that it is the least expensive - not requiring either galvo
scanners or a piezo objective positioner.  Disadvantage is the difficulty in
correctly overlapping the objective focal planes with fixed positioners and the
relatively slower stage scanning at odd angles.
 
2)  Standard single-sided system.  Light sheet from one side, emission
objective on the other.  The light sheet can be scanned using galvos to sweep
across the sample volume.  There is an emission objective piezo so the viewing
objective can be positioned to follow the light sheet as it is scanned through
the sample.
Advantages:  Rapid scanning, straight-forward set-up.
Disadvantages:  Better XY resolution than Z resolution.
 
3) Double-side system.  Light sheet excitation and emission on each side.  Both
sides have a piezo-objective positioner.  You need light sheets on each side as
well.  Dr. Shroff is currently getting very nice results from the dual system and
will be publishing a paper on this in a few months.
Advantages:  With proper post processing, XY & Z resolutions are all very good
- yielding a combination of speed and resolution that is unsurpassed for live cell
imaging.
Disadvantages:  Complicated and expensive.
 
You have choices for objectives to use for SPIM.  We have developed the
system for water-dipping objectives.   On single sided systems, the excitation
objective is usually the Nikon 10X NA 0.3 water.  Paired with this objective you
can use either the Nikon 40X NA 0.8 or, if you tilt the entire assembly a few
degrees, the Olympus 20X NA 1.0 water objective.  There are a few other
objectives that will also work, but many high NA objectives cannot be brought
into mutual focus because they bump into each other.  For Dual-sided systems,
a pair of Nikon 40X NA 0.8 water objectives are usually used.  We can provide
the correct spacers for which ever objectives you decide to use.
 
This paper may also be of help:

http://www.asiimaging.com/downloads/references/2011%20Wu%20Colon-
Ramos%20Inverted%20selective%20plane_PNAS.pdf


We hope that this is of some help and if you want to contact us off line we will
keep you posted on our progress.

Best,