Lightsheet microscopy

[Knecht, David]

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In confocal microscopy, we often run up against the problem of sample thickness limiting light penetration and objective working distance limiting capture depth when trying to image fluorescence deeper into thicker specimens. Overcoming some of those problems was the justification for 2 photon microscopy.  Now we have light sheet and I am trying to educate myself about the technology, and do not understand how (or if) that problem is overcome for specimens that are presumably somewhat larger than one would try to image with conventional confocal xyz microscopy.  I can see its utility for transparent zebrafish and you want to look at structures the size of cells, but If you have a tissue or embryo that you have trouble getting more than 100µm into with a confocal, does the same limitation exist with light sheet?  Then you have the problem of working distance and numerical aperture limiting specimen size, resolution and sensitivity.  So my question is, what are most appropriate situations where it provides a real complement to a LSCM or spinning disk confocal and what are the practical limitations of this technology.  Does it gain you anything for larger samples that are hard to image on a confocal?  Thanks- Dave

Dr. David Knecht
Professor of Molecular and Cell Biology
Core Microscopy Facility Director
University of Connecticut
91 N. Eagleville Rd.
Storrs, CT 06269
860-486-2200

[Howard Berg]

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Dave,

We recently tested Leica’s digital light sheet on plant samples (meristems, leaf tissue, root tissue, and Volvox).  There does not appear to be any benefit for penetrating deeper into tissue.  Clearing the meristems does help but we get comparable images with LSCM.  The Volvox was outstanding for light sheet’s benefits:  imaging of large samples with low laser dose.  In about 60 seconds we captured  over 400 sections through a huge Volvox cell.  Furthermore, we did time-lapse imaging of Volvox embryogenesis, 360 sections every 8 minutes for 16 hours,  and the cells remained living.  The resulting movie was really cool, showing repeated cell divisions, then inversion of the organism (as it spun), putting the previously external young Volvox inside the inverted mother cell.  My conclusion is that the utility of light-sheet is sample-dependent.
-Howard

Howard Berg
Director, Integrated Microscopy Facility
Danforth Plant Science Center
975 North Warson Road
St. Louis, MO 63132

> On Jul 30, 2015, at 8:29 PM, Knecht, David <[hidden email]> wrote:
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> In confocal microscopy, we often run up against the problem of sample thickness limiting light penetration and objective working distance limiting capture depth when trying to image fluorescence deeper into thicker specimens. Overcoming some of those problems was the justification for 2 photon microscopy.  Now we have light sheet and I am trying to educate myself about the technology, and do not understand how (or if) that problem is overcome for specimens that are presumably somewhat larger than one would try to image with conventional confocal xyz microscopy.  I can see its utility for transparent zebrafish and you want to look at structures the size of cells, but If you have a tissue or embryo that you have trouble getting more than 100µm into with a confocal, does the same limitation exist with light sheet?  Then you have the problem of working distance and numerical aperture limiting specimen size, resolution and sensitivity.  So my question is, what are most appropriate situations where it provides a real complement to a LSCM or spinning disk confocal and what are the practical limitations of this technology.  Does it gain you anything for larger samples that are hard to image on a confocal?  Thanks- Dave
>
> Dr. David Knecht
> Professor of Molecular and Cell Biology
> Core Microscopy Facility Director
> University of Connecticut
> 91 N. Eagleville Rd.
> Storrs, CT 06269
> 860-486-2200

[Davide Accardi]

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Hi David,

We have worked with the ZEISS LZ.1 for about 3 years and with a multitude of samples.
As already Howard said, the "single view" light-sheet technology does not give you higher penetration depth than single photon confocal microscopy.
However, since the ZEISS machines (and the majority of custom made systems) give you the possibility to rotate 360 degrees the spacemen and eventually combine all the images, even if you can penetrate the sample only 100 µm in one direction, you can always rotate it and acquire other 100 µm in the opposite direction.
Therefore, if your sample is 200µm thick with the LZ.1 gives you the advantage to acquire it all.
If the sample is significantly bigger and not transparent, then of course you would get as final image just the first 100µm of the out layer of your sample. Again, from 360 degrees.
Regarding the other advantages, speed and low photo toxicity and photo bleaching, Howard already said it all.

I hope this helps.

Best,

Davide
--
Dr. Davide Accardi
Light Microscopy Facility
Max Planck Institute of Molecular Cell Biology and Genetics
Pfotenhauerstrasse 108
01307 DRESDEN
Germany

Phone: [phone]




On Jul 31, 2015, at 4:20 AM, Berg, R. Howard wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Dave,
>
> We recently tested Leica’s digital light sheet on plant samples (meristems, leaf tissue, root tissue, and Volvox).  There does not appear to be any benefit for penetrating deeper into tissue.  Clearing the meristems does help but we get comparable images with LSCM.  The Volvox was outstanding for light sheet’s benefits:  imaging of large samples with low laser dose.  In about 60 seconds we captured  over 400 sections through a huge Volvox cell.  Furthermore, we did time-lapse imaging of Volvox embryogenesis, 360 sections every 8 minutes for 16 hours,  and the cells remained living.  The resulting movie was really cool, showing repeated cell divisions, then inversion of the organism (as it spun), putting the previously external young Volvox inside the inverted mother cell.  My conclusion is that the utility of light-sheet is sample-dependent.
> -Howard
>
> Howard Berg
> Director, Integrated Microscopy Facility
> Danforth Plant Science Center
> 975 North Warson Road
> St. Louis, MO 63132
>
>
>> On Jul 30, 2015, at 8:29 PM, Knecht, David <[hidden email]> wrote:
>>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> Post images on http://www.imgur.com and include the link in your posting.
>> *****
>>
>> In confocal microscopy, we often run up against the problem of sample thickness limiting light penetration and objective working distance limiting capture depth when trying to image fluorescence deeper into thicker specimens. Overcoming some of those problems was the justification for 2 photon microscopy.  Now we have light sheet and I am trying to educate myself about the technology, and do not understand how (or if) that problem is overcome for specimens that are presumably somewhat larger than one would try to image with conventional confocal xyz microscopy.  I can see its utility for transparent zebrafish and you want to look at structures the size of cells, but If you have a tissue or embryo that you have trouble getting more than 100µm into with a confocal, does the same limitation exist with light sheet?  Then you have the problem of working distance and numerical aperture limiting specimen size, resolution and sensitivity.  So my question is, what are most appropriate situations where it provides a real complement to a LSCM or spinning disk confocal and what are the practical limitations of this technology.  Does it gain you anything for larger samples that are hard to image on a confocal?  Thanks- Dave
>>
>> Dr. David Knecht
>> Professor of Molecular and Cell Biology
>> Core Microscopy Facility Director
>> University of Connecticut
>> 91 N. Eagleville Rd.
>> Storrs, CT 06269
>> 860-486-2200

[Andreas Bruckbauer]

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 Hi David,

One can think of the image formation as a two step process, first the light has to get to the position you want to image, then the excited fluorescence has to get out and to the detector. In 2-photon the longer excitation wavelength penetrates better through the sample, but a good focus is important to excite fluorescence, whereas for 1 photon lightsheet the shorter excitation wavelength does not penetrate much, but a bit of distortion of the sheet is not a problem as long as it is not completely blocked causing a shadow.
One has to understand that the image formation in normal laser scanning 2 photon microscopy is different, as the sectioning is done by the nonlinear properties of the 2-photon excitation, causing fluorescence to originate only from a small point. Scattering of the fluorescence light on the way to the detector is less of a problem as long as non-descanned large area detectors sitting close to the objective are used. Light which is scattered away and exits the objective at a slight angle is still detected and as it comes from the focal spot, we still have good sectioning capabilities. However with the light sheet illumination (even when combined with 2 photon excitation) the light still has to form an image on the CCD camera. So scattering along the way will blur the image in xy.
I recently compared 1 photon light sheet with laser scanning two photon imaging applied to lfixed lymph nodes and the 2 photon case is largely superior. However this might change with cleared samples. For live imaging in these samples, laser scanning two photon is the method of choice.
Sample rotation can also be used for other imaging modalities, LaVision BioTec has an add on which they call horizontal 2-Photon microscope which rotates the sample. http://www.photonics.com/Product.aspx?PRID=56741

best wishes

Andreas






 

-----Original Message-----
From: Davide Accardi <[hidden email]>
To: CONFOCALMICROSCOPY <[hidden email]>
Sent: Fri, 31 Jul 2015 7:45
Subject: Re: Lightsheet microscopy


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to:
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*****

Hi
David,

We have worked with the ZEISS LZ.1 for about 3 years and with a
multitude of samples.
As already Howard said, the "single view" light-sheet
technology does not give you higher penetration depth than single photon
confocal microscopy.
However, since the ZEISS machines (and the majority of
custom made systems) give you the possibility to rotate 360 degrees the spacemen
and eventually combine all the images, even if you can penetrate the sample only
100 µm in one direction, you can always rotate it and acquire other 100 µm in
the opposite direction.
Therefore, if your sample is 200µm thick with the LZ.1
gives you the advantage to acquire it all.
If the sample is significantly
bigger and not transparent, then of course you would get as final image just the
first 100µm of the out layer of your sample. Again, from 360 degrees.
Regarding
the other advantages, speed and low photo toxicity and photo bleaching, Howard
already said it all.

I hope this helps.

Best,

Davide
--
Dr. Davide
Accardi
Light Microscopy Facility
Max Planck Institute of Molecular Cell
Biology and Genetics
Pfotenhauerstrasse 108
01307 DRESDEN
Germany

Phone:
[phone]




On Jul 31, 2015, at 4:20 AM, Berg, R. Howard
wrote:

> *****
> To join, leave or search the confocal microscopy listserv,
go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images
on http://www.imgur.com and include the link in your posting.
> *****
>
>
Dave,
>
> We recently tested Leica’s digital light sheet on plant samples
(meristems, leaf tissue, root tissue, and Volvox).  There does not appear to be
any benefit for penetrating deeper into tissue.  Clearing the meristems does
help but we get comparable images with LSCM.  The Volvox was outstanding for
light sheet’s benefits:  imaging of large samples with low laser dose.  In about
60 seconds we captured  over 400 sections through a huge Volvox cell.
Furthermore, we did time-lapse imaging of Volvox embryogenesis, 360 sections
every 8 minutes for 16 hours,  and the cells remained living.  The resulting
movie was really cool, showing repeated cell divisions, then inversion of the
organism (as it spun), putting the previously external young Volvox inside the
inverted mother cell.  My conclusion is that the utility of light-sheet is
sample-dependent.
> -Howard
>
> Howard Berg
> Director, Integrated
Microscopy Facility
> Danforth Plant Science Center
> 975 North Warson Road
>
St. Louis, MO 63132
>
>
>> On Jul 30, 2015, at 8:29 PM, Knecht, David
<[hidden email]> wrote:
>>
>> *****
>> To join, leave or search the
confocal microscopy listserv, go to:
>>
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> Post images on
http://www.imgur.com and include the link in your posting.
>> *****
>>
>> In
confocal microscopy, we often run up against the problem of sample thickness
limiting light penetration and objective working distance limiting capture depth
when trying to image fluorescence deeper into thicker specimens. Overcoming some
of those problems was the justification for 2 photon microscopy.  Now we have
light sheet and I am trying to educate myself about the technology, and do not
understand how (or if) that problem is overcome for specimens that are
presumably somewhat larger than one would try to image with conventional
confocal xyz microscopy.  I can see its utility for transparent zebrafish and
you want to look at structures the size of cells, but If you have a tissue or
embryo that you have trouble getting more than 100µm into with a confocal, does
the same limitation exist with light sheet?  Then you have the problem of
working distance and numerical aperture limiting specimen size, resolution and
sensitivity.  So my question is, what are most appropriate situations where it
provides a real complement to a LSCM or spinning disk confocal and what are the
practical limitations of this technology.  Does it gain you anything for larger
samples that are hard to image on a confocal?  Thanks- Dave
>>
>> Dr. David
Knecht
>> Professor of Molecular and Cell Biology
>> Core Microscopy Facility
Director
>> University of Connecticut
>> 91 N. Eagleville Rd.
>> Storrs, CT
06269
>> 860-486-2200

 

[Julio Vazquez-2]

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*****

Not much to add to the previous posts, but for an intro to light sheet, I suggest the following video lectures:

http://www.ibiology.org/ibioeducation/taking-courses/light-sheet-sectioning.html

http://www.ibiology.org/ibioeducation/taking-courses/ibiology-microscopy-course/dual-view-inverted-selective-plane-illumination-dispim.html


Eric Betzig's talk "Imaging life at high spatio-temporal resolution" gives a great perspective on various recent microscopy techniques, including lattice light sheet, which pushes the concept one giant step further:

https://www.youtube.com/watch?v=2R2ll9SRCeo


There are also some nice examples of light sheet applications on the Zeiss web site:

http://www.zeiss.com/microscopy/en_us/products/imaging-systems/lightsheet-z-1.html#introduction




Julio Vazquez
Scientific Imaging
Fred Hutchinson Cancer Research Center
Seattle, WA 98109

http://www.fredhutch.org/en.html

--
On Jul 30, 2015, at 6:29 PM, Knecht, David wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> In confocal microscopy, we often run up against the problem of sample thickness limiting light penetration and objective working distance limiting capture depth when trying to image fluorescence deeper into thicker specimens. Overcoming some of those problems was the justification for 2 photon microscopy.  Now we have light sheet and I am trying to educate myself about the technology, and do not understand how (or if) that problem is overcome for specimens that are presumably somewhat larger than one would try to image with conventional confocal xyz microscopy.  I can see its utility for transparent zebrafish and you want to look at structures the size of cells, but If you have a tissue or embryo that you have trouble getting more than 100µm into with a confocal, does the same limitation exist with light sheet?  Then you have the problem of working distance and numerical aperture limiting specimen size, resolution and sensitivity.  So my question is, what are most appropriate situations where it provides a real complement to a LSCM or spinning disk confocal and what are the practical limitations of this technology.  Does it gain you anything for larger samples that are hard to image on a confocal?  Thanks- Dave
>
> Dr. David Knecht
> Professor of Molecular and Cell Biology
> Core Microscopy Facility Director
> University of Connecticut
> 91 N. Eagleville Rd.
> Storrs, CT 06269
> 860-486-2200

[jerie]

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Dear David,

>>what are most appropriate situations where it provides a real complement
to a LSCM or spinning disk confocal and what are the practical limitations
of this technology.  Does it gain you anything for larger samples that are
hard to image on a confocal?

Turning the sample, in LS imaging you are basically limited by the lateral
resolution of the optics and can afford lower NA to achieve the same voxel
volume resolution, which in addition is isotropic. Lower NA imaging reduces
scattering and allows larger working distance. Similar to spinning disk
confocal, the distribution of the excitation light helps to avoid
saturation. Excitation is rather confined to the area of detection,
resulting in very photon efficient imaging, which is an advantage compared
to setups using a pinhole to prevent larger parts of the signal to reach
the detector. LS allows very fast or very long term confocal imaging. It
can produce large amounts of data in short time.

my 2 cents

Abraços,
Jens

Dr. Jens Rietdorf, visiting scientist @ center for technological
development in health.
FIOCRUZ, Rio de Janeiro, Brazil.
http://br.linkedin.com/pub/jens-rietdorf/6/4a3/189/

>
>

[Raghu Parthasarathy]

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I agree with the earlier posts on the topic. I'd like to stress that the issue of lower photobleaching / phototoxicity is (arguably) the main advantage of light sheet microscopy compared to confocal in many cases. Since the "extra" light needed for confocal imaging compared to light sheet scales roughly with the number of slices in z, it is especially important for large specimens. (For a minor paper from my lab illustrating phototoxicity w/ light sheet and w/ confocal, see http://onlinelibrary.wiley.com/doi/10.1002/jbio.201200144/abstract.)

The second-most important attribute of light sheet imaging, in my opinion, is the fast acquisition speed for large fields of view. 
Tissue penetration issues are (again perhaps arguably) worse for light sheet imaging compared to confocal, since one has to worry about the penetration in the excitation direction and the (orthogonal) detection direction.

A prior email noting that multi-view imaging allows a way around this is correct, but this comes at the expense of imaging speed (since one needs specimen rotation + multiple images). One can also use complicated / tight multi-lens setups. There are other solutions, e.g. structured illumination (w/ lattices and other things), but none are trivial to implement. One can also use two-photon excitation together with light sheet microscopy:http://www.nature.com/nmeth/journal/v8/n9/abs/nmeth.1652.htmlhttp://www.nature.com/nmeth/journal/v12/n5/full/nmeth.3371.html#close
I'll echo Howard Berg's comment that light sheet's utility is specimen dependent. For our own imaging of the larval zebrafish gut, which requires fast acquisition of 3D images over a large volume, and large imaging durations, it's been crucial and enlightening! (Spatial and Temporal Features of the Growth of a Bacterial Species Colonizing the Zebrafish Gut)

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| Spatial and Temporal Features of the Growth of a Bacteri...Spatial and Temporal Features of the Growth of a Bacterial Species Colonizing the Zebrafish Gut Matthew Jemielitaa, Michael J. Taorminaa,b, Adam R. Burnsc, Jenni... |
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| View on mbio.asm.org | Preview by Yahoo |
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   I'll also mention, since it seems widely unappreciated, that there are uses for light sheet microscopy outside live-imaging, e.g.: Dancing Membranes

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| Dancing MembranesA friend of mine posts on his blog “lay summaries” of papers he writes, which I’ve always thought is a good use for a blog, though I’ve never gotten around to doing... |
|  |
| View on eighteenthelephant.w... | Preview by Yahoo |
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(Again making use of fast imaging speeds).
best wishes,
Raghu
 --
Raghuveer Parthasarathy
[hidden email]
Group web page: http:&#x2F;&#x2F;physics.uoregon.edu&#x2F;~raghu&#x2F;
Blog: http:&#x2F;&#x2F;eighteenthelephant.wordpress.com&#x2F;

Associate Professor
Department of Physics
1274 University of Oregon
Eugene, OR 97403-1274
http:&#x2F;&#x2F;physics.uoregon.edu&#x2F;~raghu&#x2F;
      From: "Knecht, David" <[hidden email]>
 To: [hidden email]
 Sent: Thursday, July 30, 2015 6:29 PM
 Subject: Lightsheet microscopy
   
*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
Post images on http://www.imgur.com and include the link in your posting.
*****

In confocal microscopy, we often run up against the problem of sample thickness limiting light penetration and objective working distance limiting capture depth when trying to image fluorescence deeper into thicker specimens. Overcoming some of those problems was the justification for 2 photon microscopy.  Now we have light sheet and I am trying to educate myself about the technology, and do not understand how (or if) that problem is overcome for specimens that are presumably somewhat larger than one would try to image with conventional confocal xyz microscopy.  I can see its utility for transparent zebrafish and you want to look at structures the size of cells, but If you have a tissue or embryo that you have trouble getting more than 100µm into with a confocal, does the same limitation exist with light sheet?  Then you have the problem of working distance and numerical aperture limiting specimen size, resolution and sensitivity.  So my question is, what are most appropriate situations where it provides a real complement to a LSCM or spinning disk confocal and what are the practical limitations of this technology.  Does it gain you anything for larger samples that are hard to image on a confocal?  Thanks- Dave

Dr. David Knecht
Professor of Molecular and Cell Biology
Core Microscopy Facility Director
University of Connecticut
91 N. Eagleville Rd.
Storrs, CT 06269
860-486-2200