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Selective Plane Illumination Microscopy Using Non-spreading Airy Beams

Leitis, Aleksandrs LU (2016) FYSM60 20161
Department of Physics
Atomic Physics
Abstract
Recently a new field of microscopy has emerged, known as selective plane illumination microscopy (SPIM). Using a SPIM setup the sample is illuminated with a thin light sheet from the side. The selective plane illumination overcomes many problems that conventional microscopes have. Conventional microscopes illuminate the sample axially and collects the light only from the focal plane of the microscope objective. Therefore parts of the sample which are out of focus are illuminated unnecessarily, leading to rapid fluorophore photobleaching and decrease of signal to noise ratio due to out of focus light. The $SPIM$ method overcomes these problems, because only the fluorophores in the focal plane of the objective are excited. Another problem... (More)
Recently a new field of microscopy has emerged, known as selective plane illumination microscopy (SPIM). Using a SPIM setup the sample is illuminated with a thin light sheet from the side. The selective plane illumination overcomes many problems that conventional microscopes have. Conventional microscopes illuminate the sample axially and collects the light only from the focal plane of the microscope objective. Therefore parts of the sample which are out of focus are illuminated unnecessarily, leading to rapid fluorophore photobleaching and decrease of signal to noise ratio due to out of focus light. The $SPIM$ method overcomes these problems, because only the fluorophores in the focal plane of the objective are excited. Another problem with conventional microscopes is that they are capable of only two-dimensional imaging. The exception are confocal microscopes, but these microscopes are slow and the field of view is restricted to few hundred micrometers. The SPIM method significantly improves the three-dimensional imaging speed and can reach up to few hundred frames per second. This imaging speed enhancement is achieved, because the whole plane is captured with a single snapshot, instead of point by point scanning as in confocal microscopes.

To take all the advantages of the SPIM method it is crucial to form a thin light sheet that extends for the whole field of view. Commercially available lasers have Gaussian beam. It is well known that tightly focused Gaussian beams spread out quickly, therefore there will be good axial resolution only in the central part of the image. Because of that the field of view will be restricted to the Rayleigh range of the Gaussian beam. In this work we present how to create non-spreading Airy beams using only two additional lens elements and how they can be implemented in the SPIM setup to significantly extend the field of view keeping high axial resolution. This is an important advancement since Airy beams would allow larger sized objects, e.g. optically cleared tissue specimens, to be imaged with high resolution. This thesis shows a novel design of a light sheet microscope that opens up new possibilities for biological and medical research. (Less)
Popular Abstract
The field of optical microscopy emerged in late 17th century, when famous Dutch draper and scientist Anton van Leeuwenhoek pioneered the techniques of microscopy. Since then the field has expanded and has greatly influenced the development of biology, chemistry, physics and medical research areas. Recently optical microscopy reached the realms of super resolution and nano scale, therefore allowing to see even single molecules. For this invention in 2014 three scientists were awarded with Nobel prize in chemistry. Overall, four Nobel prizes have been awarded for discoveries and inventions in microscopy. Most of the research in optical microscopy is done to increase the spatial resolution, neglecting the resolution in time. But obviously... (More)
The field of optical microscopy emerged in late 17th century, when famous Dutch draper and scientist Anton van Leeuwenhoek pioneered the techniques of microscopy. Since then the field has expanded and has greatly influenced the development of biology, chemistry, physics and medical research areas. Recently optical microscopy reached the realms of super resolution and nano scale, therefore allowing to see even single molecules. For this invention in 2014 three scientists were awarded with Nobel prize in chemistry. Overall, four Nobel prizes have been awarded for discoveries and inventions in microscopy. Most of the research in optical microscopy is done to increase the spatial resolution, neglecting the resolution in time. But obviously there are fast biological processes and understanding them would give significant contribution to medicine and biology.

In the last decade a new field of microscopy emerged, called selective plane illumination microscopy (SPIM). What distinguishes SPIM from conventional microscopes is that the sample is illuminated with a thin laser sheet. Therefore fluorescent light will be emitted only from the plane where the light sheet lies. And the light sheet can be made as thin as one hundredth of a human hair width. Therefore the SPIM method allows to image large samples with high temporal resolution and high spatial resolution in all 3 dimensions. With this new technique it is possible to reach up to few thousand frames per second, and it is possible to follow neuron signal propagation in real time. It is a great step forwards for research in neurology. Not only for studying cellular interactions, but also neuronal network interactions throughout the body of small animals. Furthermore bigger and older sample imaging could give better understanding of the neurological diseases that come with age, for instance Alzheimer's disease.

Besides significant increase in temporal resolution, with the SPIM technique it is possible to follow biological processes for much longer time periods compared to conventional microscopes. One can follow the evolution from larvae to fully grown organisms with single cell resolution or biological processes in cells for several days. Therefore it is now possible to follow how diseases evolve in tissue and how well the drugs can affect a disease. Thus it is possible to monitor the disease and the treatment in all stages.
This is a significant improvement compared to conventional microscopes: now it is possible to study diseases and drug efficacy like never before. (Less)
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author
Leitis, Aleksandrs LU
supervisor
organization
alternative title
Light Sheet Microscopy Using Non-spreading Airy Beams
course
FYSM60 20161
year
type
H2 - Master's Degree (Two Years)
subject
keywords
Microscopy, Selective Plane Illumination Microscopy, Light Sheet Microscopy
language
English
id
8880649
date added to LUP
2016-06-22 13:35:16
date last changed
2016-06-22 13:35:16
@misc{8880649,
  abstract     = {Recently a new field of microscopy has emerged, known as selective plane illumination microscopy (SPIM). Using a SPIM setup the sample is illuminated with a thin light sheet from the side. The selective plane illumination overcomes many problems that conventional microscopes have. Conventional microscopes illuminate the sample axially and collects the light only from the focal plane of the microscope objective. Therefore parts of the sample which are out of focus are illuminated unnecessarily, leading to rapid fluorophore photobleaching and decrease of signal to noise ratio due to out of focus light. The $SPIM$ method overcomes these problems, because only the fluorophores in the focal plane of the objective are excited. Another problem with conventional microscopes is that they are capable of only two-dimensional imaging. The exception are confocal microscopes, but these microscopes are slow and the field of view is restricted to few hundred micrometers. The SPIM method significantly improves the three-dimensional imaging speed and can reach up to few hundred frames per second. This imaging speed enhancement is achieved, because the whole plane is captured with a single snapshot, instead of point by point scanning as in confocal microscopes. 

To take all the advantages of the SPIM method it is crucial to form a thin light sheet that extends for the whole field of view. Commercially available lasers have Gaussian beam. It is well known that tightly focused Gaussian beams spread out quickly, therefore there will be good axial resolution only in the central part of the image. Because of that the field of view will be restricted to the Rayleigh range of the Gaussian beam. In this work we present how to create non-spreading Airy beams using only two additional lens elements and how they can be implemented in the SPIM setup to significantly extend the field of view keeping high axial resolution. This is an important advancement since Airy beams would allow larger sized objects, e.g. optically cleared tissue specimens, to be imaged with high resolution. This thesis shows a novel design of a light sheet microscope that opens up new possibilities for biological and medical research.},
  author       = {Leitis, Aleksandrs},
  keyword      = {Microscopy,Selective Plane Illumination Microscopy,Light Sheet Microscopy},
  language     = {eng},
  note         = {Student Paper},
  title        = {Selective Plane Illumination Microscopy Using Non-spreading Airy Beams},
  year         = {2016},
}