Light-sheet microscopy, such as ultramicroscopy, single plane illumination microscopy (SPIM) [1] and digital
scanned laser microscopy (DSLM) [2], represents a useful tool for biological investigations of thick samples.
Such techniques have been found particularly useful in developmental biology applications since they provide the
capability to perform fast imaging of living samples reducing photobleaching effects. The high signal to noise ratio
and the intrinsic optical sectioning capability provided by SPIM suggest this technique as the best choice for
imaging of thick scattering samples. Nevertheless, imaging in depth of large samples suffers from a decreasing in
the image quality due to scattering effects. Two photon excitation microscopy [3] became a popular tool to perform
imaging in turbid media since it improves the penetration depth capability and it reduces the image quality
degradation due to scattering [4] and light matter interactions. Therefore, two photon excitation within the light
sheet illumination scheme has been exploited in order to reduce scattering effects due to light-sample interactions. In
this work two photon excitation imaging in SPIM scheme has been performed in order to achieve an improvement
in the penetration depth while imaging living biological samples.
Non linear optical scanning microscopy has became a useful tool for living tissue imaging. Biological tissues
are highly scattering media and this leads to an exponential attenuation of the excitation intensity as the light
travels into the sample. While performing imaging of biological scattering tissues in non linear excitation regime,
the localization of the maximum 2PE intensity was found to shift closer to the surface1 and the 2PE imaging
depth limit appears strongly limited by near surface fluorescence.2 In this work we computed the illumination
and the photobleaching distribution3 in order to characterize the effects induced by scattering. The simulations
have been performed for different scattering coefficients and different focus depth. An experimental test has
been carried out by imaging, with 0.9 numerical aperture objective, thick scattering fluorescent immobile sample
(polyelectrolyte gel). Results confirm that under these conditions no photobleaching effects due to scattering
occur close to the surface.
Diffraction imposes for each optical system a resolution limit which could be described by using the vectorial theory of
Richards and Wolf. This theory defines the intensity distribution of a point like source imaged by a lens assuming ideal
imaging conditions. Unfortunately, these conditions can not be completely achieved in practical situations as a recorded
microscope image is always affected by noise which makes the resolution limit worse.
In this work we propose and analyze optical set-up schemes towards an image quality improvement in terms of Signal
to Noise Ratio (SNR) in linear and non-linear fluorescence microscopy. In order to reach this purpose we insert, on the
illumination arm of the microscope, a proper amplitude ring filter inducing laterally interfering beams. The effect
induced by the filter results in a shape engineering of the 3D-PSF and in a redistribution of the spatial frequencies of the
OTF. In particular, the high frequencies information are collected at improved SNR.
In order to implement such schemes we use a computational simulation mainly based on a vectorial approach analyzing
the results in both space and frequency domain to characterize the optical system response.
Analysis reveals that, although the theoretical resolution of the system is unchanged, when we impose a certain noise
level the practical imaging quality could be improved in the ring filtering scheme. The results suggest that further
improvement can be reached by the usage of the proposed annular filers in combination with image restoration.
A comparison between linear and non-linear excitation cases is presented.
KEYWORDS: Point spread functions, Optical filters, Confocal microscopy, Signal to noise ratio, Microscopy, Phase only filters, Image filtering, Optical resolution, Optical transfer functions, Image resolution
The aim of this work is to propose and analyze optical schemes to obtain an improvement of resolution in optical
fluorescence microscopy. This goal can be achieved by implementing interfering illumination beams. We start from the insertion, on the illumination arm of the confocal microscope, of appropriately phase plates inducing laterally interfering beams, and then we propose to exploit two-photon excitation, too. We plan to implement solutions for shaping also the axial component of the point spread function by use of phase-only pupil filters and binary filters. In order to implement such schemes we use a computational simulation mainly based on a vectorial approach coupled to experimental procedures utilizing ultra-thin fluorescent layers and thick gels containing immobile fluorescent molecules as 2D and 3D phantoms, respectively. As well, image processing and successive views can be recombined to get a final isotropic improvement of resolution.
Layer-by-Layer or self-assembly techniques can be used to prepare Fluorescent polymer samples on glass coverslips
serving as benchmark for two-photon excitation microscopy from conventional to 4Pi set-up, or more in general
for sectioning microscopy. Layers can be realized as ultra-thin (<< 100 nm) or thin (approx. 100 nm)
characteristics coupled to different fluorescent molecules to be used for different microscopy applications. As well, stacks hosting different fluorescent molecules can be also produce. Thanks to their controllable thickness, uniformity and fluorescence properties, these polymer layers may serve as a simple and applicable standard to
directly measure the z-response of different scanning optical microscopes. In two-photon excitation microscopy z-sectioning plays a central role and uniformity of illumination is crucial due to the non-linear behaviour of emission. Since the main characteristics of a particular image formation situation can be efficiently summarized
in a Sectioned Imaging property chart (SIPchart), we think that coupling this calibration sample with SIPchart is a very important step towards quantitative microscopy. In this work we use these polymer layers to measure the z-response of confocal, two-photon excitation and 4Pi laser scanning microscopes, selecting properly ultra-thin and thin layers. Due to their uniformity over a wide region, i.e. coverslip surface, it is possible to quantify the z-response of the system over a full field of view area. These samples are also useful for monitoring photobleaching
behavior as function of the illumination intensity. Ultrathin layers are also useful to supersede the conventional
technique of calculating the derivative of the axial edges of a thick fluorescent layer. Polymer layers can be
effciently used for real time alignment of the microscope.
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