LSFM is a technique that allows obtaining fast 2D images of biological samples. Its characteristic 90° geometry results in a highly efficient excitation and light collection of the generated signal, minimizing light dose onto the sample and reducing phototoxicity effects. Furthermore, by displacing the sample through the light sheet, high-resolution 3D images can also be obtained. Therefore, LSFM has been put forward as an interesting candidate for fast volumetric brain imaging. Here, I will present our results for 3D imaging of the spontaneous and dynamic calcium activity in primary neuron cultures in hydrogels. The obtained data is then processed to calculate the connectivity maps in the 3D neuron cultures in hydrogels and assess the topological properties of these maps such as the modules or highly connected subnetworks.
This abstract is part of the symposium: "Diagnosis and Prediction of Neurodegenerative Diseases using Artificial Intelligence"
KEYWORDS: Microscopy, 3D image processing, Functional imaging, Luminescence, Microscopes, Neurons, Signal generators, Stereoscopy, 3D scanning, Data processing
I will present a Light-sheet fluorescence microscope (LSFM) for fast volumetric imaging during extended periods of time. In this case, the observation arm of the microscope contains an electrically tunable lens (ETL) that is used to shift the focal position of the collection lens. By moving the light sheet plane in synchrony with the ETL, it is possible to scan the full 3D sample, which remains totally static, at high speeds (25 volumes/s) [2].
This system is used to image the spontaneous Ca2+ activity, as reported by GCaMP fluorescence, in 3D of primary neuron cultures in hydrogels. The field of view is of 300µm x 300µm x 1mm. The imaging speeds allows a proper sampling of the propagation of GCaMP signal in the full observation volume [4]. The obtained data is then processed to calculate the connectivity maps in the 3D neuron cultured in hydrogels.
Despite the inherent confocality and optical sectioning capabilities of multiphoton microscopy, three-dimensional (3-D) imaging of thick samples is limited by the specimen-induced aberrations. The combination of immersion objectives and sensorless adaptive optics (AO) techniques has been suggested to overcome this difficulty. However, a complex plane-by-plane correction of aberrations is required, and its performance depends on a set of image-based merit functions. We propose here an alternative approach to increase penetration depth in 3-D multiphoton microscopy imaging. It is based on the manipulation of the spherical aberration (SA) of the incident beam with an AO device while performing fast tomographic multiphoton imaging. When inducing SA, the image quality at best focus is reduced; however, better quality images are obtained from deeper planes within the sample. This is a compromise that enables registration of improved 3-D multiphoton images using nonimmersion objectives. Examples on ocular tissues and nonbiological samples providing different types of nonlinear signal are presented. The implementation of this technique in a future clinical instrument might provide a better visualization of corneal structures in living eyes.
We develop an adaptive optics (AO) multiphoton microscope by incorporating a deformable mirror and a Hartmann-Shack wavefront sensor. The AO module operating in closed-loop is used to correct for the aberrations of the illumination laser beam. This increases the efficiency of the nonlinear processes in reducing tissue photodamage, improves contrast, and enhances lateral resolution in images of nonstained ocular tissues. In particular, the use of AO in the multiphoton microscope provides a better visualization of ocular structures, which are relevant in ophthalmology. This instrument might be useful to explore the possible connections between changes in ocular structures and the associated pathologies.
A multiphoton microscope incorporating a Hartmann-Shack (HS) wavefront sensor to control the ultrafast laser beam's wavefront aberrations has been developed. This instrument allowed us to investigate the impact of the laser beam aberrations on two-photon autofluorescence imaging of human retinal tissues. We demonstrated that nonlinear microscopy images are improved when laser beam aberrations are minimized by realigning the laser system cavity while wavefront controlling. Nonlinear signals from several human retinal anatomical features have been detected for the first time, without the need of fixation or staining procedures. Beyond the improved image quality, this approach reduces the required excitation power levels, minimizing the side effects of phototoxicity within the imaged sample. In particular, this may be important to study the physiology and function of the healthy and diseased retina.
A multiphoton microscope has been developed to investigate the sources of nonlinear fluorescence (TPEF) and second
harmonic generation (SHG) in non-stained samples of ex-vivo corneas. Stacks of images from different depths are
recorded to reconstruct high-resolution 3D (volume) images of the cornea. The corneal epithelium and endothelium
provide significant TPEF signal, while the only source of SHG is the stroma. Within the stroma, the keratocytes can also
be visualized. Volumetric 3D images of the cornea combining TPEF and SHG signals are useful to characterize the
organization of the corneal collagen and to describe the distribution of keratocytes. These images will help to better
understand how different pathologies modify the corneal structure and to control the changes produced by surgical or
healing processes.
We have developed an adaptive optics multiphoton microscope. The multiphoton imaging system combines an ultrafast
high-power laser, a scanning unit, a motorized Z-scan device and a photon-counting detector. The adaptive optics
module is composed of a Hartmann-Shack wavefront sensor and a MEMS deformable mirror. The impact of
compensating the aberrations of the laser beam is shown in a number of biological and non-biological samples. As
examples, nonlinear fluorescence and second harmonic generation images of non-stained ex-vivo ocular tissues are
compared with and without adaptive optics. The correction of the beam's aberrations increases both contrast and
resolution in the non-linear microscope images.
In this study, we present the detailed imaging of the nematode Caenorhabditis elegans (C. elegans) at microscopic level
by performing Two-Photon Excitation Fluorescence (TPEF), Second-Harmonic Generation (SHG) and Third Harmonic
Generation (THG) measurements. Due to their inherent advantages in comparison with the conventional microscopy
(increased resolution, ability to section deep within tissues, minimization of photodamage and photobleaching effects),
the non-linear microscopy techniques comprise a unique and extremely powerful tool for the extraction of valuable and
unique information from biological samples. We developed a compact, reliable, inexpensive non-linear imaging system,
utilizing femtosecond laser pulses (1028nm) for the excitation of biological samples. The use of 1028nm wavelength as
excitation source minimizes photodamage effects and unwanted heating (due to the water absorption) of the biological
specimens. The emitted THG signal lies in the near UV part of the spectrum (343nm). Detailed and specific structural
and anatomical features of the worm were collected by recording THG signals. Consummative, unique information
concerning the morphology and the functions of the nematode was obtained by implementing the combination of THG,
SHG and TPEF image contrast modalities on the same microscope.
It is well known that the efficiency and selectivity of two-photon excited fluorescence (TPEF) process can
depend on various parameters of the ultrashort pulses, such as the pulse intensity and phase, which interact with the
specimen. In order to completely understand this dependence and to obtain optimal TPEF images, techniques like
Collinear Frequency Resolved Optical Gating (CFROG) arrangement can be implemented in a microscope for complete
pulse characterisation at the sample plane. However, this adds complexity that that additional forward collecting optics is
required as well as a suitable frequency doubling crystal.
Here we report a simple way to characterize the pulses within a multiphoton microscope that do not require
forward collecting optics. This is achieved by taking advantage of the fact that backward propagating second harmonic
generation (SHG) signal can be easily generated from starch granules. Since both the fluorescence and SHG signals can
be collected using the same detection scheme the experimental arrangement is considerably simplified. Starch, being a
non- toxic and non-soluble material does not affect living cells allowing the pulse characteristics to be measured in situ,
without the need to move the sample.
We obtained real-time SHG-autocorrelations traces by using a single starch granule that was placed alongside
the living HeLa cells (GFP labeled) being imaged. Furthermore by placing a spectrometer at the output port of the
microscope, a spectrally resolved SHG autocorrelation was acquired allowing complete characterisation of the pulse to
be carried out. The temporal intensity and phase profile were retrieved using CFROG technique. Marginal analysis was
carried out to ensure that the experimental data was successfully acquired.
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