3. Supercontinuum for Optical Characterization in Neuroscience

 

3.1) Supercontinuum for Optical Characterization in Neuroscience

Today we have an interview with one of the researchers using an supercontinuum laser. NanoLab laboratory at the University of Trento has an white laser for chip characterization, an essential step on the light-induced activation of an in vitro neuronal engram process.  

Clara Zaccaria graduated in Physics at University of Padua and currently in the 3rd year of her PhD at University of Trento in NanoLab laboratory headed by Lorenzo Pavesi. She explains how she is using the supercontinuum fiber laser for the BACKUP project.

 

3.2) The BACKUP Project

The aim of BACKUP is to create a memory engram in a small in vitro neuronal network using a photonic chip. This artificial engram will be created using optogenetic strategies in which patterned-light illumination will correspond to activation of group of interconnected neurons expressing channel rhodopsin along the light path. In this sense patterned light will work as an artificial learning event for generating memory engram.
This reductionist in vitro system will allow comparison of the activity and morphological changes in “engram neurons” (cells activated by light) vs “non engram neurons” (cells not activated by light) to study basic mechanisms of engram cells connectivity and to establish a link between neuronal activity and changes in the connectivity among neurons.
Clara´s  research is in the frame of the ERC BACKUP project creating hybrid platforms in which neuromorphic photonic chips communicate with biological neurons.
Within the BACKUP project she implemented two set-ups for in-vitro neuronal excitation, one using a Digital Light Projector through the microscope and one using a specifically designed photonic chip.

Iceblink, the supercontinuum source to optical characterization on a photonic chip.

They used the supercontinuum laser, Iceblink, to characterize the optical structures designed in the photonic chip.

White laser fiber laser is important for the characterization of the structures at different wavelengths: the photonics chip was designed to work at 488 nm, but it can be useful to know the behaviour of the optical structures also at higher wavelengths in order to interact with neurons in different ways.

 

3.3) Characterizing optical structures at any wavelength

Also, the laser setup is really important because they need light to be coherent in order to be used properly with the photonic chips designed in the laboratories. Moreover, they need to have a source in the visible with a large spectrum to be used along with an optical spectrum analyser. Tunable sources or broadbandsources in the visible range as it is not common due to the lack of a proper gain medium.

When they want to use a specific part of the spectrum they use filters, and some wave plates and polarizers if polarization is needed.

Advantages of supercontinuum for chip characterization

Supercontinuum source has good power delivery in the whole spectrum, from 400 up to 2000 nm.
The price is convenient because it is a very versatile laser. Indeed, it was used also by some other researches at the Nanolab for other projects.

 

3.4) The future of neuroscience

The main scientific challenges are the biocompatibility of these platforms and the design of neuromorphic circuits able to compete with biological ones.
If in the future it will be possible to realize efficient photonics platforms able to communicate with neurons, it can be possible to create devices that can replace or support malfunctioning brain sectors in diseases like amnesia or epilepsy. Moreover, these kinds of platforms is useful also to do in-vitro studies to unveil elementary processes occurring in neuronal networks, like signaling and information storing.
This kind of research is really one of the more important challenges that neuroscience is facing nowadays. This will evolve toward medical applications and knowledge for implementing Artificial Intelligence.
But the real challenge is ethical: a proper outreach is needed in order to make people open to the advantages that these kinds of technologies will bring.

 

4. Supercontinuum Laser for Light Sheet Fluorescence Microscopy

 

4.1) Advantages of elastic scattered light sheet fluorescence microscopy

Light Sheet Fluorescence Microscopy (LSFM) is a powerful imaging technique that enables fast and non-photo toxic 3D inspection of living specimens.

LSFM combines the speed of widefield imaging with moderate optical sectioning and low photobleaching. It is also referred to as SPIM, or simply “light sheet”. The defining feature of SPIM or LSFM is planar illumination of the focal plane from the side. Only a thin section of the sample is illuminated at any given time, minimizing photodamage and providing optical sectioning which improves SNR compared with widefield epifluorescence. Because the image is collected in a widefield (2D parallel) manner, light-sheet imaging is much faster than a point-scanned confocal microscope which detects only one pixel at a time.

It has become one of the most popular techniques for volumetric imaging because of three key features:

  - Photodamage is minimized because excitation is confined near the focal plane, e.g. living things stay alive for much longer.

  - Good optical sectioning is obtained, often approaching that of confocal microscopy.

  - Acquisition rates are very fast, orders of magnitude faster than a traditional confocal microscope. The main disadvantage of SPIM is that extra optics are required to generate the light sheet.

Light Sheet microscopy is usually based on fluorescence techniques, and in general, the sample under study needs to be properly labeled to be imaged.

In this article, we will go deeper into this technique and study how elastically scattered light could be used to generate images of non-labeled samples.

The main obstacle is that these images are usually affected by speckle. To solve that inconvenience, they use an supercontinuum source of low temporal coherence supercontinuum source is presented as a candidate to reduce the speckle inherent in light-sheet microscopy images from scattered light.

Pablo Loza-Alvarez, Omar Alarte, David Merino of ICFO-Institut de Ciencies Fotoniques with Diego Battista and Giannis Zacharakis of Foundation for Research and Technology-Hellas use elastic scattered light from the sample to generate images, in order to avoid the need to label the sample.

In this work, they propose a novel light sheet based optical setup which implements three strategies for dealing with speckles of elastic scattering images:

  - Polarization filtering

  - Reducing the temporal coherence of the excitation laser light

  - Reducing the spatial coherence of the light sheet.

These strategies enable pristine light-sheet elastic-scattering imaging of structural features in challenging biological samples avoiding the deleterious effects of speckle, and without relying on, but complementing fluorescent labeling.

 

4.2) Setup for polarization and coherence control in elastic scattering light sheet microscopy

The main component of their elastic scattering light sheet microscope is the supercontinuum fiber laser which emits a broadband spectrum of light from the visible to the infrared.

This source presents a very broad spectral bandwidth, at the same time, it presents very low temporal coherence. Both are desirable features in a light source when trying to reduce speckle effects in the images.

The white laser is used for Light sheet fluorescence microscopy selecting a band from 500 to 700 nm (140 nm FWHM), hence offering a lower temporal coherence for reduced speckle contrast.

Scheme of the experimental setup for polarization and coherence control in elastic scattering light-sheet microscopy. In (a) the experimental setup implemented is shown. The light sheet illumination path consists of a couple of diode lasers emitting at 515 nm and 638 nm, and a supercontinuum laser (SCL). Laser beams are expanded 10 times before entering the microscope. P1 is a half-wave plate (HWP) that controls the polarization of the three beams before passing through the cylindrical lens (CL), the galvo mirror (GM), and the illumination objective (OBJill). GM scans the beam at OBJill’s pupil generating a pivoting light sheet at the sample plane. Samples are kept within a custom-made immersion chamber (C) filled with water. The detection system is composed of a 0.5 N.A. objective lens (OBJdet), a 200 mm tube lens (for a total magnification of 20X), and a polarizer (P2).

(b) The emission spectrum of the supercontinuum laser in the band 500–700 nm (140 nm FWHM), compared to the bandwidth (1.2 nm) of the red diode laser (Red vertical band).

(c) Detail of the optical setup close to the illumination objective lens (OBJill), illustrating the pivoting light-sheet approach. The supercontinuum laser light sheet pivots around an axis located at the working distance (WD) of OBJill, i.e., at the center of the sample plane.

 

4.3) Does the supercontinuum achieve label-free structural imaging in LSFM?

The results show that Iceblink supercontinuum source presents low speckle contribution on LSM images compared to other light sources with narrower spectrum bandwidth.

In conclusion, elastic scattering light sheet microscopy is a novel light sheet imaging modality suitable for label-free structural imaging.

In order to enhance the imaging quality in this configuration They have proposed to implement:

  - Polarization control, which enables contrast selectivity and cancelling substrate background.

  - Temporal and spatial coherence reduction, which enables extracting the endogenous intrinsic contrast from the speckle noise.

Implemented in this manner, elastic scattering light sheet imaging provides useful complementary structural information to standard LSFM experiments, as shown for MCTS samples. Also, it has the potential to provide relevant morphologic details of the samples, similar to histological sections, but in a non-destructive way.

Finally, elastic scattering light sheet microscopy is a promising technique which could enable new and interesting experiments, for example, as an alternative to LSFM in applications that are limited by low SNR, such as functional imaging or fast volumetric structural imaging.

    

Images of the head of a C. Elegans worm, obtained using an elastic scattering light sheet microscopy system. a) Maximum intensity projection of a 3D stack of images of the head of the worm (image is 230×110μm in size). b) Is a detail of one of the planes of (a) obtained (image is 80×40μm). c) is the same image as (b) obtained using a 488nm CW diode laser (image is 80×40 μm).

 

SIMTRUM offers Boreal for supercontinuum lasers to choose any wavelength in the visible range (450-750 nm), which is the perfect white laser plug-in accessory for bioimaging, nanophotonic and more. Boreal offers unparalleled performance for a wide range of applications that are even impossible with other types of light sources. The module is based on filter pass band technology, allowing one to choose one wavelength at a time. The time selection, for example, measured to switch from 500 nm to 590 nm is 1.5 seconds.

With 2 options to choose from:

• Free Space: with a power transmission > 75%
• Multi-mode Fiber Output (1m) with FC/PC connector with a power transmission >50%

Model	Spectral Range	Average Power	Repetition Rate
Ultra-flat Spectrum Supercontinuum Fiber Lasers	450-2300 nm	＞3W	80MHz
Frequency-Tunable Supercontinuum Fiber Lasers	450-2300 nm	＞1.5W	1/2.5/5/10/20/40 MHz

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