Periodic Reporting for period 1 - WAVESCOPE (Wavefront shaping system for nonlinear fiber-based microscopy and endoscopy)
Reporting period: 2019-10-01 to 2021-09-30
Summary of the context and overall objectives of the project
The medical imaging systems market (spectroscopy/microscopy) is currently one of the fastest growing areas of advanced medical devices. Among all imaging systems, the WAVESCOPE project addresses the multimodal imaging market, which includes molecular spectroscopy imaging methods, as well as microscopy and endoscopy systems. The demand for multimodal imaging systems continues to grow at a fastest rate than the overall market. There is a significant demand for multimodal imaging system in different sectors. To date, it has been established that pharmacology and biology are the most demanding and strongest growth sectors in multimodal imaging systems. That is why in the WAVESCOPE proof-of-concept imaging systems we centered our development around this market segment, and more precisely around that of biological imaging of living organisms (cancerous tissues, tumors, bacteria, cells, etc.).
Spectromicroscopy and imaging applications have undergone very significant development in recent years. The innovative techniques allowing the generation of self-cleaned supercontinuum proposed by WAVESCOPE make it possible to greatly improve certain spectroscopy systems such as coherent wide-spectrum RAMAN imaging, as well as linear and nonlinear fluorescence and harmonic imaging. Laboratories and manufacturers who use imaging are increasingly expressing the need to access more complex images and with information that cannot be obtained with conventional imaging. RAMAN imaging has enabled major advances in the study of tissues and tumors, for example, and the laboratories in the sector express a very strong interest in these nonlinear imaging techniques. Among them, WAVESCOPE has demonstrated a proof-of-concept technology with the potential for an all-in-one nonlinear imaging solution comprising: i) Confocal multifluorescence microscopy including one, two and three-photon fluorescence, allowing, via labeling (or without labeling), the analysis of cell tissues, bacteria and related metabolisms; ii) Second and third harmonic generation microscopy, to target collagens and to provide a complementary analysis to fluorescence; iii) RAMAN CARS (Coherent Anti-Stokes Raman Scattering,) microscopy, to perform a molecular analysis of samples which are excited at their own vibrational frequencies. Molecular vibrations therefore appear on the spectrum in a manner similar to RAMAN but with increased sensitivity and rapidity. This technique makes it possible to overcome the defects of conventional RAMAN imaging.
Currently, traditional players in imaging offer access to these types of multimodal images only through large, very expensive and complex installations. This is why the need to which the WAVESCOPE project responds is as follows: to provide images from multiple nonlinear microscopic imaging methods (Multimodal Imaging) within a single compact machine, simple and at limited cost. The WAVESCOPE proof-of-concept device is therefore aimed both at living imaging laboratories in the fields of biophotonics and biomedical medicine, but also at industries and in particular those in the food industry, pharmacology and cosmetics. Beyond the image, allowing shape recognition or localization of particular chemical elements, classical spectroscopy, i.e. recording at a localized point of the spectral fluorescence response (linear or not), can be used to classify drugs, for example, or to identify the presence of aggressive chemical compounds.
The WAVESCOPE approach is based on exploiting the intrinsic nonlinear response of a graded-index (GRIN) MMF to high peak power optical pulses, combined with appropriate control of the input laser beam coupling conditions. By properly adjusting beam coupling at the input of the GRIN fibre, one may control in real-time the output optical beam in the spatial, spectral and temporal domains, until the condition of self-cleaning is matched, which leads to a stable and robust Gaussian-shaped beam at the fibre output. Spatial self-cleaning is a nonlinear effect, that is produced in the optical fibre at moderate optical powers: the chaotic intensity pattern produced by the fibre in a low-power regime, is recombined into a single homogeneous spot at the output of the fibre, which enables a precise focusing on the sample.
Experiments carried out by the WAVESCOPE partners have demonstrated, for the first time, that the effect of “self-cleaning” indeed leads to image resolution enhancements in multispectral multiphoton fluorescence imaging, in both microscopy and endoscopy configurations, when using a GRIN MMF to carry the laser beam onto the sample. The most important feature of the WAVESCOPE imaging system is the strong robustness of images obtained when using self-cleaned beams, as opposed to ordinary, speckled beams.
In WAVESCOPE we also exploited beam self-cleaning for improving the performances of multiplex CARS imaging with MMF. CARS is a four-wave mixing process, where the vibrational frequency of a molecular bond is excited by a light beam composed of two initial waves. The beating between the two waves creates a modulation, which matches perfectly with the molecular frequency. In this way, it is possible to obtain selective imaging, which only shows a given type of molecules. This process is implemented without any labelling, and it can be obtained in both microscopy and endoscopy configurations. The CARS signal strength is proportional to the square of the bonds’ concentration, whereas it is simply proportional to this concentration in the case of spontaneous Raman scattering. This process is obtained thanks to the mixing of a monochromatic pump wave and a wideband (or SC) Stokes beam. Thus, a portion of the initial pump beam is first spatially separated from the main pump component, which creates the SC. In a second step, the pump beam portion is resynchronized in time and spatially superimposed again with the SC, in order to permit the CARS process. This operation forces the user to introduce a delay line in the multiplex CARS device, which significantly increases the complexity and the cost of the apparatus. The use of multimode optical fibres can solve this problem, and a multiplex CARS measurement can thus be obtained directly without the separation and re-synchronisation of pump and Stokes waves. The workaround of this problem is, again, obtained thanks to the beam self-cleaning effect, which systematically feeds the pump beam by sending back all the pump wavelength stored in high-order modes towards the fundamental fiber mode, even after SC generation.
In a last step, in WAVESCOPE we developed a two-photon microendoscope system, allowing label-free high-resolution imaging recording through a standard GRIN optical fibre. The microendoscope’s transverse resolution is directly dependent on the initial multimode fibre diameter and on the scanning head, and it can reach sub-micrometre scale at 1030 nm. The endoscopic head is similar to that used in previously demonstrated endoscopes. It is based on a resonant fibre-scanner and micro-optics. The fibre-scanner is encapsulated inside a biocompatible tube. Its development has been thought in order to allow for insertion in the working channel of a clinical endoscope or in a laparoscope. Fluorescence coming from the biological sample is epi-collected by the multimode GRIN fibre, detected by a photomultiplier tube, and sent to a computer for image reconstruction.
Spectromicroscopy and imaging applications have undergone very significant development in recent years. The innovative techniques allowing the generation of self-cleaned supercontinuum proposed by WAVESCOPE make it possible to greatly improve certain spectroscopy systems such as coherent wide-spectrum RAMAN imaging, as well as linear and nonlinear fluorescence and harmonic imaging. Laboratories and manufacturers who use imaging are increasingly expressing the need to access more complex images and with information that cannot be obtained with conventional imaging. RAMAN imaging has enabled major advances in the study of tissues and tumors, for example, and the laboratories in the sector express a very strong interest in these nonlinear imaging techniques. Among them, WAVESCOPE has demonstrated a proof-of-concept technology with the potential for an all-in-one nonlinear imaging solution comprising: i) Confocal multifluorescence microscopy including one, two and three-photon fluorescence, allowing, via labeling (or without labeling), the analysis of cell tissues, bacteria and related metabolisms; ii) Second and third harmonic generation microscopy, to target collagens and to provide a complementary analysis to fluorescence; iii) RAMAN CARS (Coherent Anti-Stokes Raman Scattering,) microscopy, to perform a molecular analysis of samples which are excited at their own vibrational frequencies. Molecular vibrations therefore appear on the spectrum in a manner similar to RAMAN but with increased sensitivity and rapidity. This technique makes it possible to overcome the defects of conventional RAMAN imaging.
Currently, traditional players in imaging offer access to these types of multimodal images only through large, very expensive and complex installations. This is why the need to which the WAVESCOPE project responds is as follows: to provide images from multiple nonlinear microscopic imaging methods (Multimodal Imaging) within a single compact machine, simple and at limited cost. The WAVESCOPE proof-of-concept device is therefore aimed both at living imaging laboratories in the fields of biophotonics and biomedical medicine, but also at industries and in particular those in the food industry, pharmacology and cosmetics. Beyond the image, allowing shape recognition or localization of particular chemical elements, classical spectroscopy, i.e. recording at a localized point of the spectral fluorescence response (linear or not), can be used to classify drugs, for example, or to identify the presence of aggressive chemical compounds.
The WAVESCOPE approach is based on exploiting the intrinsic nonlinear response of a graded-index (GRIN) MMF to high peak power optical pulses, combined with appropriate control of the input laser beam coupling conditions. By properly adjusting beam coupling at the input of the GRIN fibre, one may control in real-time the output optical beam in the spatial, spectral and temporal domains, until the condition of self-cleaning is matched, which leads to a stable and robust Gaussian-shaped beam at the fibre output. Spatial self-cleaning is a nonlinear effect, that is produced in the optical fibre at moderate optical powers: the chaotic intensity pattern produced by the fibre in a low-power regime, is recombined into a single homogeneous spot at the output of the fibre, which enables a precise focusing on the sample.
Experiments carried out by the WAVESCOPE partners have demonstrated, for the first time, that the effect of “self-cleaning” indeed leads to image resolution enhancements in multispectral multiphoton fluorescence imaging, in both microscopy and endoscopy configurations, when using a GRIN MMF to carry the laser beam onto the sample. The most important feature of the WAVESCOPE imaging system is the strong robustness of images obtained when using self-cleaned beams, as opposed to ordinary, speckled beams.
In WAVESCOPE we also exploited beam self-cleaning for improving the performances of multiplex CARS imaging with MMF. CARS is a four-wave mixing process, where the vibrational frequency of a molecular bond is excited by a light beam composed of two initial waves. The beating between the two waves creates a modulation, which matches perfectly with the molecular frequency. In this way, it is possible to obtain selective imaging, which only shows a given type of molecules. This process is implemented without any labelling, and it can be obtained in both microscopy and endoscopy configurations. The CARS signal strength is proportional to the square of the bonds’ concentration, whereas it is simply proportional to this concentration in the case of spontaneous Raman scattering. This process is obtained thanks to the mixing of a monochromatic pump wave and a wideband (or SC) Stokes beam. Thus, a portion of the initial pump beam is first spatially separated from the main pump component, which creates the SC. In a second step, the pump beam portion is resynchronized in time and spatially superimposed again with the SC, in order to permit the CARS process. This operation forces the user to introduce a delay line in the multiplex CARS device, which significantly increases the complexity and the cost of the apparatus. The use of multimode optical fibres can solve this problem, and a multiplex CARS measurement can thus be obtained directly without the separation and re-synchronisation of pump and Stokes waves. The workaround of this problem is, again, obtained thanks to the beam self-cleaning effect, which systematically feeds the pump beam by sending back all the pump wavelength stored in high-order modes towards the fundamental fiber mode, even after SC generation.
In a last step, in WAVESCOPE we developed a two-photon microendoscope system, allowing label-free high-resolution imaging recording through a standard GRIN optical fibre. The microendoscope’s transverse resolution is directly dependent on the initial multimode fibre diameter and on the scanning head, and it can reach sub-micrometre scale at 1030 nm. The endoscopic head is similar to that used in previously demonstrated endoscopes. It is based on a resonant fibre-scanner and micro-optics. The fibre-scanner is encapsulated inside a biocompatible tube. Its development has been thought in order to allow for insertion in the working channel of a clinical endoscope or in a laparoscope. Fluorescence coming from the biological sample is epi-collected by the multimode GRIN fibre, detected by a photomultiplier tube, and sent to a computer for image reconstruction.