Spatio-Temporal Engineering of Light. Ultimate Multiphoton Microscopy

The development of high resolution, non-damaging imaging techniques are crucial for understanding the biological processes occurring at the cellular level. Nonlinear microscopy (NLM) is rapidly establishing as a powerful technique for high resolution imaging of living biological samples. The high peak powers and low pulse energies available from ultrashort pulses, allow an efficient excitation of nonlinear effects with reduced collateral damage when interacting with cells. Apart from peak powers and low pulse energies, ultrashort pulse light has two additional parameters that can be exploited for a more efficient light-cell interaction. i) Beam and pulse shaping, acting on the spatial and temporal intensity profile and phase of a pulse and ii) the beam's wavefront spatial distribution through the use of adaptive optics techniques. This will allow correcting aberrations for an increased transversal resolution, larger penetration depths and fields of view. In overall, this will translate in a more efficient interaction, helping to reduce any possibility of damage.

In this project we aim to join complementary expertise to perform simultaneous beam and pulse shaping and wavefront correction at the sample plane of a nonlinear microscope. The project consisted of the partnership of two world leading organisations, each of them contributing with complementary knowledge: ICFO- The Institute of Photonic Sciences, contributing with know-how in nonlinear microscopy and ultrashort pulsed lasers and Imagine Optic (IO) in adaptive optics, wavefront sensing (WFS) and correction technologies (AO). To ensure effective transfer of knowledge (ToK) of this intersectorial and interdisciplinary effort, a human resources exchange (93 person months, of which 41 PM were recruited and 52 PM seconded) plan was implemented. These PMs were distributed into 2 recruited postdocs and 3 seconded researchers (1 postdoc and two PhD students).

In optics, intensive training was performed in microscopy, ultrashort pulses, wavefront sensing and adaptive optics. In informatics, specific training was done based on Labview. In industrial engineering, training was performed on project management, and in particular the recruitment fellow at IO was in charge of mini-projects to complete with real industrial experience.

From the academic point of view, the project has resulted in the publication of 5 international journal papers (all within the first quartile in Optics) and more than 20 international conferences. It has allowed exploring new synergies within different EU and national projects (see for example the FP7 EU “Fast Dot” project) and with different research groups (USA, Mexico, France, Spain amongst others). From the industry point of view, the project has originated 2 patent submissions and the development of one prototype (an Adaptive Optics plug&play add-on for microscopes) that will be commercialized by IO in the future. Contacts were also created with major companies such as Hamamatsu, Citizen, and Nikon. In all cases, we are currently exploring fostering collaborations on the use of adaptive optics for its use in nonlinear microscopy applications.

In an effort to facilitate the ToK to young generations, a work shop was organized which was co-localized with “The 1st Europhotonics Spring School” (https://dfen.upc.edu/documents-noticies-i-esdeveniments/EurophotinicsSpringSchool.pdf). This is an Erasmus Mundus Master Course (EMMC) and Joint Doctorate (EMJD) program. STELUM contributed with 2 main speakers to the school and all the participants were encouraged to attend the STELUM workshop.

In what follows an explanation of the technical highlights of the project are detailed:

• We have demonstrated, for the first time that sample aberrations can be measured and corrected in a NLM using a wavefront sensor and a deformable mirror, in a single pass and without any modification to the sample. This aberration correction strategy was based on the development of the concept of “nonlinear guide-star” (NL-GS). This has resulted in an improved performance in terms of: i) lateral resolution, ii) field of view, and above all, iii) a better distribution of useful power at the sample plane.

• In the previous point we use a sensor-based scheme to correct aberrations. However, traditionally this is achieved using genetic algorithms (GA). These are particularly useful for correcting aberrations at large penetration depths. Although the use of these algorithms has been implemented in microscopy in the past, they are not commercially available. Here we have implemented a prototype that will allow the use of GA for microscopy applications.

• To obtain images in biological samples or living organisms at larger penetration depths, besides correcting for sample induced aberrations, the use of infrared (IR) excitation wavelengths should be explored. In particular we have demonstrated that, employing a 1550 nm femtosecond fiber laser, the third harmonic generation (THG) microscopy technique can be used for morphogenesis/ embryogenesis studies in living Caenorhabditis elegans in all the embryo development stages. Additionally, we have shown how using a compact semiconductor disk laser, operating at 970 nm, samples labeled with the widely used green fluorescent protein (GFP) can be efficiently visualized.

• We have implemented a variation on the Digitally Laser Light Scanned Microscopy (DLSM) in which it is possible to convert a Gaussian ultrashort pulsed laser beam into a Bessel one. These were used for two-photon excited fluorescence nonlinear microscopy. The advantage of this technique is that it allows for a larger field of view and larger penetration depths to be visualized without sacrificing image resolution.

• A simple methodology to temporally shape ultrashort pulses was investigated. The system employs standard pre-chirping schemes followed by a fiber bundle. With this setup, imaging of a test sample containing a human compatible dye was demonstrated. This methodology will lead towards the construction of a multiphoton microendoscope to be used in clinical applications.

• We have developed a graphic user interface to allow ultrashort pulse characterization in a nonlinear microscope in cheap, friendly, fast and reliable way. This has been done using the MEFISTO technique as it allows pulse characterization at the sample plane of a nonlinear microscope in a nonlinear microscope with minimum modifications to the microscope platform.