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Brillouin Microscopy for Mechanical Properties Measurement in Living Organisms: A Presentation by Carlo Babylakwa
0:00:02 Introduction: Carlo Babylakwa, a PhD student at the EMBL, presents his work on advancing Brillouin microscopy for measuring mechanical properties in living organisms. He worked with brilliant microscopy pioneer, Guilianno Castelli, during his Master's thesis.
0:01:33 Mechanobiology and Brillouin Microscopy: Brillouin microscopy is an emerging technique to measure mechanical properties, complementing biochemistry in understanding biological systems. It aims to bridge the gap in mechanical imaging at high resolution, offering advantages over existing techniques.
0:02:30 Presentation Outline: The presentation will cover the physical principles of Brillouin microscopy, potential biological applications, and the development of a new, improved version of the microscope, concluding with applications of this new version.
0:03:11 Mechanical Properties and Biological Processes: Mechanical properties are crucial links between forces and shape changes in biological systems, influencing processes like cell fate, proliferation, migration, and tissue organization.
0:04:11 Traditional Techniques vs. Brillouin Microscopy: Traditional techniques like AFM and micropipette aspiration are limited to surface measurements and external mechanical loading. Particle-based and image-based approaches offer some internal access but with poor resolution. Brillouin microscopy is unique due to being fully optical, non-invasive, with high resolution and 3D access.
0:05:56 Brillouin Scattering: Brillouin scattering is the interaction of light with acoustic phonons in a sample, resulting in a small portion of light undergoing Brillouin scattering. The frequency shift (Brillouin shift) is proportional to the local speed of sound, linked to the material's elastic properties.
0:07:25 Brillouin Shift & Longitudinal Modulus: The Brillouin shift is related to longitudinal modulus. Conversion requires knowing refractive index and density in 3D. Biological samples, primarily water and solids, have a proportionality between refractive index and density. In these instances, brilliance shift is used as a proxy of longitudinal modulus.
0:09:11 Longitudinal Modulus vs. Young's Modulus: The presenter contrasts the measured longitudinal modulus with the better-known Young's modulus, explaining the nuances.
0:10:25 Frequency Differences in Measurement: Notes that Brillouin microscopy probes gigahertz acoustic waves whereas AFM measures mechanical properties at a range of seconds to milliseconds, which causes differences in the material's apparent stiffness.
0:11:38 Importance of Brillouin Modulus: Concludes that the brilliant modulus of biological samples can be important and relevant to certain biological processes.
0:12:33 Implementing Brillouin Microscopy: Typically implemented by modifying a confocal microscope with a spectrometer. Challenges include high spectral resolution and suppression of elastically scattered light.
0:13:18 VIPA Spectrometers: Using two VIPAs (Virtually Image Phased Array) in a cross-axis configuration is a common approach, offering higher throughput and faster acquisition compared to scanning Fabry-Pérot interferometers.
0:14:41 Additional Instrumentation: Confocal fluorescent co-detection is also a typical feature for these microscopes, with spectral accuracy on the order of 10 MHz.
0:15:17 Limitations: Brillouin Microscopy has a mechanical resolution that's limited to around one micron.
0:15:50 The Acoustic Attenuation Problem: Optical resolution can be further affected by psf and acoustic wave, giving rise to different breathing shifts.
0:18:10 Application in Zebrafish Notochord Development: Explores the use of brilliant microscopy with zebrafish nautical development, in particular the ecm layer and vacuolated cells. There are two peaks that arise, which reflect acoustic modes that are traveling along fibers.
0:22:28 Limitation of Current Techniques and Introduction of Mouse Embryo Case Study: The presenter finds that at the excel stage, confocal imaging leads to cell death (photo damage).
0:24:18 Analogy to Fluorescent Microscopy: The presenter discusses the advantages of selective plane illumination compared to confocal fluorescent microscopy.
0:24:44 Selective Plane Illumination: Selective plane illumination offers potential advantages to improve on current limitations. However, the current iteration only allows for line illumination.
0:25:36 Challenges and Goals: Key challenges include achieving high resolution, suppressing elastically scattered light, choosing appropriate wavelengths to reduce photo damage, enabling concurrent fluorescence imaging, and maintaining physiological conditions for live imaging.
0:27:31 Solution: Inverted Geometry: The presentation uses inverted geometry to mount samples with high resolution water inversion objectives and a thin fat foil. It is mounted on a 3D translational stage and temperature regulation is in place.
0:28:51 Optical Implementations: Includes static light sheet for fluorescent images, and dye product that separates fluorescent and brilliant light.
0:29:42 Choice of Wavelength: Decided to go for 780 nm, which is near-infrared. Benefits of the near infrared wavelength includes decreased photobleaching, enhanced sensitivity, and reduced background scattering.
0:30:20 Laser Filtering: Filter out laser noise with a combination of a fabric barrel and holographic graph filters.
0:31:42 Spectrometer Specifications: The presentation uses a combination of spherical and cylindrical lens that relays light on the viper.
0:32:22 The Rubidium Cell: The Rubidium Cell used to absorb the elastically structured light.
0:33:20 Performances of Spectrometer: Discusses optical spectral and special resolutions, in particular that optical special resolution is roughly 1 micron in all directions. Notes importance of precision, which is roughly 20 MHz.
0:34:23 100x Parallel Collection: Mentions that 100 points are collected in parallel. Brings down single point acquisition to 1 millisecond.
0:34:45 Data Analysis and Computation Limitations: Needs to fit spectra with complex line shape. Was able to do this with the help of Sebastian, from the presenter's lab, who brought in gpu code for data analysis.
0:35:50 Applications and Benefits: The improved microscope enables new imaging of biological processes. Shows a mouse embryo example, showcasing that it had proper development, confirming the advancement made.
0:37:33 Implementation on Fallucia: Showcases segmentation from a recent paper, and how this could allow them to correlate gene expression and mechanical properties. They are currently able to observe up to 64 cell stages.
0:39:01 Key Observations on Asymmetric Divisions: Points out how we can observe the asymmetrical divisions in this model.
0:40:08 Observing Fast Dynamic Processes in Drosophilia Castillation: Observing fast dynamic processes, like in Drasophilia, was once more beyond their capabilities.
0:41:02 Orthogonal Geometry Limitations: High scattering in drosophila embryos makes orthogonal geometry challenging due to refraction effects.
0:41:32 A New Geometry - Epi Line Scanning: Epi line scanning is one potential new method of observation to reduce the aforementioned refraction issue.
0:41:59 Epi Linescanning Limitations: Results in confocal geometry again, but a similar trend of value relationship and the geometry works in short noise limited conditions.
0:43:16 Gastrulation Process with Juan: Went back to gastrulation process with Juan, and showcase ventral furrow. Notes significant increase of breathing shift, which has been consistent in multiple embryos.
0:45:45 Long Time Imaging in Fallucia: Showcased implementation on tail elongating infamy vacuoles made for fourteen hours. Concluded that no clear photo damage was observed.
0:46:53 Summary and Advantages: Summarized that reduced photo damage and fast process images are achieved with this version of the microscope.
0:47:55 Acknowledgements: Credits the many people and groups that were involved in the project.
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