Light-sheet fluorescence microscopy, otherwise known as selective plane illumination microscopy (SPIM), is a cutting-edge imaging technique that can be used for the visualization of biological specimens with unparalleled precision and limited photodamage.
Through the illumination and imaging of a thin plane of a sample in combination with the latest camera-based detection, light-sheet microscopy enables high-speed, three-dimensional imaging of living organisms and tissues in their native environment over prolonged periods, hours to days.
The applications of this non-invasive imaging technique are widespread, and it has facilitated groundbreaking discoveries and invaluable insights into the fundamental mechanisms governing life at the cellular and subcellular levels.
For instance, Bruker’s Luxendo light-sheet microscopes leverage industry-leading SPIM technology, renowned for its applications in fields ranging from developmental biology and neurobiology to drug discovery and plant biology.
Figure 1. Light-sheet microscopy illuminates only a thin sample section and data is collected simultaneously by an uncoupled detection objective. Image Credit: Bruker Nano Surfaces and Metrology
Bruker’s Luxendo SPIM Technology
Luxendo SPIMs facilitate precision, laser-based manipulations at particular regions of interest when the state-of-the-art photomanipulation module (PM) has been fitted. This assembly is ideal for spatially resolved biophysics and the optogenetics of cells and tissues.
Moreover, the LuxControl microscope software ensures that PM functionality is entirely integrated into the experimental workflow, making it intuitive to use. This innovative addition equips researchers with the tools to carefully evaluate intricate biophysical processes, activate fluorophores and proteins, and pursue new avenues of scientific discovery.
What is Photomanipulation?
Photomanipulation is a powerful laser experimental method used across a variety of scientific disciplines. It finds particular applications in the fields of biology, cell biology, and neuroscience. As a technique, it involves precise and controlled manipulation of biological samples, such as cells, tissues, or organisms.
Photomanipulation utilizes light beams with both high spatial and temporal accuracy and affords researchers the power to manipulate specific regions within their sample.
This method has become extremely influential for the study of cellular processes, comprehending the complicated interactions within living systems, and investigating the functionality of specific proteins and organelles.
Consequently, photomanipulation has demonstrated the capacity to be an indispensable tool that offers a deeper understanding of biological mechanisms and has helped establish an increased foundation of knowledge to facilitate innovative discoveries in modern scientific research.
The Photomanipulation Module (PM) finds application across a range of experiments such as:
- Photoablation
- Cauterization
- Photobleaching
- Fluorescence Recovery After Photobleaching (FRAP)
- Uncaging
- Optogenetics
- Photoactivation
- Photoconversion
Technical Specifications
The PM is an add-on module designed to facilitate the integration of various lasers, including visible CW (for applications like FRAP) or pulsed IR (for tasks such as cauterization), with the detection objectives of Luxendo SPIMs.
This module couples the PM laser into the detection objective lens to generate a diffraction-limited illumination spot. The size of the PM laser spot is primarily influenced by the numerical aperture (NA) of the detection objective lens.
A 3D beam scanner from the acquisition software can be controlled, allowing researchers the flexibility to easily move the illumination spot in three-dimensional space within the sample during imaging.
The PM supports the creation of complex illumination regions, such as points, circles, squares, straight lines, and freeform lines. These regions can be pre-defined as part of the experimental workflow or determined later interactively when conducting 3D imaging experiments.
This advanced functionality offers complete flexibility for intricate state-of-the-art photomanipulation experiments, facilitating precise and targeted studies of specific regions of interest within biological samples.
Figure 2. The PM laser can be shaped based on a point, line, or polygon. Importantly, it can be freely positioned in 3D in the sample. Image Credit: Bruker Nano Surfaces and Metrology
Luxendo’s PM-Compatible Light-Sheet Systems
Through a combination of innovative design and expert engineering, the add-on PM module seamlessly integrates with three of Bruker’s Luxendo SPIMs, including:
- TruLive3D Imager: Dual-sided illumination for multiplex 3D cell culture live imaging
- MuVi SPIM: Multiview design for dynamic imaging of live and cleared samples
- InVi SPIM Lattice Pro: Optimized for high-resolution, rapid imaging of single cells to 3D cell cultures or organisms
Figure 3. TruLive3D Imager SPIM without (left) and with (right) PM module. Image Credit: Bruker Nano Surfaces and Metrology
Application Examples:
Cytokine Dynamics in a Tail Wound Assay
The add-on PM facilitates the investigation of immune system responses in a zebrafish tail wound assay. Through the laser-based PM, researchers can precisely create wound sites, enabling the study of immune system responses in tissue repair and regeneration with high precision.
This method yields valuable insights into immune system dynamics and holds promise for advancing wound healing therapies for both zebrafish and higher vertebrates, including humans.
Figure 4. Timelapse of cytokine dynamics in a tail wound assay. Pseudo-Brightfield and il1b:GFP (green) merged. Captured every 5 min for 12 hours. Sample courtesy of Dr. Elizabeth Jerison, Stanford University. Image Credit: Bruker Nano Surfaces and Metrology
Tissue Morphogenesis in Drosophila
In developmental biology, photomanipulation offers key insights into the role of biophysical forces in tissue morphogenesis. The Bruker webinar “Studying Morphogenetic Waves with Photo Manipulation Coupled to Multi-View Light-Sheet Microscopy” presents revolutionary research that surpasses conventional 2D embryo imaging techniques.
During the webinar, Dr. Matteo Rauzi, a guest speaker from the University Côte d’Azur, discussed his laboratory’s work using a groundbreaking computational model based on light-sheet imaging, photomanipulation, and multidimensional image analysis.
The webinar underscores how photomanipulation opens new avenues for exploring critical processes such as Drosophila embryo gastrulation, neurulation, and the shaping of the animal body.1
Figure 5. Dr. Rauzi’s research showing multi-view light-sheet microscopy for in toto imaging and big data processing to obtain digital embryonic carpets1. Image Credit: Bruker Nano Surfaces and Metrology
Dr. Rauzi’s novel approach has facilitated discoveries related to epithelial furrowing, challenging previous notions regarding its role in embryo development and exposing the molecular signals and mechanical forces responsible for this process.1,2
Microglia Response to Axonal Damage
IR laser ablation conducted with a Luxendo MuVi SPIM enables the selective dissection of a neuron’s axon in the zebrafish brain. Subsequently, researchers can monitor the movement of microglia to the damaged axon following ablation.³
Figure 6. After ablation, it took approximately thirty minutes for four microglia to reach the damaged axon. Image used with permission under Creative Commons Attribution (CC-BY 4.0 DEED).³
Mouse Peri-Implantation
To understand more about mouse peri-implantation, a pulsed infrared (IR) laser at 1040 nm, 200 fs pulse length, and 1.5 W output power (Spectra-Physics, HighQ-2) was paired with the detection objective.
Results from this experimental assembly revealed that the polar trophectoderm (pTE) cells were cut short in one direction (apically) and extended in another (along their apicobasal axis), which provides details into how these cells develop.4
This study highlights the practicalities of using advanced imaging and laser manipulation techniques to better understand embryonic development.
Figure 7. Kymographs of GFP-Myh9 signal along with blue and red lines in (F), and measurement of the apico-basal length of pTE cells upon laser ablation. Used with permission under Creative Commons Attribution (CC BY 4.0).4
Groundbreaking Investigations with the PM Module
This article outlines the advanced capabilities of Bruker’s Luxendo light-sheet microscopes, particularly when paired with the photomanipulation module. These integrated technologies enable precise, laser-based manipulations of specific regions of interest, making them ideal for non-invasive imaging of living organisms and tissues in their native environments.
The compatibility of the PM module add-on with three of Bruker’s Luxendo SPIMs extends its utility and sets the stage for pioneering investigations in developmental biology, neurobiology, and beyond.
Acknowledgments
Based on materials originally authored by Dr. Elisabeth Kugler, External Marcom Specialist, and Melissa Martin, Life Science Writer at Bruker.
References
- Bruker webinar. “Studying Morphogenetic Waves with Photo Manipulation Coupled to Multi-View Light-Sheet Microscopy.” (2023). https://www.bruker.com/en/news-and-events/webinars/2023/studying-morphogenetic-waves-with-photo-manipulation-coupled-to-multi-view-light-sheet-microscopy.html
- Popkova, A., Pagnotta, S., Rauzi, M. “A mechanical wave travels along a genetic guide to drive the formation of an epithelial furrow.” bioRxiv, 12.08.518365, (2022). https://doi.org/10.1101/2022.12.08.518365. (preprint).
- Medeiros, G., Kromm, D., Balazs, B., et al. “Cell and tissue manipulation with ultrashort infrared laser pulses in lightsheet microscopy.” Sci Rep, 10, 1942, (2020). https://www.nature.com/articles/s41598-019-54349-x.
- Ichikawa, T., Zhang, H.T., Panavaite, L. An ex vivo system to study cellular dynamics underlying mouse peri-implantation development.” Developmental Cell, 57(3), 373-386, (2022). https://doi.org/10.1016/j.devcel.2021.12.023.
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Whatever your measurement and analysis needs, whatever your material or scale of investigation, Bruker has a specialized high-performance solution for you.
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