Developmental biologists are often required to study embryogenesis in different stages and acquire this information by imaging their samples at periodic intervals. These samples are commonly labeled with fluorescent dyes which emit a specific wavelength of light after excitation with a specific wavelength of light source, such as a laser. Though this is a universal practice, the dyes are often easily photobleached due to excessive exposure to light. This results in the loss of signal intensity and a high background (non-specific signals arising out of autofluorescence), which ultimately limits the quality of images.
Photobleaching is known to cause irreparable photodamage to samples and thus prohibits the study of Biophysical phenomena (that are known to occur in the range of milli-to-micro second timescale). This stands as a major roadblock in the field of developmental biology and calls for rapid volume imaging techniques. Now, scientists at the Indian Institute of Science, Bangalore, have developed a novel method to overcome this drawback.
Researchers at the Nanobioimaging Laboratory, Department of Instrumentation and Applied Physics at IISc, researchers have reported a novel method to eliminate photobleaching. They have discovered that by reducing the number of 2D angular views for imaging, which would later be used to reconstruct a 3D volume image of the specimen, the risk of photobleaching may be eliminated, paving the path for high-resolution fluorescence based imaging. Analogous to CT-scan imaging, commonly used by clinicians to visualize internal organs with enhanced clarity, this technique involves illumination of the specimen by a sheet-of-light and rotating it through a specific angle to capture several images, which would later be assembled together to construct a 3D image. The purpose of moving the sample is to capture the different ‘slices’ or portions of the samples as regular 2D images and later stack these images together to form a complete 3D picture, a process known as 3D reconstruction.
The team conducted experiments by embedding the Zebrafish embryo (obtained from DBG lab headed by Prof. Upendra Nongthomba) in an agar gel held in a capillary tube and placed on a sample holder which can rotate and/or translate. An expanded beam of appropriate wavelength was generated to illuminate the sample in the form of a sheet and a sensitive detector is used to record the fluorescence light. Since the images are acquired by rotating the specimen, the recorded images (in cylindrical coordinate system) are then transformed to Cartesian coordinate system using computer programs.
“The contrast for 18 view based 3D reconstruction is marginal as compared to 3D SPIM image. This shows that, there is a trade-off between the image quality (i.e., contrast) and the number of angular views required for reconstructing 3D image. A balanced choice on specific application may enable the best image quality within a reasonable time”, says Rasmi, one of the members of the team.
The new technique resulted in 10-fold faster imaging. This reduces the radiation-dose that cause photobleaching and also eliminates the necessity for translational motion of the specimen that is central to state-of-the-art SPIM microscopy. The image contrast decreased linearly when the angular views were reduced from 18 to 6. However, this reduction in contrast is marginal when 18-view 3D reconstructed image is compared to the currently in-vogue SPIM imaging strategy.
This study enables researchers to rapidly image in-vivo specimens (Zebrafish Embryo, tumor or small mammals) with reduced photobleaching effects that play havoc for experiments requiring periodical imaging. This technique is a step closer to temporal super-resolution and is well suited for rapid non-invasive imaging of small organisms.
About the Authors:
Partha Pratim Mondal and Chelur K.Rasmi are from the Nanobioimaging Laboratory, Department of Instrumentation and Applied Physics, Indian Institute of Science, Bangalore. Upendra Nongthomba and Mani Madhangi are from MRDG, IISc, Bangalore.
About the Research:
This research work was published in Microscopy Research and Technique journal (vol. 79, pp. 455-458) titled, “Curtailed light sheet microscopy for rapid imaging of macroscopic biological specimens”.