Scientists from the Medical Institute of Howard Hughes perfected the technique of fluorescence microscopy so that now it is possible to remove in high resolution the dynamic processes occurring in a living organism. The results of their work, scientists shared in the journal Science, and visualization of dynamic processes, which made shooting the movement of immune cells in the embryo fish, was presented in the article of the journal Nature.
Due to the current capabilities of light microscopy, particularly fluorescence microscopy, high-resolution, scientists may even consider a three-dimensional fragments of living tissue. Earlier work on overcoming the diffraction resolution limit of light microscope and the development of methods for non-invasive fluorescence imaging belong to Eric Betchu, Stefan Hello and William Marner. For that scientists was awarded the Nobel prize in chemistry in 2014.
The researchers decided not to stop. So? one of the latest developments of Bettiga became fluorescent microscopy of in-plane illumination (light sheet fluorescence microscopy), allowing to visualize the volume of live biological samples for a long time. And its modification, microscopy light-sheet illumination with a discrete (lattice light sheet microscopy — LLSM), allows visualization of rapid dynamic processes. Based on the two methods lies a quick scan of the sample a thin, flat beam of light, allowing to accumulate a large number of two-dimensional images, which are then combined into a three-dimensional model.
These methods, however, has its limitations. For example, the heterogeneity of the surrounding tissue introduces a distortion when determining signal which reduces the picture resolution. Even in the absence of distortion and a high resolution requires high intensity irradiation, resulting in a living sample can get damaged. Obtaining the highest quality images still required fixation and special preparation of the samples.
A team of Bettega were able to overcome these limitations and presented a combined microscopy technique with which scientists were able to observe many different processes directly inside a living organism. The researchers received motion image latinovich bubbles, the dynamics of cellular organelles, the growth processes of nerve cells in the forming spinal cord and movement of immune cells in the embryo of the model fish, zebrafish. In the video below, for example, shows migration of immune cells in perilymphatic space of the inner ear of the embryo.
For the modernization of the LLSM method the researchers used adaptive optics techniques used in the creation of ground-based telescopes used for astronomy. A team of Bettega measured the amount of distortion in the determination of the special fluorescent labels and adjust them using the variable shape of the adaptive mirror. The combined technique has a name AO-LLSM. To reduce phototoxicity beam managed with limit lighting only a thin plane of the specimen without irradiating it to a basic volume.
Scientists hope that their development will help to substantially advance the study of cells in their natural environment. The team is now thinking about how to reduce the cost of such a microscope, and also on how to make it more compact. Currently, the installation of the microscope is a three-meter table.