Today : Monday, September 25, 2017
New microscope makes movies of tiny live specimens

A new microscopy technique allowing biologists to see more detail in living specimens without damaging them with intense light has been developed by researchers in the US, Europe and Japan. The method uses interacting beams of light to create an ultrathin "light sheet" that passes through the sample, illuminating only the part of the specimen that is being imaged. This minimizes tissue damage caused by unnecessary illumination, and has allowed the team to make "movies" showing the time evolution of live specimens.

The team is led by Eric Betzig of the Howard Hughes Medical Institute in Virginia, who earlier this month shared the 2014 Nobel Prize for Chemistry for the invention of super-resolution microscopy, which allows researchers to build up nanometre-scale images of static biological samples. However, Betzig has now moved away from the technique. "There's only so much you're going to ever learn – even with the highest resolution picture – from static images," he explains. "The only way you're going to understand what connects inanimate objects to animate life is by seeing movies."

Studying tiny living cells in real time is a major challenge because the strong illumination needed to obtain multiple, rapid, high-resolution images can alter biological and chemical processes or even kill the cells. Traditional imaging techniques such as confocal microscopy send a cone of light through the sample to a tiny focus in the image plane. The focus is then swept across the image plane and light propagates back out of the sample to be collected. The problem is that the light cone illuminates – and therefore potentially damages – much of the sample to image a tiny spot.

Thin sheet of light

Light-sheet microscopy gets round this problem by illuminating the sample with a thin sheet of light perpendicular to the imaging direction. The sheet is swept through the sample to produce a 3D image. The technique was popularized by Ernst Stelzer and colleagues at the European Molecular Biology Laboratory in Heidelberg, Germany, who first used it on fly embryos. Unfortunately, it is difficult to produce sheets of light thinner than about 5 μm, whereas the depth of focus of a high-resolution microscope is about 1 μm. This means that most of the light passing through the sample blurs the image instead of enhancing it.

Betzig and colleagues addressed this problem by illuminating the sample with plane waves from multiple directions. Interference between these waves produces a thin optical lattice of standing waves similar to lattices used to trap ultracold atoms. To achieve images at the highest possible resolution, the researchers took multiple images in each plane, gradually shifting the lattice across the plane, before combining the images electronically. They also developed a technique to acquire images much more quickly and with less light going into the sample. This involves shifting the lattice back and forth rapidly, resulting in the effective uniform illumination of the plane with a light sheet only 1 μm thick. Using this latter technique, the researchers we able to record at up to 1000 image frames per second.

Moving pictures

The researchers used their optical-lattice microscope to gain new insights into a variety of biological processes, such as the behaviour of mitochondrial fragments and chromosomes during cell division and the development of fly and worm embryos. By adjusting the frame rate of their microscope, the researchers were able to monitor both slow embryonic processes such as the localization of a particular protein as the embryo divides and grows, and faster processes that occur just before it hatches.

Stelzer, now director of the Buchmann Institute for Molecular Life Sciences in Frankfurt, is impressed, saying that the technique "allows us to work with smaller objects such as cells and get a really good resolution, while, at the same time, maintaining the viability of the specimen".

Developmental biologist Pavel Tomancak of the Max Planck Institute of Molecular Cell Biology and Genetics in Dresden agrees. "It's a kind of incremental improvement of the light-sheet paradigm," he says, "but the paper sells itself by showing these spectacular applications. Looking at the movies, I would say it's breathtaking!"

The research is published in Science.

Copyright © 2014 Bahonar University of Kerman - All rights reserved

Developer by Creative Thinking Company