q&more
My watch list
my.chemie.de  
Login  

News

New approach doubles 3-D resolution of fluorescence microscopy

Yicong Wu, National Institute of Biomedical Imaging and Bioengineering

By adding a third objective lens, researchers were able to capture previously neglected fluorescence, improving image resolution in three dimensions. The schematic on the right shows the new approach as applied to a type of light-sheet microscopy.

16-Aug-2016: Researchers have developed a new fluorescence microscopy approach that significantly improves image resolution by acquiring three views of a sample at the same time. Their new method is particularly useful for watching the dynamics of biological processes, which can provide insights into how healthy cells work and what goes wrong when diseases occur.

The researchers apply their multi-view technique in two microscopy modes and use it to image several types of biological samples. For both modes, the researchers demonstrated a volumetric resolution of up to 235 by 235 by 340 nanometers, double the volumetric resolution of traditional methods.

Biologists commonly use fluorescence microscopy to study everything from embryo development to the intricate processes within living cells. However, most fluorescence microscopy methods fail to capture much of the fluorescence emitted from the sample, which not only represents lost information but also reduces image resolution.

"In our work, we captured this previously neglected fluorescence and fused it with the traditional views used in conventional microscopy," said Yicong Wu, staff scientist at the National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health, Maryland, USA. "This increases resolution without compromising either temporal resolution or adding additional light to the sample."

Adding a third objective lens

The new multi-view approach helps improve a technique the researchers previously developed called dual-view plane illumination microscopy (diSPIM). Scientists around the world employ commercial versions of diSPIM, which uses a thin sheet of light and two objectives lenses to excite and detect fluorescence.

"The main motivation of this new research was that the resolution in diSPIM was limited by the numerical aperture of the upper lenses, and fluorescence emitted in the direction of the coverslip is not captured," explained Hari Shroff, leader of the research team. "We reasoned that if we could simultaneously image this neglected signal by adding a higher numerical aperture lens that acquired the bottom view, then we could boost the lateral resolution."

In the improved diSPIM microscopy technique, each light sheet is tilted at a 45-degree angle relative to an additional lower objective lens. In its current design, the researchers swept the lower objective's plane of focus through the sample to image the previously unused fluorescence, but this mechanical scanning could be replaced with a passive optic in future versions of the microscope. Using the multi-view approach improved the lateral, or horizontal, resolution of diSPIM to about 235 nm.

The researchers also implemented the new technique in wide-field mode by scanning the three objectives through a sample simultaneously to produce three individual 3D views. With this mode, the multi-view method improved axial, or Z-axis, resolution, to about 340 nm, an increase of 45%.

Merging three views into one

Whether acquired in wide-field or light-sheet mode, the three views must be precisely aligned and also cleaned up with an image processing technique known as deconvolution.

"One helpful trick was to deconvolve each view first to increase image quality, contrast, and so forth, which then allowed accurate registration of the three views," said Wu. "In wide-field mode, we further aided registration of the images by adding fluorescent beads to the samples as point of reference." He added that collaboration with Patrick La Riviere's research group at the University of Chicago was essential in thinking through and testing this deconvolution method.

The researchers demonstrated the multi-view technique by imaging biological samples and were able to see detailed features not typically observable. For example, the wide-field multi-view microscope clearly resolved the spherical protein shell present when Bacillus subtilis forms a spore and also allowed the researchers to observe the dynamics of organelles inside cells. In light-sheet mode, they clearly saw the 3D dynamic nature of tiny protrusions on living white blood cells when they acquired 150 triple-view images over 40 minutes.

Although other methods have been used to capture multiple views sequentially, this new method improves spatial resolution without introducing additional illumination or compromising temporal resolution relative to conventional imaging. This is important because additional light can be damaging, even deadly to living cells, and the temporal resolution is needed to capture fast processes.

The research team is now exploring additional biological applications for the new system and is working to extend the method to other microscope modalities, such as confocal microscopy.

Original publication:
Yicong Wu, Panagiotis Chandris, Peter W. Winter, Edward Y. Kim, Valentin Jaumouillé, Abhishek Kumar, Min Guo, Jacqueline M. Leung, Corey Smith, Ivan Rey-Suarez, Huafeng Liu, Clare M. Waterman, Kumaran S. Ramamurthi, Patrick J. La Riviere, and Hari Shroff; "Simultaneous multiview capture and fusion improves spatial resolution in wide-field and light-sheet microscopy"; Optica; 2016

Facts, background information, dossiers

  • dual-view plane ill…
  • biomedical imaging

More about National Institutes of Health

  • News

    Combination Pack Battles Cancer

    For efficient cancer therapy with few side effects, the active drug should selectively attain high concentration in the tumor. Scientists have now introduced a new approach, in which two synergistic drug components are combined into a dimer. This dimer can be incorporated into polymeric nan ... more

    Clever use of mirrors boosts performance of light-sheet microscope

    Using a simple "mirror trick" and not-so-simple computational analysis, scientists affiliated with the Marine Biological Laboratory (MBL) have considerably improved the speed, efficiency, and resolution of a light-sheet microscope, with broad applications for enhanced imaging of live cells ... more

More about OSA

  • News

    New glass for solar cells and LEDs

    Using nanoscale grass-like structures, researchers at the University of Pittsburgh , Pennsylvania have created glass that lets through a large amount of light while appearing hazy. This is the first time that glass has been made with such high levels of haze and light transmittance at the s ... more

    New screen coating makes reading in sunlight a lot easier

    Screens on even the newest phones and tablets can be hard to read outside in bright sunlight. Inspired by the nanostructures found on moth eyes, researchers have developed a new antireflection film that could keep people from having to run to the shade to look at their mobile devices. The a ... more

    Optical approach offers faster and less expensive method for carbon dating

    Researchers from Istituto Nazionale di Ottica (INO), within Consiglio Nazionale delle Ricerche (CNR), Italy have demonstrated a new compact spectroscopic instrument that offers a highly sensitive optical method for detecting radiocarbon dioxide concentration, which can be used to carbon dat ... more

q&more – the networking platform for quality excellence in lab and process

The q&more concept is to increase the visibility of recent research and innovative solutions, and support the exchange of knowledge. In the broad spectrum of subjects covered, the focus is on achieving maximum quality in highly innovative sectors. As a modern knowledge platform, q&more offers market participants one-of-a-kind networking opportunities. Cutting-edge research is presented by authors of international repute. Attractively presented in a high-quality context, and published in German and English, the original articles introduce new concepts and highlight unconventional solution strategies.

> more about q&more

q&more is supported by:

 

Your browser is not current. Microsoft Internet Explorer 6.0 does not support some functions on Chemie.DE