Fluorescence Microscopy With Doubled 3D Resolution

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 (Source: Y. Wu, NIH)

By adding a third objective lens, researchers were able to capture previously neglected fluorescence, improving image resolution in three dimensions. The schematic shows the new approach as applied to a type of light sheet microscopy (Source: Y. Wu, NIH)

Researchers at the National Institute of Biomedical Imaging and Bioen­gineering, National Institutes of Health, Maryland, USA, 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. They 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. “This increases reso­lution without com­promising either temporal reso­lution or adding additional light to the sample.”

The new multi-view approach helps improve a technique the researchers previously developed, thedual-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 %.

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. (Source: OSA)

Reference: Y. Wu et al: Simultaneous multi-view capture and fusion improves spatial resolution in wide-field and light-sheet microscopy, Optica, 3, 897 (2016). DOI: 10.1364/optica.3.000897

Link: National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health, Maryland, USA

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