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Researchers say they have developed a fluorescence microscopy technique that improves image resolution by acquiring 3 views of a sample at the same time.
The team applied their technique in 2 microscopy modes and used it to image several types of biological samples.
For both modes, the technique demonstrated a volumetric resolution of up to 235 by 235 by 340 nanometers, double the volumetric resolution of traditional methods.
The researchers believe this technique will prove particularly useful for watching the dynamics of biological processes, which can provide insights into the workings of healthy and diseased cells.
They described the technique in the journal Optica.
The researchers noted that most fluorescence microscopy methods fail to capture much of the fluorescence emitted from a sample, which represents lost information and reduces image resolution.
“In our work, we captured this previously neglected fluorescence and fused it with the traditional views used in conventional microscopy,” said study author Yicong Wu, PhD, of the National Institute of Biomedical Imaging and Bioengineering at the National Institutes of Health in Bethesda, Maryland.
“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 2 objective 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 study author Hari Shroff, PhD, also of the National Institute of Biomedical Imaging and Bioengineering.
“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 3 objectives through a sample simultaneously to produce 3 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 3 views into 1
Whether acquired in wide-field or light-sheet mode, the 3 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 3 views,” Dr Wu said. “In wide-field mode, we further aided registration of the images by adding fluorescent beads to the samples as point of reference.”
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, the team 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, the researchers said 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 and even deadly to living cells, and the temporal resolution is needed to capture fast processes.
The researchers are now exploring additional biological applications for the new system and are working to extend the method to other microscope modalities, such as confocal microscopy. 
Researchers say they have developed a fluorescence microscopy technique that improves image resolution by acquiring 3 views of a sample at the same time.
The team applied their technique in 2 microscopy modes and used it to image several types of biological samples.
For both modes, the technique demonstrated a volumetric resolution of up to 235 by 235 by 340 nanometers, double the volumetric resolution of traditional methods.
The researchers believe this technique will prove particularly useful for watching the dynamics of biological processes, which can provide insights into the workings of healthy and diseased cells.
They described the technique in the journal Optica.
The researchers noted that most fluorescence microscopy methods fail to capture much of the fluorescence emitted from a sample, which represents lost information and reduces image resolution.
“In our work, we captured this previously neglected fluorescence and fused it with the traditional views used in conventional microscopy,” said study author Yicong Wu, PhD, of the National Institute of Biomedical Imaging and Bioengineering at the National Institutes of Health in Bethesda, Maryland.
“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 2 objective 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 study author Hari Shroff, PhD, also of the National Institute of Biomedical Imaging and Bioengineering.
“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 3 objectives through a sample simultaneously to produce 3 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 3 views into 1
Whether acquired in wide-field or light-sheet mode, the 3 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 3 views,” Dr Wu said. “In wide-field mode, we further aided registration of the images by adding fluorescent beads to the samples as point of reference.”
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, the team 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, the researchers said 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 and even deadly to living cells, and the temporal resolution is needed to capture fast processes.
The researchers are now exploring additional biological applications for the new system and are working to extend the method to other microscope modalities, such as confocal microscopy.
Researchers say they have developed a fluorescence microscopy technique that improves image resolution by acquiring 3 views of a sample at the same time.
The team applied their technique in 2 microscopy modes and used it to image several types of biological samples.
For both modes, the technique demonstrated a volumetric resolution of up to 235 by 235 by 340 nanometers, double the volumetric resolution of traditional methods.
The researchers believe this technique will prove particularly useful for watching the dynamics of biological processes, which can provide insights into the workings of healthy and diseased cells.
They described the technique in the journal Optica.
The researchers noted that most fluorescence microscopy methods fail to capture much of the fluorescence emitted from a sample, which represents lost information and reduces image resolution.
“In our work, we captured this previously neglected fluorescence and fused it with the traditional views used in conventional microscopy,” said study author Yicong Wu, PhD, of the National Institute of Biomedical Imaging and Bioengineering at the National Institutes of Health in Bethesda, Maryland.
“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 2 objective 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 study author Hari Shroff, PhD, also of the National Institute of Biomedical Imaging and Bioengineering.
“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 3 objectives through a sample simultaneously to produce 3 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 3 views into 1
Whether acquired in wide-field or light-sheet mode, the 3 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 3 views,” Dr Wu said. “In wide-field mode, we further aided registration of the images by adding fluorescent beads to the samples as point of reference.”
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, the team 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, the researchers said 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 and even deadly to living cells, and the temporal resolution is needed to capture fast processes.
The researchers are now exploring additional biological applications for the new system and are working to extend the method to other microscope modalities, such as confocal microscopy.