Limits...
Direct imaging of phase objects enables conventional deconvolution in bright field light microscopy.

Hernández Candia CN, Gutiérrez-Medina B - PLoS ONE (2014)

Bottom Line: Polystyrene nanoparticles and microtubules (biological polymer filaments) serve as the pure phase point and line objects, respectively, that are imaged with high contrast and low noise using standard microscopy plus digital image processing.Our experimental results agree with a proposed model for the response functions, and confirm previous theoretical predictions.Finally, we use the measured phase point-spread function to apply conventional deconvolution on the bright field images of living, unstained bacteria, resulting in improved definition of cell boundaries and sub-cellular features.

View Article: PubMed Central - PubMed

Affiliation: Program in Molecular Biology, Instituto Potosino de Investigación Científica y Tecnológica, San Luis Potosí, Mexico.

ABSTRACT
In transmitted optical microscopy, absorption structure and phase structure of the specimen determine the three-dimensional intensity distribution of the image. The elementary impulse responses of the bright field microscope therefore consist of separate absorptive and phase components, precluding general application of linear, conventional deconvolution processing methods to improve image contrast and resolution. However, conventional deconvolution can be applied in the case of pure phase (or pure absorptive) objects if the corresponding phase (or absorptive) impulse responses of the microscope are known. In this work, we present direct measurements of the phase point- and line-spread functions of a high-aperture microscope operating in transmitted bright field. Polystyrene nanoparticles and microtubules (biological polymer filaments) serve as the pure phase point and line objects, respectively, that are imaged with high contrast and low noise using standard microscopy plus digital image processing. Our experimental results agree with a proposed model for the response functions, and confirm previous theoretical predictions. Finally, we use the measured phase point-spread function to apply conventional deconvolution on the bright field images of living, unstained bacteria, resulting in improved definition of cell boundaries and sub-cellular features. These developments demonstrate practical application of standard restoration methods to improve imaging of phase objects such as cells in transmitted light microscopy.

Show MeSH

Related in: MedlinePlus

Main characteristics of the PSF and comparison with theory.(A) False-color, vertical slice () of the experimental PSF. (B) Superimposed lateral cross-section profiles (light gray). (C) Axial cross-section profiles. (D) Vertical slice () of the theoretical PSF. (E) Lateral cross-section profiles. (F) Axial cross-section profiles. The intensity of the theoretical model was multiplied by an arbitrary factor for comparison with experiment. Profiles highlighted in black correspond to the maximum positive intensity peak of the PSF, used to determine the PSF main spot size.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3928359&req=5

pone-0089106-g002: Main characteristics of the PSF and comparison with theory.(A) False-color, vertical slice () of the experimental PSF. (B) Superimposed lateral cross-section profiles (light gray). (C) Axial cross-section profiles. (D) Vertical slice () of the theoretical PSF. (E) Lateral cross-section profiles. (F) Axial cross-section profiles. The intensity of the theoretical model was multiplied by an arbitrary factor for comparison with experiment. Profiles highlighted in black correspond to the maximum positive intensity peak of the PSF, used to determine the PSF main spot size.

Mentions: The emerging 3D PSF (see Figure 1B) and a representative central x-z slice (Figure 2A) feature a high peak SNR (100, see Materials and Methods), allowing for several interference-diffraction fringes to be clearly distinguished. Although the hourglass-like aspect is similar to its fluorescence counterpart, the BF-PSF has distinctive characteristics, showing negative and positive intensity count values, together with a main central lobe that changes from negative to positive amplitude as the -position is changed (i.e. as the microscope is defocused). This last effect is, as expected, due to the 100-nm polystyrene bead acting as an effective phase object being small () and mostly transparent at the wavelength range involved. For reference purposes, we define here a 3D coordinate system where x = 0, y = 0 locates the center of the bead on the image plane, and z = 0 locates the axial position where the phase object is the least visible. Using these definitions, pixel count vs. x, z profiles from the central x-z slice of the PSF further show good fringe visibility (see Figure 2B) and an (axially) asymmetrical central lobe with a larger positive amplitude (see Figure 2C). The widths of the main spot are nm and nm, along the x- and z-axis, respectively (see Figure S1).


Direct imaging of phase objects enables conventional deconvolution in bright field light microscopy.

Hernández Candia CN, Gutiérrez-Medina B - PLoS ONE (2014)

Main characteristics of the PSF and comparison with theory.(A) False-color, vertical slice () of the experimental PSF. (B) Superimposed lateral cross-section profiles (light gray). (C) Axial cross-section profiles. (D) Vertical slice () of the theoretical PSF. (E) Lateral cross-section profiles. (F) Axial cross-section profiles. The intensity of the theoretical model was multiplied by an arbitrary factor for comparison with experiment. Profiles highlighted in black correspond to the maximum positive intensity peak of the PSF, used to determine the PSF main spot size.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3928359&req=5

pone-0089106-g002: Main characteristics of the PSF and comparison with theory.(A) False-color, vertical slice () of the experimental PSF. (B) Superimposed lateral cross-section profiles (light gray). (C) Axial cross-section profiles. (D) Vertical slice () of the theoretical PSF. (E) Lateral cross-section profiles. (F) Axial cross-section profiles. The intensity of the theoretical model was multiplied by an arbitrary factor for comparison with experiment. Profiles highlighted in black correspond to the maximum positive intensity peak of the PSF, used to determine the PSF main spot size.
Mentions: The emerging 3D PSF (see Figure 1B) and a representative central x-z slice (Figure 2A) feature a high peak SNR (100, see Materials and Methods), allowing for several interference-diffraction fringes to be clearly distinguished. Although the hourglass-like aspect is similar to its fluorescence counterpart, the BF-PSF has distinctive characteristics, showing negative and positive intensity count values, together with a main central lobe that changes from negative to positive amplitude as the -position is changed (i.e. as the microscope is defocused). This last effect is, as expected, due to the 100-nm polystyrene bead acting as an effective phase object being small () and mostly transparent at the wavelength range involved. For reference purposes, we define here a 3D coordinate system where x = 0, y = 0 locates the center of the bead on the image plane, and z = 0 locates the axial position where the phase object is the least visible. Using these definitions, pixel count vs. x, z profiles from the central x-z slice of the PSF further show good fringe visibility (see Figure 2B) and an (axially) asymmetrical central lobe with a larger positive amplitude (see Figure 2C). The widths of the main spot are nm and nm, along the x- and z-axis, respectively (see Figure S1).

Bottom Line: Polystyrene nanoparticles and microtubules (biological polymer filaments) serve as the pure phase point and line objects, respectively, that are imaged with high contrast and low noise using standard microscopy plus digital image processing.Our experimental results agree with a proposed model for the response functions, and confirm previous theoretical predictions.Finally, we use the measured phase point-spread function to apply conventional deconvolution on the bright field images of living, unstained bacteria, resulting in improved definition of cell boundaries and sub-cellular features.

View Article: PubMed Central - PubMed

Affiliation: Program in Molecular Biology, Instituto Potosino de Investigación Científica y Tecnológica, San Luis Potosí, Mexico.

ABSTRACT
In transmitted optical microscopy, absorption structure and phase structure of the specimen determine the three-dimensional intensity distribution of the image. The elementary impulse responses of the bright field microscope therefore consist of separate absorptive and phase components, precluding general application of linear, conventional deconvolution processing methods to improve image contrast and resolution. However, conventional deconvolution can be applied in the case of pure phase (or pure absorptive) objects if the corresponding phase (or absorptive) impulse responses of the microscope are known. In this work, we present direct measurements of the phase point- and line-spread functions of a high-aperture microscope operating in transmitted bright field. Polystyrene nanoparticles and microtubules (biological polymer filaments) serve as the pure phase point and line objects, respectively, that are imaged with high contrast and low noise using standard microscopy plus digital image processing. Our experimental results agree with a proposed model for the response functions, and confirm previous theoretical predictions. Finally, we use the measured phase point-spread function to apply conventional deconvolution on the bright field images of living, unstained bacteria, resulting in improved definition of cell boundaries and sub-cellular features. These developments demonstrate practical application of standard restoration methods to improve imaging of phase objects such as cells in transmitted light microscopy.

Show MeSH
Related in: MedlinePlus