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Optimal resolution in Fresnel incoherent correlation holographic fluorescence microscopy.

Brooker G, Siegel N, Wang V, Rosen J - Opt Express (2011)

Bottom Line: An important improvement from our previous FINCH configurations capitalizes on the polarization sensitivity of the SLM so that the same SLM pixels which create the spherical wave simulating the microscope tube lens, also pass the plane waves from the infinity corrected microscope objective, so that interference between the two wave types at the camera creates a hologram.This advance dramatically improves the resolution of the FINCH system.Results from imaging a fluorescent USAF pattern and a pollen grain slide reveal resolution which approaches the Rayleigh limit by this simple method for 3D fluorescent microscopic imaging.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Engineering, Johns Hopkins University, 9605 Medical Center Drive, Rockville, Maryland 20850 USA. gbrooker@jhu.edu

ABSTRACT
Fresnel Incoherent Correlation Holography (FINCH) enables holograms and 3D images to be created from incoherent light with just a camera and spatial light modulator (SLM). We previously described its application to microscopic incoherent fluorescence wherein one complex hologram contains all the 3D information in the microscope field, obviating the need for scanning or serial sectioning. We now report experiments which have led to the optimal optical, electro-optic, and computational conditions necessary to produce holograms which yield high quality 3D images from fluorescent microscopic specimens. An important improvement from our previous FINCH configurations capitalizes on the polarization sensitivity of the SLM so that the same SLM pixels which create the spherical wave simulating the microscope tube lens, also pass the plane waves from the infinity corrected microscope objective, so that interference between the two wave types at the camera creates a hologram. This advance dramatically improves the resolution of the FINCH system. Results from imaging a fluorescent USAF pattern and a pollen grain slide reveal resolution which approaches the Rayleigh limit by this simple method for 3D fluorescent microscopic imaging.

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Comparison of widefield and FINCH holographic imaging as a function of fluorescence emission bandwidth. The specimen was the USAF test pattern, imaged with an Olympus 20x 0.75 NA objective with a SLM-CCD distance of 400 mm. Columns I, II, and III respectively are widefield images, FINCH reconstructed images, and fluorescence emission spectra taken with varying emission filter combinations, as described in the text. Images and spectra in A, were taken with both a long pass and a standard emission bandpass filter, in B were taken with a standard emission bandpass filter and in C are were taken with only a long pass filter. The FWHM fluorescence emission (in nm) was ~17 nm for the narrow bandwidth (Row A), ~38 nm for the normal bandwidth (row B) and >50 nm bandwidth with a > 50 nm tail (Row C) for the wide bandwidth.emission fluorescence. The widefield images were obtained with input and output polarizers set at 0° with a 400 mm focal length diffractive lens pattern displayed on the SLM. The FINCH holograms were obtained with input and output polarizers set at 60° with an 800 mm focal length diffractive lens pattern displayed on the SLM. Best focus images were calculated from the holograms.
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g010: Comparison of widefield and FINCH holographic imaging as a function of fluorescence emission bandwidth. The specimen was the USAF test pattern, imaged with an Olympus 20x 0.75 NA objective with a SLM-CCD distance of 400 mm. Columns I, II, and III respectively are widefield images, FINCH reconstructed images, and fluorescence emission spectra taken with varying emission filter combinations, as described in the text. Images and spectra in A, were taken with both a long pass and a standard emission bandpass filter, in B were taken with a standard emission bandpass filter and in C are were taken with only a long pass filter. The FWHM fluorescence emission (in nm) was ~17 nm for the narrow bandwidth (Row A), ~38 nm for the normal bandwidth (row B) and >50 nm bandwidth with a > 50 nm tail (Row C) for the wide bandwidth.emission fluorescence. The widefield images were obtained with input and output polarizers set at 0° with a 400 mm focal length diffractive lens pattern displayed on the SLM. The FINCH holograms were obtained with input and output polarizers set at 60° with an 800 mm focal length diffractive lens pattern displayed on the SLM. Best focus images were calculated from the holograms.

Mentions: The effect of the fluorescence emission bandwidth on widefield images and those generated by FINCH holography was examined. The emission bandwidth of the Semrock GFP filter set used in this study produced an emission bandwidth from the USAF slide of about ~38 nm (500 nm – 538 nm) FWHM (Fig. 10 BFig. 10


Optimal resolution in Fresnel incoherent correlation holographic fluorescence microscopy.

Brooker G, Siegel N, Wang V, Rosen J - Opt Express (2011)

Comparison of widefield and FINCH holographic imaging as a function of fluorescence emission bandwidth. The specimen was the USAF test pattern, imaged with an Olympus 20x 0.75 NA objective with a SLM-CCD distance of 400 mm. Columns I, II, and III respectively are widefield images, FINCH reconstructed images, and fluorescence emission spectra taken with varying emission filter combinations, as described in the text. Images and spectra in A, were taken with both a long pass and a standard emission bandpass filter, in B were taken with a standard emission bandpass filter and in C are were taken with only a long pass filter. The FWHM fluorescence emission (in nm) was ~17 nm for the narrow bandwidth (Row A), ~38 nm for the normal bandwidth (row B) and >50 nm bandwidth with a > 50 nm tail (Row C) for the wide bandwidth.emission fluorescence. The widefield images were obtained with input and output polarizers set at 0° with a 400 mm focal length diffractive lens pattern displayed on the SLM. The FINCH holograms were obtained with input and output polarizers set at 60° with an 800 mm focal length diffractive lens pattern displayed on the SLM. Best focus images were calculated from the holograms.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3368318&req=5

g010: Comparison of widefield and FINCH holographic imaging as a function of fluorescence emission bandwidth. The specimen was the USAF test pattern, imaged with an Olympus 20x 0.75 NA objective with a SLM-CCD distance of 400 mm. Columns I, II, and III respectively are widefield images, FINCH reconstructed images, and fluorescence emission spectra taken with varying emission filter combinations, as described in the text. Images and spectra in A, were taken with both a long pass and a standard emission bandpass filter, in B were taken with a standard emission bandpass filter and in C are were taken with only a long pass filter. The FWHM fluorescence emission (in nm) was ~17 nm for the narrow bandwidth (Row A), ~38 nm for the normal bandwidth (row B) and >50 nm bandwidth with a > 50 nm tail (Row C) for the wide bandwidth.emission fluorescence. The widefield images were obtained with input and output polarizers set at 0° with a 400 mm focal length diffractive lens pattern displayed on the SLM. The FINCH holograms were obtained with input and output polarizers set at 60° with an 800 mm focal length diffractive lens pattern displayed on the SLM. Best focus images were calculated from the holograms.
Mentions: The effect of the fluorescence emission bandwidth on widefield images and those generated by FINCH holography was examined. The emission bandwidth of the Semrock GFP filter set used in this study produced an emission bandwidth from the USAF slide of about ~38 nm (500 nm – 538 nm) FWHM (Fig. 10 BFig. 10

Bottom Line: An important improvement from our previous FINCH configurations capitalizes on the polarization sensitivity of the SLM so that the same SLM pixels which create the spherical wave simulating the microscope tube lens, also pass the plane waves from the infinity corrected microscope objective, so that interference between the two wave types at the camera creates a hologram.This advance dramatically improves the resolution of the FINCH system.Results from imaging a fluorescent USAF pattern and a pollen grain slide reveal resolution which approaches the Rayleigh limit by this simple method for 3D fluorescent microscopic imaging.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Engineering, Johns Hopkins University, 9605 Medical Center Drive, Rockville, Maryland 20850 USA. gbrooker@jhu.edu

ABSTRACT
Fresnel Incoherent Correlation Holography (FINCH) enables holograms and 3D images to be created from incoherent light with just a camera and spatial light modulator (SLM). We previously described its application to microscopic incoherent fluorescence wherein one complex hologram contains all the 3D information in the microscope field, obviating the need for scanning or serial sectioning. We now report experiments which have led to the optimal optical, electro-optic, and computational conditions necessary to produce holograms which yield high quality 3D images from fluorescent microscopic specimens. An important improvement from our previous FINCH configurations capitalizes on the polarization sensitivity of the SLM so that the same SLM pixels which create the spherical wave simulating the microscope tube lens, also pass the plane waves from the infinity corrected microscope objective, so that interference between the two wave types at the camera creates a hologram. This advance dramatically improves the resolution of the FINCH system. Results from imaging a fluorescent USAF pattern and a pollen grain slide reveal resolution which approaches the Rayleigh limit by this simple method for 3D fluorescent microscopic imaging.

Show MeSH
Related in: MedlinePlus