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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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Best plane of focus reconstruction from holograms of the fluorescent USAF test slide using the constant phase mask technique and the polarizers method. (a) Static mask. (b) Input and output polarizers at 60 degrees. Olympus 20 × 0.75 NA objective. Bars labeled “a” are 1.6 microns thick and there is 2.5 microns distance between each of the three bars. Full bin 1 camera field of view, 300 microns square.
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g006: Best plane of focus reconstruction from holograms of the fluorescent USAF test slide using the constant phase mask technique and the polarizers method. (a) Static mask. (b) Input and output polarizers at 60 degrees. Olympus 20 × 0.75 NA objective. Bars labeled “a” are 1.6 microns thick and there is 2.5 microns distance between each of the three bars. Full bin 1 camera field of view, 300 microns square.

Mentions: The optimal conditions for imaging the fluorescent USAF slide with both the constant phase mask method and the polarization method were compared. The results demonstrate the superiority of the polarization method. Figure 6Fig. 6


Optimal resolution in Fresnel incoherent correlation holographic fluorescence microscopy.

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

Best plane of focus reconstruction from holograms of the fluorescent USAF test slide using the constant phase mask technique and the polarizers method. (a) Static mask. (b) Input and output polarizers at 60 degrees. Olympus 20 × 0.75 NA objective. Bars labeled “a” are 1.6 microns thick and there is 2.5 microns distance between each of the three bars. Full bin 1 camera field of view, 300 microns square.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

g006: Best plane of focus reconstruction from holograms of the fluorescent USAF test slide using the constant phase mask technique and the polarizers method. (a) Static mask. (b) Input and output polarizers at 60 degrees. Olympus 20 × 0.75 NA objective. Bars labeled “a” are 1.6 microns thick and there is 2.5 microns distance between each of the three bars. Full bin 1 camera field of view, 300 microns square.
Mentions: The optimal conditions for imaging the fluorescent USAF slide with both the constant phase mask method and the polarization method were compared. The results demonstrate the superiority of the polarization method. Figure 6Fig. 6

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