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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 using a constant phase mask (a) versus the polarization method (b) to select and separate the plane and spherical waves in FINCH holography. Notice that when the polarization method is used, all the pixels on the SLM are used to create the diffractive lens pattern.
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g004: Comparison of using a constant phase mask (a) versus the polarization method (b) to select and separate the plane and spherical waves in FINCH holography. Notice that when the polarization method is used, all the pixels on the SLM are used to create the diffractive lens pattern.

Mentions: FINCH creates holograms in a single beam system as a result of interference between a plane wave and a spherical wave originating from every object point. In our previous reports we created a random constant phase mask so that with a phase-only SLM, the plane wave from an infinity corrected microscope objective could be directed to the camera along with the spherical wave created by the SLM. The use of a constant phase mask presents certain disadvantages in that it requires half the pixels on the SLM and also degrades the resolution of the mask which creates the spherical wave. Because only one linear polarization state on the liquid crystal based SLM can change the phase of incoming light, half of the randomly polarized fluorescent light striking the device can have quadratic phase modulation whereas the other half is shifted by a constant phase, as shown in Fig. 4(a)Fig. 4


Optimal resolution in Fresnel incoherent correlation holographic fluorescence microscopy.

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

Comparison of using a constant phase mask (a) versus the polarization method (b) to select and separate the plane and spherical waves in FINCH holography. Notice that when the polarization method is used, all the pixels on the SLM are used to create the diffractive lens pattern.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

g004: Comparison of using a constant phase mask (a) versus the polarization method (b) to select and separate the plane and spherical waves in FINCH holography. Notice that when the polarization method is used, all the pixels on the SLM are used to create the diffractive lens pattern.
Mentions: FINCH creates holograms in a single beam system as a result of interference between a plane wave and a spherical wave originating from every object point. In our previous reports we created a random constant phase mask so that with a phase-only SLM, the plane wave from an infinity corrected microscope objective could be directed to the camera along with the spherical wave created by the SLM. The use of a constant phase mask presents certain disadvantages in that it requires half the pixels on the SLM and also degrades the resolution of the mask which creates the spherical wave. Because only one linear polarization state on the liquid crystal based SLM can change the phase of incoming light, half of the randomly polarized fluorescent light striking the device can have quadratic phase modulation whereas the other half is shifted by a constant phase, as shown in Fig. 4(a)Fig. 4

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