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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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Microscope configuration for SLM testing and alignment. A Coherent DPSS 532 nm or Thorlabs 633 nm laser passed through a Glan-Thompson polarizer and 20 × beam expander. The expanded laser beam was confirmed to be coherent and collimated with a shearing plate interferometer. The beam was directed to the microscope through a beam splitting cube mounted on the microscope turret which allowed the expanded laser beam to enter the microscope, reflect off the SLM and be directed to the camera or in some cases a power meter. Moving the turret to another position with a microscope objective made it possible to first obtain precision alignment of the microscope using the lasers and then to switch to imaging mode with objectives. The distance between the camera and SLM (zh) was varied by moving the camera along a precision track to confirm the focal lengths and characteristics of diffractive lens patterns.
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g002: Microscope configuration for SLM testing and alignment. A Coherent DPSS 532 nm or Thorlabs 633 nm laser passed through a Glan-Thompson polarizer and 20 × beam expander. The expanded laser beam was confirmed to be coherent and collimated with a shearing plate interferometer. The beam was directed to the microscope through a beam splitting cube mounted on the microscope turret which allowed the expanded laser beam to enter the microscope, reflect off the SLM and be directed to the camera or in some cases a power meter. Moving the turret to another position with a microscope objective made it possible to first obtain precision alignment of the microscope using the lasers and then to switch to imaging mode with objectives. The distance between the camera and SLM (zh) was varied by moving the camera along a precision track to confirm the focal lengths and characteristics of diffractive lens patterns.

Mentions: The laser beams were directed into the microscope through a beam splitting cube attached to the microscope turret as shown in Fig. 2Fig. 2


Optimal resolution in Fresnel incoherent correlation holographic fluorescence microscopy.

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

Microscope configuration for SLM testing and alignment. A Coherent DPSS 532 nm or Thorlabs 633 nm laser passed through a Glan-Thompson polarizer and 20 × beam expander. The expanded laser beam was confirmed to be coherent and collimated with a shearing plate interferometer. The beam was directed to the microscope through a beam splitting cube mounted on the microscope turret which allowed the expanded laser beam to enter the microscope, reflect off the SLM and be directed to the camera or in some cases a power meter. Moving the turret to another position with a microscope objective made it possible to first obtain precision alignment of the microscope using the lasers and then to switch to imaging mode with objectives. The distance between the camera and SLM (zh) was varied by moving the camera along a precision track to confirm the focal lengths and characteristics of diffractive lens patterns.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

g002: Microscope configuration for SLM testing and alignment. A Coherent DPSS 532 nm or Thorlabs 633 nm laser passed through a Glan-Thompson polarizer and 20 × beam expander. The expanded laser beam was confirmed to be coherent and collimated with a shearing plate interferometer. The beam was directed to the microscope through a beam splitting cube mounted on the microscope turret which allowed the expanded laser beam to enter the microscope, reflect off the SLM and be directed to the camera or in some cases a power meter. Moving the turret to another position with a microscope objective made it possible to first obtain precision alignment of the microscope using the lasers and then to switch to imaging mode with objectives. The distance between the camera and SLM (zh) was varied by moving the camera along a precision track to confirm the focal lengths and characteristics of diffractive lens patterns.
Mentions: The laser beams were directed into the microscope through a beam splitting cube attached to the microscope turret as shown in Fig. 2Fig. 2

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