TY - JOUR

T1 - Dispersion, aberration and deconvolution in multi-wavelength fluorescence images

AU - Scalettar, B. A.

AU - Swedlow, J. R.

AU - Sedat, J. W.

AU - Agard, D. A.

N1 - Publisher Copyright:
© 1996 The Royal Microscopical Society.

PY - 1996/4

Y1 - 1996/4

N2 - The wavelength dependence of the incoherent point spread function in a wide-field microscope was investigated experimentally. Dispersion in the sample and optics can lead to significant changes in the point spread function as wavelength is varied over the range commonly used in fluorescence microscopy. For a given sample, optical conditions can generally be optimized to produce a point spread function largely free of spherical aberration at a given wavelength. Unfortunately, deviations in wavelength from this value will result in spherically aberrated point spread functions. Therefore, when multiple fluorophores are used to localize different components in the same sample, the image of the distribution of at least one of the fluorophores will be spherically aberrated. This aberration causes a loss of intensity and resolution, thereby complicating the localization and analysis of multiple components in a multiwavelength image. We show that optimal resolution can be restored to a spherically aberrated image by constrained, iterative deconvolution, as long as the spherical aberration in the point spread function used for deconvolution matches the aberration in the image reasonably well. The success of this method is essentially independent of the initial degree of spherical aberration in the image. Deconvolution of many biological images can be achieved by collecting a small library of spherically aberrated and unaberrated point spread functions, and then choosing a point spread function appropriate for deconvolving each image. The co-localization and relative intensities of multiple components can then be accurately studied in a multi-wavelength image.

AB - The wavelength dependence of the incoherent point spread function in a wide-field microscope was investigated experimentally. Dispersion in the sample and optics can lead to significant changes in the point spread function as wavelength is varied over the range commonly used in fluorescence microscopy. For a given sample, optical conditions can generally be optimized to produce a point spread function largely free of spherical aberration at a given wavelength. Unfortunately, deviations in wavelength from this value will result in spherically aberrated point spread functions. Therefore, when multiple fluorophores are used to localize different components in the same sample, the image of the distribution of at least one of the fluorophores will be spherically aberrated. This aberration causes a loss of intensity and resolution, thereby complicating the localization and analysis of multiple components in a multiwavelength image. We show that optimal resolution can be restored to a spherically aberrated image by constrained, iterative deconvolution, as long as the spherical aberration in the point spread function used for deconvolution matches the aberration in the image reasonably well. The success of this method is essentially independent of the initial degree of spherical aberration in the image. Deconvolution of many biological images can be achieved by collecting a small library of spherically aberrated and unaberrated point spread functions, and then choosing a point spread function appropriate for deconvolving each image. The co-localization and relative intensities of multiple components can then be accurately studied in a multi-wavelength image.

KW - Dispersion

KW - Fluorescence microscopy

KW - Iterative deconvolution

KW - Point-spread function

KW - Spherical aberration

UR - http://www.scopus.com/inward/record.url?scp=0002378571&partnerID=8YFLogxK

U2 - 10.1046/j.1365-2818.1996.122402.x

DO - 10.1046/j.1365-2818.1996.122402.x

M3 - Article

C2 - 8632447

AN - SCOPUS:0002378571

VL - 182

SP - 50

EP - 60

JO - Journal of Microscopy

JF - Journal of Microscopy

SN - 0022-2720

IS - 1

ER -