Flat-top illumination profile in an epi-fluorescence microscope by dual micro lens arrays

Type:
Peer-reviewed article
Authors: 
F.A.W. Coumans, E. van der Pol, and L.W.M.M. Terstappen
Date:
Published March 5, 2012
Journal:
Cytometry Part A
Volume:
81
Issue:
4
Pagination:
324-31
DOI:
10.1002/cyto.a.22029
Keywords:
Microlens array, coefficient of variation, epifluorescence microscopy, homogeneous illumination profile, beam shaping, flat top
Attachment:
Coumans 2012 Cytometry A Flat-top illumination.pdf (1,859 kB)
Supplements: 
Model for double MLA in mercury based 10x epi fluorescence microscope:
Coumans 2012 Cytometry A Flat-top illumination Supp1.pdf (384 kB)
MATLAB m-file:
Coumans 2012 Cytometry A Flat-top illumination Supp2.pdf (147 kB)
Guideline for modifying an epi fluorescence microscope with double MLA:
Coumans 2012 Cytometry A Flat-top illumination Supp3.pdf (370 kB)
Raw data files flow cytometer:
Coumans 2012 Cytometry A Flat-top illumination Supp4.txt (83 kB)

Abstract

Low uniformity in illumination across the image plane impairs the ability of a traditional epifluorescence microscope to quantify fluorescence intensities. Two microlens arrays (MLAs) were introduced into the illumination path of two different epifluorescence microscope systems to improve the uniformity of the illumination. Measurements of the uniformity of illumination were performed with a CCD camera in the focal plane and with fluorescent beads in the image plane. In semi critical alignment, a uniformity of illumination of 15–23% was found compared with 1–2% in the modified system. Coefficient of variation (CV) of fluorescent beads measured on the unmodified system was 20.4% ± 5.3% in semi critical alignment and 10.8% ± 1.3% in Koehler alignment. On the MLA systems, CV was 7.9% ± 2.0% and on a flow cytometer, the CV was 6.7% ± 0.7%. Implementation of MLAs in an epifluorescence microscope improves the uniformity of illumination, thereby reducing the variation in detection of fluorescent signals of the measured objects and becomes equivalent to that of flow cytometry.

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