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Mie light scattering calculations for an Indian age-related nuclear cataract with a high density of multilamellar bodies.

Gilliland KO, Johnsen S, Metlapally S, Costello MJ, Ramamurthy B, Krishna PV, Balasubramanian D - Mol. Vis. (2008)

Bottom Line: Multilamellar bodies (MLBs) are lipid-coated spheres (1-4 microm in diameter) found with greater frequency in the nuclear region of human age-related cataracts compared with human transparent lenses.Pairwise distribution analysis has revealed that MLBs even in this exceptional case are found with a distribution that appears to be random.Based on the extremely high frequency, as well as their spherical shape, large size, and apparent random distribution, the MLBs are predicted according to Mie light scattering calculations to cause high amounts of forward scattering sufficient to produce nuclear opacity.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Developmental Biology, University of North Carolina, Chapel Hill, NC 27599, USA.

ABSTRACT

Purpose: Multilamellar bodies (MLBs) are lipid-coated spheres (1-4 microm in diameter) found with greater frequency in the nuclear region of human age-related cataracts compared with human transparent lenses. Mie light scattering calculations have demonstrated that MLBs are potential sources of forward light scattering in human age-related nuclear cataracts due to their shape, size, frequency, and cytoplasmic contents, which often differ in refractive index from their surroundings. Previous studies have used data from several non-serial tissue sections viewed by light microscopy to extrapolate a volume and have assumed that MLBs are random in distribution. Currently, confocal microscopy is being used to examine actual tissue volumes from age-related nuclear cataracts and transparent lenses collected in India to confirm MLB shape, size, frequency, and randomness. These data allow Mie scattering calculations to be done with directly observed MLBs in intact tissue.

Methods: Whole Indian donor lenses and Indian lens nuclei after extracapsular cataract extraction were immersion-fixed in 10% formalin for 24 h and in 4% paraformaldehyde for 24 h before sectioning with a Vibratome. The 160 microm thick sections were stained for 24 h in the lipid dye DiI (1,1'-dilinoleyl-3,3,3',3' tetramethylindocarbocyanine, 4-chlorobenzenesulfonate), washed, stabilized in Permount under coverslips and examined with a Zeiss LSM 510 confocal microscope. Individual volumes of tissue (each typically 500,000 microm(3)) were examined using a plan-apochromat 63X oil (NA=1.4) lens. Other lenses were prepared for electron microscopy and histological examination using previously described procedures.

Results: Analysis of tissue volumes within Indian age-related nuclear cataracts and transparent lenses has confirmed that most MLBs are 1-4 microm in diameter and typically spherical with some occurring as doublets or in clusters. Most Indian cataracts and transparent lenses are similar to samples obtained in the United States. One cataract contained as many as 400,000 MLBs per mm(3) -100 times more than in cataracts collected in the United States. Pairwise distribution analysis has revealed that MLBs even in this exceptional case are found with a distribution that appears to be random. Mie calculations indicate that more than 90% of the incident light could be scattered by the high density of MLBs.

Conclusions: An important finding was that one advanced Indian cataract contained many more MLBs than cataracts examined from India and previously from the United States. This indicates that specific conditions or susceptibilities may exist that promote the formation of excessive MLBs. Based on the extremely high frequency, as well as their spherical shape, large size, and apparent random distribution, the MLBs are predicted according to Mie light scattering calculations to cause high amounts of forward scattering sufficient to produce nuclear opacity.

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Distribution of MLBs. A: An example of one of the many volumes examined is shown. This particular volume from an age-related nuclear cataract is 73 µm (x) by 73 µm (y) by 16 µm (z) for a total volume of approximately 85,000 µm3. The z distance of 16 µm resulted from the stacking of 40 optical sections of 0.4 µm each. MLBs appear as bright spots as their lipid coatings are labeled well by the lipophilic DiI. B: To facilitate the viewing of MLBs, a sphere has been placed manually over each MLB with the aid of thresholding to locate most of the MLBs. C: With the membranes removed, it can be seen that 64 MLBs are located within this volume of 85,000 µm3 – a frequency of 753,000 MLBs per mm3.
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f6: Distribution of MLBs. A: An example of one of the many volumes examined is shown. This particular volume from an age-related nuclear cataract is 73 µm (x) by 73 µm (y) by 16 µm (z) for a total volume of approximately 85,000 µm3. The z distance of 16 µm resulted from the stacking of 40 optical sections of 0.4 µm each. MLBs appear as bright spots as their lipid coatings are labeled well by the lipophilic DiI. B: To facilitate the viewing of MLBs, a sphere has been placed manually over each MLB with the aid of thresholding to locate most of the MLBs. C: With the membranes removed, it can be seen that 64 MLBs are located within this volume of 85,000 µm3 – a frequency of 753,000 MLBs per mm3.

Mentions: While many Indian age-related cataracts contained MLBs with the same frequency as those previously analyzed in the United States [32,33], the one Indian lens examined with an exceptionally high number of MLBs is emphasized here. The MLBs within a volume similar to that described above to establish the random distribution mathematically can be used to directly visualize the MLB distribution. The MLBs appear as bright spots because their lipid coatings, which are labeled by the lipophilic dye, DiI, are displayed in a reconstructed volume of approximately 85,000 µm3 (Figure 6A). To facilitate the viewing and counting of MLBs, a sphere has been placed over each MLB (Figure 6B). After the membranes are removed by thresholding, it can be seen that 64 MLBs are located within this volume of 85,000 µm3 – a frequency of 753,000 MLBs per mm3. This frequency, while exceptional even among Indian cataracts, is much greater than that of the most advanced cataract analyzed in the United States.


Mie light scattering calculations for an Indian age-related nuclear cataract with a high density of multilamellar bodies.

Gilliland KO, Johnsen S, Metlapally S, Costello MJ, Ramamurthy B, Krishna PV, Balasubramanian D - Mol. Vis. (2008)

Distribution of MLBs. A: An example of one of the many volumes examined is shown. This particular volume from an age-related nuclear cataract is 73 µm (x) by 73 µm (y) by 16 µm (z) for a total volume of approximately 85,000 µm3. The z distance of 16 µm resulted from the stacking of 40 optical sections of 0.4 µm each. MLBs appear as bright spots as their lipid coatings are labeled well by the lipophilic DiI. B: To facilitate the viewing of MLBs, a sphere has been placed manually over each MLB with the aid of thresholding to locate most of the MLBs. C: With the membranes removed, it can be seen that 64 MLBs are located within this volume of 85,000 µm3 – a frequency of 753,000 MLBs per mm3.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2275208&req=5

f6: Distribution of MLBs. A: An example of one of the many volumes examined is shown. This particular volume from an age-related nuclear cataract is 73 µm (x) by 73 µm (y) by 16 µm (z) for a total volume of approximately 85,000 µm3. The z distance of 16 µm resulted from the stacking of 40 optical sections of 0.4 µm each. MLBs appear as bright spots as their lipid coatings are labeled well by the lipophilic DiI. B: To facilitate the viewing of MLBs, a sphere has been placed manually over each MLB with the aid of thresholding to locate most of the MLBs. C: With the membranes removed, it can be seen that 64 MLBs are located within this volume of 85,000 µm3 – a frequency of 753,000 MLBs per mm3.
Mentions: While many Indian age-related cataracts contained MLBs with the same frequency as those previously analyzed in the United States [32,33], the one Indian lens examined with an exceptionally high number of MLBs is emphasized here. The MLBs within a volume similar to that described above to establish the random distribution mathematically can be used to directly visualize the MLB distribution. The MLBs appear as bright spots because their lipid coatings, which are labeled by the lipophilic dye, DiI, are displayed in a reconstructed volume of approximately 85,000 µm3 (Figure 6A). To facilitate the viewing and counting of MLBs, a sphere has been placed over each MLB (Figure 6B). After the membranes are removed by thresholding, it can be seen that 64 MLBs are located within this volume of 85,000 µm3 – a frequency of 753,000 MLBs per mm3. This frequency, while exceptional even among Indian cataracts, is much greater than that of the most advanced cataract analyzed in the United States.

Bottom Line: Multilamellar bodies (MLBs) are lipid-coated spheres (1-4 microm in diameter) found with greater frequency in the nuclear region of human age-related cataracts compared with human transparent lenses.Pairwise distribution analysis has revealed that MLBs even in this exceptional case are found with a distribution that appears to be random.Based on the extremely high frequency, as well as their spherical shape, large size, and apparent random distribution, the MLBs are predicted according to Mie light scattering calculations to cause high amounts of forward scattering sufficient to produce nuclear opacity.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Developmental Biology, University of North Carolina, Chapel Hill, NC 27599, USA.

ABSTRACT

Purpose: Multilamellar bodies (MLBs) are lipid-coated spheres (1-4 microm in diameter) found with greater frequency in the nuclear region of human age-related cataracts compared with human transparent lenses. Mie light scattering calculations have demonstrated that MLBs are potential sources of forward light scattering in human age-related nuclear cataracts due to their shape, size, frequency, and cytoplasmic contents, which often differ in refractive index from their surroundings. Previous studies have used data from several non-serial tissue sections viewed by light microscopy to extrapolate a volume and have assumed that MLBs are random in distribution. Currently, confocal microscopy is being used to examine actual tissue volumes from age-related nuclear cataracts and transparent lenses collected in India to confirm MLB shape, size, frequency, and randomness. These data allow Mie scattering calculations to be done with directly observed MLBs in intact tissue.

Methods: Whole Indian donor lenses and Indian lens nuclei after extracapsular cataract extraction were immersion-fixed in 10% formalin for 24 h and in 4% paraformaldehyde for 24 h before sectioning with a Vibratome. The 160 microm thick sections were stained for 24 h in the lipid dye DiI (1,1'-dilinoleyl-3,3,3',3' tetramethylindocarbocyanine, 4-chlorobenzenesulfonate), washed, stabilized in Permount under coverslips and examined with a Zeiss LSM 510 confocal microscope. Individual volumes of tissue (each typically 500,000 microm(3)) were examined using a plan-apochromat 63X oil (NA=1.4) lens. Other lenses were prepared for electron microscopy and histological examination using previously described procedures.

Results: Analysis of tissue volumes within Indian age-related nuclear cataracts and transparent lenses has confirmed that most MLBs are 1-4 microm in diameter and typically spherical with some occurring as doublets or in clusters. Most Indian cataracts and transparent lenses are similar to samples obtained in the United States. One cataract contained as many as 400,000 MLBs per mm(3) -100 times more than in cataracts collected in the United States. Pairwise distribution analysis has revealed that MLBs even in this exceptional case are found with a distribution that appears to be random. Mie calculations indicate that more than 90% of the incident light could be scattered by the high density of MLBs.

Conclusions: An important finding was that one advanced Indian cataract contained many more MLBs than cataracts examined from India and previously from the United States. This indicates that specific conditions or susceptibilities may exist that promote the formation of excessive MLBs. Based on the extremely high frequency, as well as their spherical shape, large size, and apparent random distribution, the MLBs are predicted according to Mie light scattering calculations to cause high amounts of forward scattering sufficient to produce nuclear opacity.

Show MeSH
Related in: MedlinePlus