Polarization Microscopy - Biomedical Optical Phase Microscopy and Nanoscopy - page 318

(A) (B) (C) (D)

60

62

61

62

56

1.3

69

nm

461 nm 2
62 MTs

0

0 µ m 3.5

Figure 15.9
Meiosis I spindle in a spermatocyte of the crane fly Nephrotoma suturalis imaged with the LC-
PolScope. (A) and (B) are two sections (19 and 23 out of 43) from a series of optical sections
made through the cell at focus steps of 0.26 m. Each section shows chromosomes as areas of

reduced birefringence and the high birefringence of the dense arrays of MTs forming the
kinetochore fibers. In addition, the birefringence of all the parallel spindle MTs superimposes on
the birefringence of the k-fibers, more than doubling the measured k-fiber retardance. In viewing
these LC-PolScope images, brightness represents the retardance (black

μ

0 nm and white 2.5 nm

5 5

m. (C) is a
duplicate image of (B) and includes a rectangle that indicates the location of the image data that
were used to generate the retardance plot in the lower half of the panel. The shaded area in the
plot is the retardance associated with the k-fiber, which was further evaluated for the number of
kinetochore MTs. (D) The metaphase positions of the three bivalent chromosomes in this cell
upon projection of all images within the Z-focus series to make a 2D profile. Two dots indicate
the positions of the flagellar basal bodies within the centrosomes at the two spindle poles.
The numbers on each kinetochore fiber indicate the number of kinetochore MTs in that fiber,
based on the retardance analysis. Source: Reproduced from Ref. [30] .

retardance), irrespective of the orientation of the slow axis. The bar in panel A is 5 μ

as explained in the caption to Figure 15.9 , the retardance measured in areas adjacent to the
k-fiber can be used to estimate the spindle retardance at the location of the k-fiber and
subtract its contribution from that of the k-fiber [30] .

In the previous example, simple subtraction of the spindle retardance leads to a correct
estimate of the k-fiber retardance because the slow axes of the k-fiber and spindle
birefringence are co-aligned. The superposition of birefringent structures whose slow axes
are not parallel to each other is complex and their individual retardance values do not
simply add. The effect of one birefringent structure, whose image overlaps with that of
another birefringent structure, depends on their mutual orientation. So far, we lack a reliable
method to unambiguously identify the individual contributions of birefringent structures that
are separated along the optical axis of the microscope.