Page 336 - Fundamentals of Light Microscopy and Electronic Imaging

ANSWER KEY TO PROBLEM SETS 319

Chapter 11

(1) Rhodamine bleeds through the long-pass filter set intended for fluorescein. (2) Rho-
damine is stimulated by the 490 nm excitation wavelength for fluorescein, and since the
long-pass emission filter transmits red light, the fluorescence emission of rhodamine is
also transmitted by this set. (3) The fluorescein set with narrow bandpass filter is not
completely effective in transmitting fluorescein-specific fluorescence. Rhodamine is
weakly excited by the band of wavelengths at 490 nm used to excite fluorescein and its
emission spectrum overlaps the region of peak fluorescence by fluorescein. It is
unavoidable that some rhodamine fluorescence is transmitted by the fluorescein-narrow
set. (4) A fluorescein set with long-pass emission filter is useful when fluorescein is the
only label, fluorescence emission is weak, and it is desirable to transmit the maximum
amount of emitted fluorescent light. (5) To reduce bleed-through, reduce the amount of
rhodamine label or use an illuminator such as a xenon arc lamp that does not stimulate
rhodamine 10 times the amount it does fluorescein (as is the case for a mercury arc
lamp).

Chapter 12

(1) Removing a neutral density filter or increasing laser power provides more fluores-
cent light, allowing one to reduce the gain setting or scan at a faster rate; opening the
pinhole diaphragm increases the thickness of the optical section in the specimen;
increasing the gain on the PMT makes the image look grainy; decreasing the offset on
the PMT can make the image look flat and washed out; reducing the scan speed
increases the rate of photobleaching; increasing the zoom factor improves spatial reso-
lution but increases the rate of photobleaching. (2) The relationship between pinhole
size and the thickness of an optical section is nonlinear, being greater at small pin-
hole diameters. (3) Generally, the phenomenon of bleed-through is asymmetric—long-
wavelength fluorochromes bleed more into filter sets for short-wavelength fluo-
rochromes than occurs the other way around. This is because excitation of short-
wavelength fluorochromes also excites longer-wavelength fluorochromes, and since
fluorescence emission spectra begin at the excitation wavelength, some of the short-
wavelength fluorescence emission of the longer-wavelength fluorochrome can pass
through the filter set for the short-wavelength fluorochrome.

Chapter 13

(1) Video is suitable for time-lapse sequences lasting several minutes to hours. Mem-
brane ruffling, organelle movements, cell migration, and mitosis are ideally suited for
video time-lapse recording. (2) The horizontal cutoff frequency is calculated as f
H
M objective M relay N /1.2D, where M represents the magnification of the objective and
H
relay lens, N is the lines of horizontal or vertical resolution at N 800, and D is the
H
diagonal measurement of the target in the video electron tube in mm. For a D of 15.875