microscopic techniques can visualize objects ranging in size from millimeters to
nanometers.
This chapter addressed the basic functions of confocal microscopes,fluorescence
microscopy, multiphoton microscopy, Raman microscopy, light sheet microscopy,
and super-resolutionfluorescence microscopy. Applications of microscopy instru-
ments and techniques are found in all branches of biomedicine and biophotonics,
for example, analyses of biological samples, brain research, cancer research,
cytology, detection and assessment of diseases, drug discovery, food analyses,
gynecology, healthcare, hematology, and pathology. As such, microscopic tech-
niques are fundamental tools for the fields of spectroscopy and for imaging
modalities, which are the topics of the next two chapters, respectively.
8.10 Problems.
8 :1 Write a simple Java applet to show how the size and angular aperture of
objective light cones change with numerical aperture.
8 :2 Suppose that the half-angle of the light cone captured by an objective lens is
20°. Show that the NA is 0.45, 0.50, and 0.52 for an immersion medium of
water, glycerin, and immersion oil, which have refractive indices of 1.33, 1.47,
and 1.51, respectively.
8 :3 Consider a microscope objective that has NA = 0.60. Show that the half-angle
of the light cone captured by an objective lens with an immersion medium of
water is 26.8°.
8 :4 Consider an objective lens that has a 30X magnification and which is used
with an eyepiece offield number 7. (a) Show that thefield of view is 0.23 mm.
(b) Suppose that only half of an object that is viewed by this eyepiece and
objective combinationfits across the FOV (i.e., only half of the object is seen
in the FOV). Show that the size of the object is 0.46 mm.
8 :5 Consider two objective lenses that have NAs of 0.20 and 0.55, respectively.
Show that the depths offield at a wavelength of 650 nm are 24.5 and 3.37μm,
respectively, if the immersion material is oil with an index n = 1.51.
8 :6 Consider two microscope setups being used at a 480 nm wavelength. Suppose
for one setup NAobj1= 0.80 and for the other setup NAobj2= 1.25. (a) Show
that the resolution is 366 and 234 nm, respectively. (b) How do these reso-
lution values compare with a test setup at 700 nm?
8 :7 The key feature of a confocal microscope is the use of image-forming con-
jugate planes, which are paired sets of in-focus planes. Using reference texts or
Web resources, use about one page to describe the operating principle of
conjugate planes and show in a diagram where these sets of planes are located
in a confocal microscope.
8 :8 Consider the optical fiber-based confocal microscope system shown in
Fig.8.5. Here the light from a confocal pinhole aperture is collimated and sent
through a beam splitting mirror to a xy laser scanning mechanism. This
256 8 Microscopy