Order Fluorescence Microscopy Discussion

Order Fluorescence Microscopy Discussion
7.1 In the inset of Figure 7.1 the characteristic dimensions of a confocal detection volume are defined by the axial and lateral resolution, z0 and r0,
respectively. What are the most important factors that determine the magnitude of these two parameters?
7.2 What is the diffraction-limited resolution of a microscope objective with a
numerical aperture of 1.2 when using light of 400, 600 and 800 nm? How
does the resolution change when using a microscope objective with a
numerical aperture of 1.0 or 1.33? What are the collection efficiencies of
microscope objectives with a numerical aperture of 1.0, 1.2 or 1.33?
Assume that a water immersion objective is used and the refractive index
of water is 1.33.
7.3 Explain why different angles of collimated beams entering or exiting the
back aperture of a microscope objective (Figure 7.5) correspond to a lateral
shift of the excitation or emission focus, respectively. Describe the microscope objective as a single lens and use ray optics for your explanation.
Discuss the beam path for both excitation and emission. In what direction
does the focus shift if the excitation beam is not collimated but is slightly
divergent or focused?
Order Fluorescence Microscopy Discussion
7.4 Explain why slightly focusing the excitation beam for wide-field fluorescence microscopy allows illumination of a larger area in the focal region
but still enables us to detect the fluorescing object with diffraction-limited
resolution.
7.5 In a distinct TIRF microscope an excitation wavelength of λ ∼ 488 nm is
used and the angle of the reflected excitation light with the reflecting surface is α=20°. Calculate the distance from the surface by which the intensity of the evanescent field drops to 50% when assuming for the refractive
indices of glass and water n1 ∼ 1.52 and n2=1.33, respectively.
7.6 In light-sheet microscopy there exists a trade-off between the size of the
field-of-view in which the sheet’s thickness remains sufficiently uniform
and the minimal central thickness of the sheet. Calculate the central thickness of the light sheet when a lense with a numerical aperture of 0.1 and a
focal length of f ∼ 30 mm, an excitation wavelength of λ ∼ 532 nm and a