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3.6  Basic Fluorescence Microscopy Illumination Modes

3.6  BASIC FLUORESCENCE MICROSCOPY ILLUMINATION MODES

There are several fluorescence microscopy methods available that allow fluorophores in

labeled biomolecules to be excited and detected. These not only include camera-​imaging

methods of wide-​field illumination modes comprising approaches such as epifluorescence

and oblique epifluorescence, as well as narrower illumination modes such as Slimfield and

narrow-​field, used normally in combination with a high-​quantum-​efficiency EMCCD

camera detector, but also include spectroscopic approaches such as fluorescence correlation

spectroscopy and scanning confocal microscopy.

3.6.1  WIDE-​FIELD MODES OF EPIFLUORESCENCE AND OBLIQUE

EPIFLUORESCENCE

Wide-​field microscopy is so called because it excites a laterally “wide” field of view of the

sample (Figure 3.5a). Epifluorescence is the most standard form of fluorescence microscopy

illumination and involves focusing a light beam into the back focal plane of an objective lens

centered on its optical axis. This generates an excitation field that is uniform with height z

into the sample from the microscope slide/​coverslip surface, but with a radially symmetrical

2D Gaussian profile in the xy lateral plane. The full width at half maximum of this intensity

field in xy is 30–​60 μm. Epi refers to excitation and emission light being routed through the

same objective lens in opposite directions. Trans fluorescence illumination, in which the

excitation and emission beam travel in the same direction, is possible but not used in prac­

tice since the amount of unblocked excitation light entering the objective lens is significantly

higher, thus reducing the contrast.

A laser beam can be tightly focused into the back aperture of an objective lens, unlike spa­

tially extended sources such as arc lamps or LEDs, and so there is a room to translate the focus

laterally away from the optic axis, allowing the angle of incidence to be adjusted. Beyond the

critical angle, TIRF excitation is generated. However, an angle of incidence between zero and

the critical angle results in oblique epifluorescence (Figure 3.5b), also known as variable-​angle

epifluorescence, oblique epi illumination, pseudo-​TIRF, quasi-​TIRF, near-​TIRF, leaky TIRF,

and highly inclined and laminated optical sheet illumination (HILO) (see Tokunaga et al.,

2008). Oblique epifluorescence results in uniform excitation intensity parallel to the excita­

tion field wave vector but has lower back scatter from cellular samples and from the surface

of the microscope slide/​coverslip, which can increase the contrast compared to standard epi­

fluorescence by almost an order of magnitude.

3.6.2  TOTAL INTERNAL REFLECTION FLUORESCENCE

TIRF microscopy (for a comprehensive discussion, see Axelrod et al., 1984) generates wide-​

field excitation laterally in the focal plane of a few tens of microns diameter but utilizes a near-​

field effect (i.e., an optical phenomenon over a length scale of less than a few wavelengths of

light) parallel to the optic axis of the microscope objective lens to generate a fluorescence

excitation field very close to a glass microscope slide/​coverslip of ~100 nm characteristic

depth (Figure 3.5c). TIRF is an enormously powerful and common biophysical technique,

and so we cover several technical aspects of its use here. TIRF usually utilizes laser excita­

tion, such that a beam of wavelength λ is directed at an oblique supercritical angle θ to the

interface between a glass microscope coverslip and a water-​based solution surrounding a bio­

logical sample. Total internal reflection of the incidence beam occurs at angles of incidence,

which are greater than a critical angle θc:

(3.37)

θc

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