Page 197 - Fundamentals of Light Microscopy and Electronic Imaging

180 FLUORESCENCE MICROSCOPY

the basis for fluorescence microscopy, so we will take a moment to examine the physi-
cal basis of the phenomenon.
When a fluorescent molecule absorbs a photon of the appropriate wavelength, an
electron is excited to a higher energy state and almost immediately collapses back to its
initial ground state. In the process of energy collapse the molecule can release the
absorbed energy as a fluorescent photon. Since some energy is lost in the process, the
emitted fluorescent photon typically exhibits a lower frequency of vibration and a
longer wavelength than the excitatory photon that was absorbed. The situation is
depicted graphically in what is known as a Jablonski diagram (Fig. 11-2), which shows
a series of increasing energy states as a stack of horizontal lines. Each energy level is in

Excited
singlet
states

Excited
states

Intersystem
crossing
Absorption, 10 –15 sec Fluorescence, 10 –9 – 10 –12 sec Internal conversion Internal conversion Excited triplet
Phosphorescence, 10 2 – 10 –2 sec
state

Ground
state

Figure 11-2
Jablonski diagram showing energy levels occupied by an excited electron within a
fluorescent molecule (chlorophyll a). Chlorophyll a is unique in absorbing blue and red
wavelengths of the visual spectrum. Blue photons are excited to a higher energy level than
are red ones (straight upward arrows, left), but the collapse to the ground state by an
electron excited by either wavelength can occur through any of the following three pathways:
Chlorophyll can give off a photon (fluorescence emission, straight downward pointing arrow);
it can release vibrational energy as heat without photon emission (internal conversion, wavy
downward pointing arrows); or its electron can enter an excited triplet state (intersystem
crossing, dotted downward arrow), which can make the molecule chemically reactive.
Electrons in the triplet excited state can return to the ground state through internal
conversion or by emission of phosphorescence. Refer to the text for details.