Chapter 3 – Making Light Work in Biology  101

some mobility, for example, diffusion coefficients in the range 1−2 × 10⁻³ μm ²

s⁻¹ have been measured, so, for example, during the ~20 ms “binding site off”

traversal time, a DNA-​bound LacI might have a root mean squared displacement

of up to ~90 nm, which is then roughly a half of the confocal volume focal waist

radius—​so knowing whether a molecule is bound or not is not a trivial task (see

Chapter 7 for analytical methods to assist here).

3.6.5  ENVIRONMENTAL FLUORESCENCE MICROSCOPY

Fluorescence microscopy can be adapted to provide information about the local physical and

chemical environment in the vicinity of a fluorescent dye label. For example, confocal micros­

copy can be utilized to probe the intracellular environment by precise measurement of the life­

time of a fluorophore attached to a specific biological molecule using a technique called FLIM,

which is spatially dependent upon physical and chemical parameters such as the local pH and

the concentration of certain ions, especially chloride (Cl⁻). The lifetime of the excited fluores­

cence state typically varies in the range 10⁻⁹ to 10⁻⁷s and can be measured by synchronizing

the fluorescence detection to the excitation using a rapidly pulsed laser at least tens of mega­

hertz frequency. Usual confocal-​type imaging is applied with lateral xy scanning of the sample

through the confocal volume resulting in a 2D map of fluorescence lifetimes across the extent of

the cell, unlike conventional fluorescence imaging that generates maps of fluorescence intensity.

Confocal microscopy allows images at different values of height z to be generated, thus in prin­

ciple enabling the fluorescence lifetime volume in a cell to be reconstructed. The same principle

can also be used applying multiphoton excitation to further improve the spatial resolution.

The fluorescence lifetime can be expressed as the reciprocal of the sum of all the rate

constants for all the different fluorescence decay processes present. It can be measured using

either a frequency or time domain method. For the time-​domain method, a high-​bandwidth

detector such as an APD or PMT is used to perform time-​correlated single-​photon counting

(TCSPC) to count the arrival time of a photon after an initial laser excitation pulse. To

improve sampling speed, multiple detection (usually in the range 16–​64) can be employed.

The arrival times are modeled as a Poisson distribution, and after this process is repeated

several times, sufficient statistics are acquired to estimate the fluorescence lifetime from

binned arrival time histograms using an exponential fit. Some older FLIM equipment still in

operation use instead a gating optical intensifier that is only activated after a small proportion

of the fluorescence light is directed onto a photodiode via a dichroic mirror, such that photon

detection is only possible after a programmed electronic delay. By performing detection

across a range of delay times thus similarly allows the fluorescence lifetime to be estimated,

but with poorer time resolution compared to TCSPC and lower photon collection efficiency.

For the frequency domain approach, the fluorescence lifetime is estimated from the phase

delay between a high-​frequency modulated light source such as an LED or laser modulated

using an AOD (see Chapter 6), coupled to a fast intensified camera detector. An independent

estimate of the lifetime may also be made from the modulation ratio of the y components of

the excitation and fluorescence. If these values do not agree within experimental error, it may

indicate the presence of more than one lifetime component. Frequency domain methods

are faster than time domain approaches due to the use of camera detection over slow lateral

scanning, and thus are the most commonly used for dynamic cellular studies.

Molecular interactions may also be monitored by using a variant of the technique called

FLIM–​FRET. This technique utilizes the fact that the lifetime of a fluorophore will change if

energy is non-​radiatively transferred to, or from it, from another fluorophore in a very close

proximity via FRET. Separate lifetime measurements can be made on both the FRET donor

dye at shorter wavelengths and FRET acceptor dye at longer wavelengths (FRET effects are

discussed fully in Chapter 4) to infer the nanoscale separation of two different biological

molecules separately labeled with these two different dyes. FLIM–​FRET therefore can gen­

erate cellular molecular interaction maps.