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3.5  Fluorescence Microscopy: The Basics

εr and ε0 are the relative electrical permittivity and absolute electrical permittivity in

a vacuum

a is the QD radius

When light interacts with a QD, an electron–​hole exciton pair is created. An exciton

has an associated length scale called the Bohr radius, such that beyond this length scale,

the probability of exciton occurrence is very low. For CdSe QDs, the Bohr radius is

~5.6 nm, and thus quantum confinement effects occur at QD diameters that are less than

~11.2 nm. The aforementioned equation predicts that the energy state of the confined

exciton decreases with increasing QD radius. In other words, smaller QDs are blue, while

larger QDs are red.

QDs are characterized by a broad absorption spectrum and narrow emission spectrum—​

this means that they can be excited using a range of different lasers whose wavelength of

emission does not necessarily correspond to an absorption peak in the fluorophore as is the

case for organic dyes, with the tightness of the spectral emission meaning that emissions can

be relatively easily filtered without incurring significant loss of signal (Figure 3.4d).

This narrowness of spectral emission means that several different colored QDs can be

discriminated in the same sample on the basis of their spectral emission, which is useful if

each different colored QD tags a different biomolecule of interest. QDs are brighter than

their corresponding organic dyes at similar peak emission wavelength; however, their relative

brightness is often overstated (e.g., a single QD emitting in the orange-​red region of the VIS

light spectrum at the corresponding excitation wavelengths and powers is typically only six

to seven times brighter than a single molecule of the organic dye rhodamine).

QDs undergo a photophysical phenomenon of blinking (Figure 3.4e). Many different types

of fluorophores also undergo blinking. Blinking is a reversible transition between a photo­

active (light) and an inactive (dark) state; the dye appears to be bright and then momentarily

dark in a stochastic manner (i.e., random with respect to time), but, in general, these are

prevalent more at excitation intensities higher than would normally be used for fluorescence

imaging with dark state dwell times <10 ms, which is often sufficiently fast to be averaged out

during typical fluorescence microscopy sampling time windows of ~100 ms or more. QDs

blink more appreciably at lower excitation intensities with longer dwell times of dark states

more comparable to the time scale of typical fluorescence imaging, which can make it dif­

ficult to assess if what you see from one image to a consecutive image in a kinetic series of

images is a continuous acquisition of the same tagged biomolecule or a different one that has

diffused in to the field of view from elsewhere.

The actual functional diameter of a QD can be more like 15–​20 nm since the core is fur­

ther coated with a solvent-​protective shell (typically zinc sulfide) and a polymer matrix for

chemical functionalization. The size can increase further to ~30 nm if an antibody is also

attached to the QD to allow more specific binding to a given biological molecule. A diameter

of 30 nm is an order of magnitude larger than a typical biomolecule, which can result in sig­

nificant steric hindrance effects, which can inhibit native biological processes and also make

it challenging to deliver the QD into a cell. However, there have been several applications of

cellular imaging developed using QD fluorophores, going back to the turn of the century (see

Michalet et al., 2005).

An alternative metal-​based fluorophore involves lanthanides. The lanthanides are a group

of 15 4f-​orbital metals in the periodic table between atomic number element 57 (lanthanum)

to 71 (lutetium). They form unique fluorescent complexes when bound via their 3+​ ion states

with an organic chelating agent, such as a short random coil sequence of a given protein to

be labeled. This can confer significant stability to the fluorophore state, since the chelation

complex is protected from water and so exhibits limited free radical photobleach damage

(Allen and Imperiali, 2010).

Fluorescent nanodiamond (FND) is emerging as a valuable probe for biological samples

since it has a high photostability manifest as no photobleaching or blinking, is biocom­

patible, and has spectral properties that are relatively insensitive to the surrounding fluid

environment and are spectrally distinct from the normal range of cellular autofluorescence.

A nanodiamond is a synthetic nanoscale-​sized particle composed of carbon atoms bound