Chapter 3 – Making Light Work in Biology  99

The axial spatial resolution in confocal microscopy is worse than the lateral resolution

w by the same factor of ~2.5 for the relative stretching of the PSF parallel to optic axis

to generate a roughly ellipsoidal volume. A modification to standard confocal imaging

is used in confocal theta microscopy in which juxtaposed confocal volumes are angled

obliquely to each other (ideally perpendicularly) to interfere at the level of the sample that

generates a reduced size of confocal volume with a resultant small improvement to axial

resolution.

The z range of confocal microscopy illumination is sometimes limited by diffractive effects

of the confocal beam. The standard illumination intensity of a confocal microscope laser

beam incident on the back aperture of the objective lens has a Gaussian profile, resulting in a

divergent confocal volume that, as we have seen, has an aspect ratio parallel to the optic axis

of ~1:3, implying that a wide area of sample can obstruct the incident beam en route to the

focal waist if imaging reasonably deep into a large cell or multicellular tissue sample. The use

of Bessel beam illumination can circumvent this problem. A Bessel beam is nondiffractive

(discussed fully, in the context of their application in OTs, in Chapter 6), which means that it

is relatively nondivergent and insensitive to minor obstructions in the beam profile. A Bessel

beam can be used as an alternative source of scanning illumination for exciting fluorophores

in the sample (see Planchon et al., 2011), but note that since the beam is nondivergent the

ability to optically section in z is much reduced compared to standard confocal microscopy.

Another application of confocal illumination is to monitor the diffusion of biomolecules

in tissues, or even single cells. This can be achieved using the technique of fluorescence

recovery after photobleaching (FRAP). Here, a relatively intense confocal excitation volume is

generated for the purpose of photobleaching dye molecules of a specific fluorescently labeled

biomolecule in that region of space. If the laser intensity is high enough, then relatively little

diffusion will occur during the rapid photobleach process. Before and after imaging in fluor­

escence then shows a dark region indicative of this photobleaching. However, if subsequent

fluorescence images are acquired, then fluorescence intensity may recover in this bleached

region, which is indicative of diffusion of photoactive dye-​labeled biomolecules back into this

bleached area. This can be used to determine rates of diffusion of the biomolecule, but also

rates of biomolecule turnover in distinct molecular complexes (see Chapter 8).

A related technique to FRAP is fluorescence loss in photobleaching (FLIP). This experi­

mental photobleaching method is similar, but fluorescence intensity measurements are instead

made at positions outside of the original bleach zone. Here, the diffusion of photobleach dye-​

labeled biomolecules results in a decrease in fluorescence intensity in surrounding areas.

FLIP gives similar information to FRAP but can also yield more complex features of hetero­

geneous diffusion between the point of photobleaching and the physically distant point of

fluorescent intensity measurement.

Worked Case Example 3.2: Using Confocal Microscopy

A fluorescence microscopy experiment was performed on a live Caenorhabditis elegans

embryo (see Chapter 7 for a discussion on the use of the C. elegans worm as a model

organism) at room temperature to investigate stem cells near the worm’s outer surface,

whose nuclei are ~2 μm in diameter using a confocal microscope with high numerical

aperture objective lens of NA 1.4. A bacterial protein called “LacI” was tagged with GFP

and inserted into the worm, and the C. elegans DNA sequence was modified to create

a LacO binding site for LacI inside the nucleus of the stem cells that could be switched

on (i.e., binding site accessible by LacI) or off (i.e., binding site not accessible by LacI) by

external chemical control. With the binding site switched off, a laser at a wavelength of

473 nm was focused on the sample at the same height as the center of the nucleus to

generate a confocal excitation volume, and consecutive images were acquired at a rate of

1000 fps without moving the sample relative to the confocal volume, with an observation

of single fluorescent particles diffusing through the confocal volume whose brightness

was consistent with single molecules of GFP.