98

3.6  Basic Fluorescence Microscopy Illumination Modes

at oblique nonzero angles of incidence the excitation field projects onto the focal

plane as an ellipse whose major axis is longer than w by a factor cos θg. From part

(b), the initial fluorescence intensity of the dye is ~411 − 110 =​ 301 counts, but the

intensity due to the camera noise will be insensitive x; thus,

I0

0 8 5 3

0 46

301

21

≈(

)×

×

(

) ≈

. / .

.

counts per pixel

3.6.4  CONFOCAL MICROSCOPY

An in vivo sample in a light microscope can often encapsulate a height equivalent to tens

of equivalent depth of field layers, which can generate significant background noise on the

image. The most robust standard biophysical tool to limit this effect is that of confocal micros­

copy, which uses a combination of two pinholes, in front of the sample and the detector

(Figure 3.5g) to delimit the detected intensity to that emerging from the focal plane, resulting

in significant increases in fluorescence imaging contrast. Laser light is focused to a volume

of just 1 femtoliter (fL), 10⁻¹⁸ m³, onto the sample that is either raster scanned across the

sample or the sample stage raster scanned relative to the laser focus. Fluorescence emissions

acquired during the analog raster scanning are then digitized during software reconstruction

to create a 2D pixel array image.

The confocal volume can be approximated as a 3D Gaussian shape, roughly like an egg,

with its long vertical axis parallel to the microscope optic axis that is longer than the lateral

width w in the focal plane by a factor of a of typically ~2.5, giving a volume V:

(3.53)

V

a

w

= π^{3 2}

3

/

Photon emissions are ultimately focused onto a sensitive detector, typically a PMT, which can

then be reconstituted from the raster scan to form the 2D image. Slow speed is the primary

disadvantage, limited to ~100 fps. Improvements have involved high-​speed spinning disk (or

Nipkow disk) confocal microscopy comprising two coupled spinning disks scanning ~1000

focused laser spots onto a sample at the same time allowing imaging of ~1000 fps. The prin­

cipal issue with such fast confocal imaging methods is that the extra exposure to light can

result in significant photodamage effects on living biological samples.

The lateral width w is determined by the PSF of the microscope. Note that this value is

identical to the optical resolution limit in diffraction-​limited light microscopy, discussed fully

in Chapter 4. For a circular aperture objective lens of numerical aperture NA:

(3.54)

w

NA

= ^{0 61}

.

λ

For determining the excitation volume size in confocal imaging, this formula can be also

used with the radius of the confocal volume in the focal plane equal to w. For determining

the resolution of the fluorescence emission images for VIS light fluorescence, the wavelength

λ is normally in the range 500–​700 nm, low-​magnification light microscopy allows fields

of view of several hundred microns or more in diameter to be visualized in a single image

frame, w can be as low as 1–​2 μm (essentially the length scale of subcellular organelles in a

eukaryotic tissue, or single bacterial cells within a biofilm), whereas the highest magnifica­

tion light microscopes have the smallest values of w of ~250–​300 nm. The Nyquist criterion

indicates that pixel edge length in a digitized confocal microscopy image should be less than

half the size of the smallest resolvable length scale, that is, w/​2, in the sample to overcome

undersampling, and in practice, pixel edge lengths equivalent to 50–​100 nm in the sample

plane are typical.