Chapter 3 – Making Light Work in Biology  81

TOTHER is a combination of all of the other transmission spectra of the other optical

components on the emission path of the microscope

Ecamera is the efficiency of photon detection of the camera

The total SNR for fluorescence emission detection is then

(3.30)

SNR

G

N

n N

N

N

i

n

EM

EX

AF

CAM

=

+

+

=

∑

1

2

2

2

where NEM is the number of detected fluorescence emission photons per pixel, with their

summation being over the extent of a fluorophore image consisting of n pixels in total (i.e.,

after several capture and transmission losses, through the microscope) from a camera whose

gain is G (i.e., for every photon detected the number of electron counts generated per pixel

will be G) and readout noise per pixel is NCAM, with NEX photons transmitted over an equiva­

lent region of the camera over which the fluorophore is detected.

3.5.3  MULTICOLOR FLUORESCENCE MICROSCOPY

A useful extension of using a single type of fluorophore for fluorescence microscopy is to

use two or more different fluorophore types that are excited by, and which emit, different

characteristic ranges of wavelength. If each different type of fluorophore can be tagged onto

a different type of biomolecule in an organism, then it is possible to monitor the effects of

interaction between these different molecular components and to see where each is expressed

in the organism at what characteristic stages in the lifecycle and how different effects from

the external environment influence the spatial distributions of the different molecular

components. To achieve this requires splitting the fluorescence emission signal from each

different type of fluorophore onto a separate detector channel.

The simplest way to achieve this is to mechanically switch between different fluorescence

filter sets catered for the different respective fluorophore types and acquire different images

using the same region of sample. One disadvantage with this is that the mechanical switching

of filter sets can judder the sample, and this coupled to the different filter set components

being very slightly out alignment with each other can make it more of a challenge to cor­

rectly coalign the different color channel images with high accuracy, necessitating acquiring

separate bright-​field images of the sample for each different filter set to facilitate correct

alignment (see Chapter 8).

A more challenging issue is that there is a time delay between mechanically switching

filter sets, at least around a second, which sets an upper limit on the biological dynamics

that can be explored using multiple fluorophore types. One way round this problem is to

use a specialized multiple band-​pass dichroic mirror in the filter set, which permits exci­

tation and transmission of multiple fluorophore types, and then using one more additional

standard dichroic mirrors and single band-​pass emission filters downstream from the filter

set to then split the mixed color fluorescence signal, steering each different color channel

to a different camera, or onto different regions of the same camera pixel array (Figure 3.4a).

Dual and sometimes triple-​band dichroic mirrors are often used. The main issue with having

more bands is that since the emission spectrum of a typical fluorophore is often broad, each

additional color band results in losing some photons to avoid cross talk between different

bands by bleed-​through of the fluorescence signal from one fluorophore type into the detec­

tion channel of another fluorophore type. Having sufficient brightness in all color channels

sets a practical limit on the number of channels permitted, though quantum dots (QDs) have

much sharper emission spectra compared to other types of fluorophores and investigations

can be performed potentially using up to seven detection bands across the VIS and near IR

light spectrum.