Chapter 3 – Making Light Work in Biology  73

metal-​oxide semiconductor (MOS) pixels. MOS pixels act as micron length scale capacitors

with a voltage bias set just above the threshold for inversion, which thus generates electrons

and holes on absorption of incoming photons.

Each MOS capacitor accumulates an electric charge proportional to the light absorbed,

and control circuitry transfers this charge to its neighbor along each 1D line of the pixel, such

that the last MOS capacitor in the line dumps its charge into the MOS pixel in the next line

up or down, ultimately with all the charge transferred into an amplifier. This serial voltage

data stream can then be subsequently reconstructed as a 2D image. A variant on the CCD

includes the intensified CCD (ICCD) that comprises an initial detection step on a phosphor

screen, with this phosphor light image then detect by a CCD behind it, which improves the

ultimate photon detection efficiency to >90%.

Many cheaper cameras utilize a CMOS chip (these are now found ubiquitously in

webcams, mobile phone cameras, and also in microprocessors in nonimaging applications).

The core feature of a CMOS chip is a symmetrical back-​to-​back combination of n-​ and p-​type

MOS field effect transistors, requiring less additional circuitry with greater power efficiency

compared to CCD pixels, manifest ultimately substantially faster imaging speeds. A scientific

CMOS camera has an inferior photon collection efficiency of ~50% compared to ICCDs or

EMCCDs, but can acquire data faster by an order of magnitude or more, equivalent to several

thousand image frames per second.

An EMCCD utilizes a solid-​state electron-​multiplying step at the end of each line of

CCD pixels. This amplifies relatively weak electrical signal above any readout noise that is

added from the final output amplification step. This electron multiplication has normally

a few hundred stages during which electrons are transferred by impact ionization, which

generates multiple secondary electrons to amplify the signal. The resultant amplification is

up to ~10³, which compares favorably to APDs and ICCDs but with a much-​reduced readout

noise. EMCCDs are currently the photon detection tool of choice for low-​light microscopy

investigations in biology, having up to ~95% photon detection efficiency, for example, applied

to single-​molecule fluorescence detection, and have a reasonable sampling speed equivalent

to ~1 ms per image frame for small pixel arrays of ~100 pixels of edge length, relevant to

many fast biological processes.

3.4  NONFLUORESCENCE MICROSCOPY

Basic light microscopy is invaluable as a biophysical tool. However, its biggest weakness is

poor image contrast, since most of the material in living organisms is water, on average ~60%.

Since cells are surrounded by a fluid environment, which is largely water, the signal obtained

from VIS light scattered from cellular object is small. However, there are several adaptations

to basic light microscopy that can be applied to enhance image contrast.

3.4.1  BRIGHT-​FIELD AND DARK-​FIELD MICROSCOPY

Bright-​field microscopy relies on measuring the differences in the absorbed or scattered inten­

sity of light as it passes through different features of the sample. Incident light is generated

usually from either a tungsten halogen filament broadband source or a bright LED, which is

captured by short focal length collector lens. Light is then directed through a condenser lens

using a Köhler illumination design that involves forming an image of the light source in the

back focal plane of the condenser (Figure 3.2b). This results in a collimated beam incident on

the sample and a uniform illumination intensity in the focal plane of the microscope.

A cell on a microscope coverslip/​slide whose shape is broadly symmetrical on either

side of a plane parallel to a focal plane taken through its midheight will exhibit minimal

bright-​field image contrast between the foreground cellular material and the background

cell media. A simple approach to increase the contrast for the outline of the cell is to use

defocusing microscopy. Negative defocusing, for which the focal plane is moved below the

midheight level of the cell closer to the object lens, generates an image of a dark cell body