Spectral Imaging: Methods, Design, and Applications - Biomedical Optical Imaging Technologies: Design and Applications - page 117

Tabl e 4. 1 Typical parameters of spectral imaging systems

Category Property Typical

Imaging Spatial resolution 250 nm (in plane) at D 500 nm

50 m (high magnification) 1; 000 1; 000
pixels or more
Dynamic range 8-16 bits (256-65,536 intensity levels)
Lowest detectable signal Shot-noise limited
Field of view depends on detector area and foreoptics
Frame rate 10-400 [fps]
Spectroscopy Spectral resolution 1-20 nm (may depend on ) can be a variable
parameter
Spectral range 400-900 nm
Spectral response 60-70% at 500-600 nm 80-100% for
back-illuminated chips
The Typical column shows the common value that is achievable but greatly depends on the specific
dispersion method that is used and the quality of the system elements (e.g., high NA objective lens,
high quantum efficiency QE )

Field of view

emitted spectrum. Different effects such as saturation and bleaching may disrupt
an expected linearity of the signal and should be tested for each specific case.
In bright-field microscopy, reflection and scattering microscopy, the sample is
illuminated with an external light source and the detector measures that very same
light after interaction with the sample. Spectral analysis of the data must take
into account the light source spectrum in order to accurately extract the desired
sample's spectral properties. In bright-field microscopy, the measured signal may
not be directly proportional to the concentration of the observed molecules but to its
logarithm.
In order to measure a spectrum, the intensity at each wavelength is measured.
There are different methods to disperse the light and almost all of them are used in
various spectral imaging systems, as will be described below. Some of the important
characteristics of a spectrum include (see also Table 4.1 ):

1. Spectral resolution
Spectral resolution determines the smallest separation of two peaks in the
spectrum that can still be distinguished. Typically, spectral imaging systems
can resolve down to several nanometers. In general, the spectral resolution is
a function of the method in which light is dispersed, the fore-optics and other
parameters, where in some systems it is a tunable parameter and in some not.
2. Spectral range
The spectral range refers to the wavelength band in which spectra can be
measured. It is governed by the same factors mentioned in the preceding term in
combination with the detector's capabilities which differ in different wavelength
bands.
3. Lowest detectable signal and dynamic range
Lowest detectable signal defines the smallest measurable signal, while dynamic
range dictates the number of distinguishable levels in a given measurement.