Biomedical Engineering Reference
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half the wavelength,
λ
. This means, if the light used has a wavelength of about
500 nm, the observable structures in the specimen should be larger than 250 nm.
With low Signal to Noise Ratio (SNR), even the Abbe resolution limit (Eq.
4.1
)is
not realized. However, fluorescent proteins like Green Fluorescent Protein (GFP)
are approximately 2
.
5 nm in diameter and classical dyes like 4',6-diamidino-2-
phenylindole (DAPI), a DNA specific dye, are even lesser in size, and at least
100 times smaller than the resolution limit! The situation can be compared to a
hypothetical case of trying to observe a rice grain but being limited to the size of a
rugby ball! We will discuss more on this limit in Sect.
4.2.1.2
. Increasing the NA can
improve the resolution, but this is not practical. Higher NA means larger objective
lenses that translates into additional cost. This also lowers the working distance
between the objective lens and the specimen. The main limit on the NA is imposed
by the refractive index of the specimen. For example, specimens immersed in water
(refractive index 1
.
33) can only really support objectives of NAs up to 1
.
25.Onthe
other hand oil at a refractive index of 1
.
515, can still support objectives of 1
.
45.
If we use a shorter wavelength, it can induce more light scattering and it will also
damage the viability of the biological specimen.
Since Abbe, the resolution of a microscope was considered to be limited by
this barrier of half a wavelength. Once suitable image sensors and fast computers
became available, it was clear that the resolution of the acquired image could be
improved by twofold in the radial direction and sometimes fourfold in the axial
direction by using computational methods such as deconvolution [
67
]. Computers
can thus be considered as a secondary lens system, and when combined with
a microscope optical system, the system is known as
deconvolution microscope
[
45
,
72
].
Although there are many kinds of microscopes in the market, we will restrict our
discussion to the resolution improvement of the Wide-field microscope (WFM) and
the Confocal laser scanning microscope (CLSM). In the process, it is our intention
to take our readers through the fascinating and beautiful world of fluorescence
microscopy, and also walk them through the development of this
auxiliary com-
putational lens
.
4.1.4
Chapter Overview
Public
While writing this chapter, we aimed to reach a wide range of readers, graduate stu-
dents to cell biologists, interested in an introduction to deconvolution microscopy.
We have assumed that the reader has some prior exposure to linear system theory
and digital image processing, as are found in [
4
,
11
]. For the basics of digital
image processing in microscopy we refer to the articles [
29
,
58
,
69
]. For the basic
principles of fluorescence, we suggest [
21
,
43
], and to know more about fluorescent
compounds (or
fluorophores
) that are used for the specific labeling of components
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