Biomedical Engineering Reference
In-Depth Information
range enhanced with numerical processing for retrieving the full 3D sample information.
Several applications have risen up from this kind of microscopy allowing 3D imaging with
micrometer resolution
[25]
. Some examples include underwater observations
[26
28]
,
tracking of moving objects and particles
[29,30]
, and study of erosion processes in coastal
sediments
[31,32]
.
As in conventional microscopy, resolution in DIHM is a combination of several connected
parameters. First, the wavelength of the illumination source used in the setup affects the
resulted resolution since resolution (
R
) is proportional to that value (
R
B
λ
/NA, being
λ
the
illumination wavelength and NA the numerical aperture). Second, the pinhole diameter
(playing the role of both the illumination diaphragm and the condenser lens in the
illumination module of a microscope) controls the spatial coherence, the useful amount of
light and the divergence of the illumination beam. Third, the NA (classically defined by
the microscope objective) is determined in DIHM by both the transversal dimensions of the
electronic recording device and the sample-to-detection plane distance. To optimize the
experimental setup in terms of light efficiency, the pinhole diameter is adjusted to provide a
cone of light according with the NA defined by the electronic device. Typical achievable
NA values in DIHM are in the range of 0.4
[33]
. And fourth, the characteristics of the
electronic recording device (number of pixels, pixel size, and dynamic range) affect the
sampling of the interferometric fringes and the noise level in the recorded in-line hologram.
For fixed illumination wavelength, there are two key points in DIHM when trying to
achieve high NA, and thus a low spatial resolution limit. On one hand, the first one
concerns the definition of a high optical magnification in the experimental setup as a
requirement to circumvent the limitation imposed by the sampling incoming from the pixel
size of the electronic device. Since magnification (
M
) in DIHM can be defined as the
geometrical projection at the detection plane of the sample being illuminated from the
pinhole, a highly magnified diffraction pattern will be recorded by controlling the distance
between the pinhole and the sample (
z
S
) which will be much lower than the distance
between the sample and the detection plane (
z
D
) in the form of
M
5
(
z
S
1
z
D
)/
z
S
. Typical
values for
z
S
and
z
D
are below 1 mm and in the cm range, respectively, and a high
M
value
can be easily fulfilled. Nevertheless, the high magnification implies a restriction
proportional to
M
2
in the reconstructed object field of view provided by the method.
On the other hand, the second point is related to the digital processing of the recorded
hologram and becomes the hardest task in DIHM, being nowadays basically resolved thanks
to the optimum power of available computers. Once the in-line hologram of the sample is
recorded (sample hologram:
U
S
), the sample is removed from the setup and another in-line
hologram is stored at the computer's memory only considering the illumination beam
(reference hologram:
U
R
). Then, a synthetic hologram (
U
0
S
) is derived from the previously
recorded holograms in the form of
U
0
S
5
(
U
S
2
U
R
)/
U
R
1/2
. In addition, a coordinate
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