[ Digital holographic microscopy ] Phil Saunders/ spacechannel.org
Digital holographic microscopy
(DHM) integrated with a microfluidic
device for 3-D imaging, identification
and dynamic analysis of cells and
microorganisms. For Gabor mode
DHM, the reference arm of the interferometer is removed.
Conventional bright-field optical microscopes typically
map optical absorption across the specimen and dismiss the
phase information. Due to the low absorption coefficient of
cytoplasm (the jelly-like substance that fills the cell) and most
cellular components, the resulting 2-D bright-field images of
cells are often low-contrast and produce few useful details.
One of the typical practices used to increase the contrast
of semitransparent cells is to stain and fix them with high
absorption coefficient dyes. Unfortunately, this is an invasive
process that often terminates the cell’s life cycle. However, cell
biologists typically need to monitor live cells to study their
behavior and dynamics. Fortunately, light phase is very sensitive to the refractive index mismatch between the cytoplasmic
content and the surrounding medium. This property has been
leveraged in conventional phase contrast as well as differential
interference contrast (DIC) microscopic techniques to create
high-contrast intensity images.
However, such techniques do not provide optical thickness
information about cells. Although phase contrast and DIC
techniques have been very useful in the past, the qualitative
data that are generated by such techniques does not lend itself
to quantitative characterization and the automatic recognition
of cells and microorganisms. In addition, in phase contrast and
DIC modalities, one needs to mechanically scan the specimen in order to bring different sections into focus. This adds
to the imaging time and forbids real-time cell inspection and
dynamic events over time.
DHM is a quantitative phase imaging method that directly
and noninvasively provides the optical thickness of cells in live,
dynamic conditions. Optical thickness is related to cell thickness and its 3-D shape through the cell refractive index.
Thus, when combined with appropriate computational
algorithms, DHM is an attractive candidate for non-invasive
real-time 3-D imaging and for identifying cells and microorganisms. A digital hologram contains rich, quantitative
information about the 3-D structure of the specimen, which
can be captured in one exposure and used to identify cells and
recognize objects. To this end, one can leverage the wealth of
pattern recognition techniques that have been developed for
automatic object recognition and used in applications in medicine, military, robotics and various industries.
Recently, researchers have proposed using 3-D sensing and
imaging systems for noninvasive automated identification of
cells based on their 3-D structure and dynamics. Real-time,
automated screening, analysis and identification of biological specimens can aid in disease diagnostics, environmental
monitoring and early detection of pandemics. By contrast,
conventional biological characterization methods are typically invasive, labor-intensive and time-consuming. They also
require staining, which can be very detrimental to the cells.
Moreover, they might not be effective in large-scale deployment. In addition, specimen analysis based on 2-D features
such as the shape, size and morphology of a biological specimen is not always conclusive.
Therefore, the integration of 3-D computational imaging,
information optics and DHM bears promise for a reliable,
automated and low-cost tool for rapid sensing, visualization
and identification of cells (for example, blood or stem cells),
which can be used to detect and track disease states.
In single-exposure DHM, one records a digital Fresnel hologram from the specimen in either on-axis or off-axis configurations. In on-axis mode, the real and conjugate images
are superimposed on the digital hologram. Separating them
requires a spatial or temporal phase-shifting method, which
adds to the complexity and capture time. By introducing a
small angular separation between the two interfering beams in
off-axis mode, one effectively separates the real and conjugate
images in a single exposure at the expense of suboptimal use
of sensor space-bandwidth product. This separation allows one
to discard the virtual image and perform quantitative phase
measurements based on the real image (or vice versa).
However, single-exposure, on-axis DHM is simpler to
implement, and it allows for the use of partially coherent light
instead of a fully coherent source. In the partially coherent
case, there is no need for a separate reference beam, since the
semitransparent specimen transmits part of the incident wave
unperturbed. In other words, the reference and object arms
of the interferometer can be collapsed into one. This mode is
referred to as Gabor holography.
In addition, the microscope objective that is used for magnifying the field can be removed in a lensless implementation.
The divergence of the incident beam allows for some geometrical
magnification, and the numerical processing of the hologram