Non-Scanning Holographic Fluorescence Microscopy

Justine Dupere, Yamil Nieves, David C. Clark, Dept. of Physics, Univ. of South Florida
Initial Holograms

Final Holograms
Differential hologram focused at
stationary plane
Stationary plane (zh= -370mm)

Abstract:

Digital holographic microscopy is a growing field

because it is a powerful tool in three-dimensional (3D) imaging.
The importance of incorporating digital holography is the ability to
obtain the 3D complex optical field from a single image capture,
however this requires coherence.
We use self-interference
incoherent digital holography (SIDH) combined with non-scanning
fluorescence microscopy to create a coherent hologram from an
incoherent source, fluorescence. The purpose is to obtain a digital
hologram of fluorescence micro-beads and in the future image cells
for biological purposes. We demonstrate how a two-dimensional
commercial microscope can be converted into a device that gives
three-dimensional information to the user. This can easily be
adapted to a standard fluorescence microscope. This is extremely
useful for imaging in the medical and biological fields because
current 3D scanning techniques can be photo-toxic and damage a
specimen by repeated or extended exposure to intense
illumination. In the future we will work towards imaging cells
tagged with fluorophores. This research is very important because
it provides 3D information without scanning, which means it can
easily be integrated into biological fields.

Background:

(a)
(b)

(a)
Initial plane (zh= -1mm)

(c)

Final plane (zh= -9mm)

(b)
FluoSpheres Polystyrene Microspheres were
used from LifeTechnologies. Theyre 1 m and
excite/emit at 540/560nm. The beads were
mixed with a polyacrylamide gel to not only
prevent the beads from moving and clumping
together but to create a 3D matrix. (a), (b), (c)
and (c) show particles coming into focus at
different planes.

(d)

(e)

Differential hologram focused at particle plane

Experiments/Results:

Coherent light, such as a laser, is a beam of photons that all have

the same frequency. The waves are identical and in phase.
Incoherent light, such as an LED or flashlight, has random
differences in the phase. Fluorescence is also incoherent.
Fluorescence is the result of the absorption of light at a certain
bandwidth by a molecule and the emission of light at a lower
energy.

Experiments were performed using differential holography.

Holograms were recorded at two different times. The imaged space
consisted of a stationary bead matrix and a slightly translated bead
plane. A differential hologram was calculated by subtracting the
initial and final holograms, which cancelled out the stationary
information, showing only the particles that moved.

Differential Holography:

Apparatus:

(f)

(g)

The fluorescent beads were shifted [-30.0x, -50.8y, 25.4z] m. The final
x,y position is indicated by the red circles in the dynamic plane-propagated
holograms. The bottom layer is a gel matrix and the top is a translating
layer, both with fluorescent beads. (a), (b), and (c) are the initial, final, and
differential holograms, respectively, at the stationary plane. (d) is the initial
position of the beads and (e) is the final position at the translated planes.
(f) and (g) are the differential holograms. The initial and final holograms
were subtracted, cancelling out the stationary information leaving only the
initial and final positions of the beads. So, the difference hologram is
focused to the stationary plane (c), the initial plane (f), and the final plane
(g).

Future Work:

A modified Michelson interferometer is positioned on top of a

commercial microscope and is used to create a hologram of a sample
using fluorescence. A 532 nm laser beam is angled onto the stage and
excites the fluorophores in the sample. A 560nm bandpass filter only
allows the emitted light to pass through and transmit toward the CCD
camera. The filter is used to ensure that only the emitted, fluorescing,
light passes through, which means the illumination is incoherent. A
translational stage moves the samples with micrometer resolution.

(c) 2015 MK Kim

Differential holographic microscopy: (a) is the initial hologram

focused on a USAF 1951 resolution target and (b) is the final
hologram. The resolution target was stationary. (c) is the
differential hologram propagated to the resolution target plane. (d)
is the initial and (e) the final hologram propagated to the particle
system plane. Notice the particles were moved to the left. (f) is the
differential complex hologram at the particle plane. (g), (h), and (i)
are the imaginary component representations at the particle plane.
The initial position of the particles are darker than the final positions
because the signs were preserved.

3D images of fluorescent tagged,

biological tissues such as (a) fibroblast
cells, (b) drosophila eggs, and (c) bone
sections
3D images of shifts to a bead matrix
caused by the traction forces of cells
exhibited at the surface of a gel.
Image dynamic cellular activities such as
mitosis
by
differential
fluorescent
holography

(a)

(b)

(c)