Holography Fluorescence Microscopy

Yamil A. Nieves*, Juan S. Gomez*, Justine Dupere, Myung Kim, David C. Clark, Dept. of Physics, Univ. of South Florida
*Corresponding authors: ynieves@mail.usf.edu, jgomezvelez@mail.usf.edu

Abstract:

Digital holography is a fast-growing research field that

has drawn increasing attention leading to a myriad of new technological
applications due to its powerful three-dimensional (3D) imaging
capacity. Previously, fluorescent microscopy has been limited by the
need of coherent light sources or two dimensional scanning. Recent
developments in digital holography, including self-interference
incoherent digital holography (SIDH), provide highly versatile
capabilities for 3D holographic imaging with incoherent light that can
remove the barrier between fluorescence and holography. Current
progress in fluorescence cell imaging is presented using an SIDH
module attached to a hand-built customized fluorescence microscope.
The module does not employ the use of coherent light sources or
scanning devices. Methods are proven to be effective through imaging
of fluorescent beads (fluorophores) and preliminary images of fibroblast
cells. Future work will include holography fluorescence microscopy of
cellular processes, such as mitosis and motility of cells tagged with
fluorescent proteins.

Background:

(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. 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).

(a)

Fibroblast cells:
Using the apparatus described, we were able to obtain a holographic image
(Fig. 6 (b)) of fibroblast cells fluorescing in DAPI and Alexa 488 conjugated
secondary antibody that recognizes primary antibody against tubulin (Fig. 6
(a)).

(b)
Fig. 5: 1m FluoSpheres Polystyrene Microspheres were used from
LifeTechnologies with excitation & emission at 540nm & 560nm
respectively. The beads were mixed with a polyacrylamide gel to
create a 3D matrix, and prevent the beads from moving and
clumping together.
(a), (b), and (c) show particles coming into focus at different
planes.

(c)

Experiments/Results:
Differential Holography:
Fig. 1: Coherent light wave pattern

Fig. 2: Absorption & emission spectra

Fig. 3: Incoherent light wave pattern

Coherence is one of the unique properties of laser light. It arises from

the stimulated emission process which provides the amplification. Since
a common stimulus triggers the emission events which provide the
amplified light, the emitted photons are "in step" and have a definite
phase relation to each other (Fig. 1). However, since the transitions
between energy levels during the fluorescence process is a completely
random process (Fig. 2), we have no control over when an atom is
going to lose energy in the form of radiation. This means that
fluorescence is an incoherent source. Incoherent sources emit light with
frequent and random changes of phase between the photons (Fig. 3).

In differential holography, two holograms are acquired by SIDH, while

some structures or components change in the object scene. Holograms
were recorded at two different times. The imaged space consisted of a
stationary bead matrix and a slightly translated bead plane. Subtraction of
one hologram by the other results in another hologram that contains
three-dimensional dynamics of the object volume. Differential fluorescence
holographic microscopy of cellular process can be studied, such as in
mitosis or motility of cells tagged with fluorescent proteins.
Initial Holograms

(b)

(c)

(a) Initial, (b) final, and (c) differential holograms at stationary plane ( = 370)

(d)

(b)
Fig. 6: Direct image and hologram of a fluorescent fibroblast cell
(a) Direct Image, and (b) Holographic image ( = 320) of the fibroblast cell.

Future Work:

Final Holograms

Apparatus:
(a)

(a)

(e)

3D images of fluorescent tagged, biological

tissues such as (a) drosophila eggs and (b)
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
and
motility
by
differential
fluorescent holography

(a)

(b)

(d) Initial plane = 1 and (e) Final plane ( = 9)

References:

Fig. 4: Hand-built SIDH fluorescence microscope

A modified Michelson interferometer is positioned on top of a hand-built

fluorescence microscope and is used to create a hologram of a sample
using fluorescence. A 490nm Blue LED is used to excite the
fluorophores in the sample. An FITC (fluorescein) filter configuration
(absorption, dichroic, and emission filters) only allows the emitted to
transmit toward the CCD camera.

(c) 2015 MK Kim

(f)

(g)

(f) And (g) Differential hologram focused at particle plane

Fig. 5: Fluorescent beads 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.

Kim, M. K., Principles and techniques of digital holographic microscopy, SPIE Reviews 1(1), 018005 (2010).
Lohmann, A. W.,Wavefront reconstruction for incoherent objects, J. Opt. Soc. Am. 55, 15551556
(1965).
Kim, M. K., Full color natural light holographic camera, Optics Express 21 (8), 9636-9642 (2013).
Myung K Kim, Incoherent digital holographic adaptive optics, Appl. Opt. 52, A117-A130 (2013).
C Jang, J Kim, DC Clark, S Lee, B Lee, & MK Kim, Holographic fluorescence microscopy with
incoherent digital holographic adaptive optics, J. Biomed. Optics, 20, 111204-1~8 (2015).
DC Clark, MK Kim, Nonscanning three-dimensional differential holographic fluorescence microscopy,
J. Electron. Imaging. 24, 043014 (2015).