holographic fluorescence microscopy
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7/23/2019 Holographic Fluorescence Microscopy
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(c) 2015 MK Kim
Abstract:Digital holography is a fast-growing research field thathas drawn increasing attention leading to a myriad of new technologicalapplications due to its powerful three-dimensional (3D) imagingcapacity. Previously, fluorescent microscopy has been limited by theneed of coherent light sources or two dimensional scanning. Recentdevelopments in digital holography, including self-interferenceincoherent digital holography (SIDH), provide highly versatilecapabilities for 3D holographic imaging with incoherent light that canremove the barrier between fluorescence and holography. Currentprogress in fluorescence cell imaging is presented using an SIDHmodule attached to a hand-built customized fluorescence microscope.The module does not employ the use of coherent light sources orscanning devices. Methods are proven to be effective through imagingof fluorescent beads (fluorophores) and preliminary images of fibroblastcells. Future work will include holography fluorescence microscopy ofcellular processes, such as mitosis and motility of cells tagged withfluorescent proteins.
Holography Fluorescence MicroscopyYamil A. Nieves*, Juan S. Gomez*, Justine Dupere, Myung Kim, David C. Clark, Dept. of Physics, Univ. of South Florida*Corresponding authors: [email protected]
Apparatus:
A modified Michelson interferometer is positioned on top of a hand-builtfluorescence microscope and is used to create a hologram of a sampleusing fluorescence. A 490nm Blue LED is used to excite thefluorophores in the sample. An FITC (fluorescein) filter configuration(absorption, dichroic, and emission filters) only allows the emitted totransmit toward the CCD camera.
Fig. 5:1m FluoSpheres Polystyrene Microspheres were used fromLifeTechnologies with excitation & emission at 540nm & 560nmrespectively. The beads were mixed with a polyacrylamide gel tocreate a 3D matrix, and prevent the beads from moving andclumping together.
(a), (b), and (c) show particles coming into focus at differentplanes.
Experiments/Results:
Differential Holography:
Background:
Future Work:Initial Holograms Final Holograms
(a)Initial, (b)final, and (c)differential holograms at stationary plane ( = 370)
(d) Initial plane = 1 and (e) Final plane ( = 9)
(f) And (g) Differential hologram focused at particle plane
3D images of fluorescent tagged, biologicaltissues such as (a) drosophila eggs and (b)bone sections
3D images of shifts to a bead matrixcaused by the traction forces of cellsexhibited at the surface of a gel.
Image dynamic cellular activities such asmitosis and motility by differentialfluorescent holography
(a) (b) (c)
(d) (e)
(f) (g)
(a) (b)
(a)
(b)
(c)
Coherence is one of the unique properties of laser light. It arises fromthe stimulated emission process which provides the amplification. Sincea common stimulus triggers the emission events which provide theamplified light, the emitted photons are "in step" and have a definitephase relation to each other (Fig. 1).However, since the transitionsbetween energy levels during the fluorescence process is a completelyrandom process (Fig. 2), we have no control over when an atom isgoing to lose energy in the form of radiation. This means thatfluorescence is an incoherent source. Incoherent sources emit light withfrequent and random changes of phase between the photons(Fig. 3).
Fig. 1: Coherent light wave pattern Fig. 3: Incoherent light wave patternFig. 2: Absorption & emission spectra
Fig. 4: Hand-built SIDH fluorescence microscope
Fig. 5: Fluorescent beads shifted [-30.0x, -50.8y, 25.4z]m. The final x, y position is indicated by thered 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.
References:
Fibroblast cells:
Kim, M. K.,Principlesand 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 K im, 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).
(a) (b)
Fig. 6: Direct image and hologram of a fluorescent fibroblast cell(a) Direct Image, and (b) Holographic image ( = 320)of the fibroblast cell.
(a), (b), and (c) are the initial, final, and differential holograms,respectively, at the stationary plane. (d)is the initial position of the beadsand (e)is the final position at the translated planes. The initial and finalholograms were subtracted, cancelling out the stationary information leavingonly the initial and final positions of the beads. So, the difference hologramis focused to the stationary plane (c), the initial plane (f), and the finalplane (g).
Using the apparatus described, we were able to obtain a holographic image(Fig. 6 (b)) of fibroblast cells fluorescing in DAPI and Alexa 488 conjugatedsecondary antibody that recognizes primary antibody against tubulin (Fig. 6(a)).
In differential holography, two holograms are acquired by SIDH, whilesome structures or components change in the object scene. Holograms
were recorded at two different times. The imaged space consisted of astationary bead matrix and a slightly translated bead plane. Subtraction ofone hologram by the other results in another hologram that containsthree-dimensional dynamics of the object volume. Differential fluorescenceholographic microscopy of cellular process can be studied, such as inmitosis or motility of cells tagged with fluorescent proteins.
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