Multimodal Imaging

A wide range of optical imaging modalities based on different contrast mechanisms have emerged in recent years. While many of these techniques are in use for biological research and medical applications, the ability to combine these different techniques will enable the full potential of these technologies to be realized. However, integrating different imaging modalities is often challenging due to the different hardware requirements for various imaging modalities. One aim of the Biophotonics Imaging Laboratory is to develop technology for integrating multiple imaging modalities and to explore applications of these techniques.

Integrated optical coherence and multiphoton microscopy

We have developed an integrated microscope that can perform structural and functional imaging of cells in 3-D and for extended periods of time. This integrated microscope combines optical coherence microscopy (OCM), multi-photon microscopy (MPM), and second harmonic generation (SHG) microscopy in a single instrument. Spectral-domain OCM data provides structural information based on the optical scattering from the sample, without the need for exogenous contrast agents to label structures. MPM provides functional information by using auto fluorescence, exogenous fluorophores or genetically-expressed fluorescent proteins that are linked to cell function or physiology. With simultaneous acquisition, perfect image registration is possible, providing spatiotemporal relationships between cell and tissue structure and function.

Experimental setup of the integrated spectral domain optical coherence and multiphoton microscope. Region A is the dual spectrum laser source while region B is the microscope. Abbreviations: BS, beam splitter; DG, diffraction grating; DM, dichroic mirror; F, emission filter; HWP, half-wave plate; M, mirror; MMF, multi-mode fiber; OBJ, objective lens; P, polarizer; PCF, photonic crystal fiber; PMT, photomultiplier tube; TS, translation stage.

Vinegoni C, Ralston TS, Tan W, Luo W, Marks DL, Boppart SA.  Integrated structural and functional optical imaging combining spectral-domain optical coherence and multiphoton microscopy.  Appl. Phys. Lett., 88:053901, 2006. n/a PDF


OCM and MPM typically require different laser source characteristics in order to be performed optimally. For MPM, tunable Ti-Sapphire lasers are most commonly used as they allow fluorescent markers with different excitation spectra to be targeted. OCM requires a laser source with a broad spectral bandwidth in order to achieve strong optical sectioning. To facilitate the integration of OCM and MPM and to make their simultaneous use the most flexible, we have developed a single-laser, dual-spectrum laser source.

The dual spectrum source consists of a tunable mode-locked Ti-Sapphire laser with a portion of its output spectrally broadened via continuum generation in a photonic crystal fiber. The continuum-broadened beam allows for enhanced optical sectioning with OCM while the unbroadened beam from the ultrashort-pulse Ti-Sapphire laser optimally excites fluorescent markers for MPM. This source effectively implements a tunable, broadband source which meets the requirements for both OCM and MPM.

Wavelength-dependent characteristics of continuum generation in the dual-spectrum source. Plot (a) shows the input (dashed line) and output (solid line) power of the photonic crystal fiber. The fiber output is the available power for OCM. Plot (b) shows the spectral bandwidth of the broadened beam. The bandwidth of the pump laser is 5-10 nm, depending on wavelength.

Multiphoton and optical coherence microscopy images acquired at three different center wavelengths. The first, second, and third column corresponds to 750 nm, 850 nm, and 920 nm center wavelengths, respectively. (a-c) MPM images. (d-f) OCM images. (g-i) MPM images overlaid on OCM images to show spatial correspondence.

Graf BW, Jiang Z, Tu H, Boppart SA. Dual-spectrum laser source based on fiber continuum generation for integrated optical coherence and multiphoton microscopy. J Biomed Opt, 14(3):034019, 2009. PubMed Abstract PDF


Structural and Functional Imaging

The unique configuration of this integrated microscope allows for the simultaneous acquisition of both anatomical (structural) and functional imaging information with particular emphasis for applications in the fields of tissue engineering and cell biology. This microscope has been used to visualize functional changes in cells on various substrates, in 3D scaffolds, and under static and dynamic culture conditions. The use of spectral-domain OCM not only provides structural information of the substrate (e.g., micropeg structure) or the extracellular matrix (e.g., microfibers), but also shows cell morphology. Simultaneously, MPM offers complementary information on cell adhesions as well as subcellular structures.

Multimodal microscopy of fibroblasts on a planar substrate. (a-d) Fibroblasts from a GFP-mouse, and (e-h) 3T3 fibroblasts with GFP-vinculin are imaged in this 2-D culture. (a,e) MPM and (b,f) OCM image channels are combined (c,g) for multimodal visualization to demonstrate the relationship between cell morphology, cell adhesion activity, and nuclei (blue channel). (d,h) Phase contrast microscopy of similar cultures is shown for comparison. The light-dark banding observed in (b,f) is due coherent interference effects from reflections off of the planar substrate and the cell membrane. The scale bars represent 20 µm.

Functional interactions between GFP-vinculin fibroblasts and microtextured substrate following mechanical stretching of the elastic polymer substrate. Substrate was stretched in the directions indicated by the arrows. (a) Multimodal image combining OCM and MPM data. Inset shows the MPM channel. (b,c) OCM and multimodal images of boxed region in (a) at en face planes 6 µm and 12 µm, respectively, above the plane in (a). Note the increase in GFP-vinculin signal-expression, which is present over a larger area of the cells. The images in (c) illustrate the depth-dependent optical sectioning of this instrument. At this plane, signal from the planar substrate is decreased (darker), while the upper peg surface reflection is increased. MPM signals from the cells are decreased at the upper en face imaging planes of these cells. The scale bar represents 20 µm.

Three-dimensional multimodal images of fibroblasts from a transgenic GFP mouse cultured in Matrigel under mechanical stimulation. 3-D culture was subjected to 5% cyclic uniaxial stretching for 18 hours, in the directions indicated by the arrows. (a) 3-D multimodal images of cells, with corresponding projections in three orthogonal planes. Color channels correspond to: Red OCM, Blue nuclei, Green GFP. (b) Phase-contrast and (c) fluorescence microscopy images of the same 3-D cultures are shown for comparison. The scale bars represent 20 µm.

Multimodal OCM/MPM images of GFP-vinculin fibroblasts showing structural and functional relationships between cells and substrate/scaffold.

Liang X, Graf B, Boppart SA.  Imaging engineered tissues using structural and functional optical coherence tomography.  Article and Cover Figure.  J. Biophotonics, 2:643-655, 2009. PubMed


Intravital Imaging

The primary advantage of the integrated microscope over other imaging techniques is that it is well suited for imaging live, intact tissue. This ability enables high resoultion visualization of live tissue which is of great interest for both biological research and medical diagnostics. The figures below show MPM auto-fluorescence and OCM images taken from the skin of a live human subject. Many features of the skin are visible such as individual cells, skin folds and hair follicles/shafts.

OCM and MPM images from different depths of live human skin. Many different skin features are visible such as hair follicles, skin folds as well as individual cells.

Multimodal Skin Imaging

The high resolution and multimodal framework make the integrated microscope uniquely suited for in vivo skin imaging in a label-free manner.  These advantages allow specific biological events that occured in the skin, such as wound healing, apoptosis, and the effect of pharmaceutical treatments to be characterized longitudinally.  A variety of skin components can be visualized with the multimodal system, including collagen structure (second harmonic generation microscopy, SHG), endogenous/labeled fluorophores (two-photon excitation fluorescence, TPF), surface structure (OCT/OCM), vasculature network (phase-variance OCT, PV-OCT), and metabolic activity (fluorescence lifetime imaging microscopy, FLIM).

Wound Healing

Multimodal in vivo en face images of mouse ear wound, showing the collagen structure (blue), GFP bone marrow-derived cells (green), surface structure (gray), and vasculature (red).  Scale bar: 500 µm.

Graf BW, Chaney EJ, Marjanovic M, Adie SG, De Lisio M, Valero MC, Boppart MD, Boppart SA.  Long-term time-lapse multimodal intravital imaging of regeneration and bone-marrow-derived cell dynamics in skin.  Technology 1, 8-19, 2013.    


Left - In vivo longitudinal tracking of collagen remodeling (cyan) and localization of stem cells (red) in diabetic wound show that stem cell-treated skin wounds experienced faster remodeling rate.  Right - Co-localization of stem cells and collagen fibers in diabetic wound.

Li J, Pincu Y, Marjanovic M, Bower AJ, Chaney EJ, Jensen T, Boppart MD, Boppart SA.  In vivo evaluation of adipose- and muscle-derived stem cells as a treatment for nonhealing diabetic wounds using multimodal microscopy.  J. Biomed. Optics 21, 086006, 2016.    


In vivo apoptosis

Single cell analysis of in vivo apoptotic keratinocytes in the mouse skin using FLIM, demonstrating longitudinal tracking of different lifetime components of individual cells.

Bower AJ, Marjanovic M, Zhao Y, Li J, Chaney EJ, Boppart SA.  Label-free in vivo cellular-level detection and imaging of apoptosis.  J. Biophotonics 10, 143-150, 2017.    


Effects of pharmaceutical treatments

Left - Longitudinal FLIM imaging of steroid-treated animals on different days, which illustrates the effect of steroid on the metabolic activity in skin.  Right - Analysis of dermal collagen reorganization following topical steroid treatment using SHG: untreated (a) vs. two types of steroids (b & c).

Bower AJ, Arp Z, Zhao Y, Li J, Chaney EJ, Marjanovic M, Hughes-Earle A, Boppart SA.  Longitudinal in vivo tracking of adverse effects following topical steroid treatment.  Exp. Dermatol. 25, 362-367, 2016.    


PV-OCT demonstrates the positive effect of a topical treatment on vasculature regeneration in diabetic skin.  Scale bar: 500 µm.

Optical Parametric Amplifier (OPA)

High-resolution imaging in turbid media has been limited by the intrinsic compromise between the gating efficiency (removal of multiply-scattered light background) and signal strength in the existing optical gating techniques, which leads to shallow depths due to the weak ballistic signal, and/or degraded resolution due to the strong multiply-scattering background.  This challenge can be addressed utilizing nonlinear optics based OPA, which can simultaneously enhance both the imaging depth and the spatial resolution in turbid media.  The coherent process makes the OPA potentially useful as a general-purpose optical amplifier applicable to a wide range of optical imaging techniques.

Schematic of the OPA setup for optical imaging.  BBO: beta-barium borate crysta, BPF: band-pass filter, BS: beam splitter, DC: dichroic mirror, DL: delay line, NA: numerical aperture, NDF: neutral density filter, SA: sapphire crystal, SHG: second harmonic generation.

OPA gain and imaging. (a) Supercontinuum spectra measured with (blue) and without (pink) the presence of the OPA pump.  Imaging of the onion skin (c-e) illustrates the difference in image quality with (c) and without (d & e) the OPA gain.  Scale bar: 100 µm.

Imaging of fresh rat muscle tissue at different depths below the surface. (a) OPA imaging. (b) conventional confocal imaging.  Scale bar: 100 µm.

Zhao Y, Adie SG, Tu H, Liu Y, Graf BW, Chaney EJ, Marjanovic M, Boppart SA. Optical parametrically gated microscopy in scattering media.  Opti. Express 22, 22547-22560, 2014.