1.

Introduction

Optical coherence tomography (OCT) is a noninvasive imaging modality that uses low-coherence interferometry to detect optical scattering from tissue, cells, or objects with lateral spatial resolutions of a few microns up to several millimeters deep into tissue.^1,2 Thus, a noninvasive technology like OCT can be very useful for longitudinal studies of the cellular and molecular foundations of diseased conditions. Because of the lack of an OCT based imaging system that is capable of providing information via functionalized contrast agents, our knowledge is currently limited to visual clues obtained with conventional OCT images of anatomical structures and/or anomalies within tissues. However, these anomalies are merely the outcome of complex molecular and cellular interactions. Without the benefit of enhanced molecular contrast in OCT imaging, it is difficult to determine the role of the aforementioned complex structural anomalies in a diseased condition. Here we seek to optimize an OCT-based imaging technique coupled with functionalized contrast agents as a multifaceted technology for imaging single cells and blood vessels in vivo inside the mouse retina noninvasively at subnanomolar sensitivity.

Following our development of numerous imaging agents for specifically labeling target proteins of interest in living mice,^3–6 we recently designed a new OCT-based imaging system for the retina by developing nanoparticles based on gold nanorods (GNRs), which produce a strong OCT signal. GNRs have been recently used to enhance OCT signals in tissue mimicking phantoms,^7–11 excised porcine eyes,⁸ vitreous of mice eyes,¹² anterior chambers and cornea of mice eyes,¹³ and in mouse mammary tumors.¹⁴ Additionally, using our custom synthesized GNRs ($\sim 100\times 30\mathrm{nm}$), we have achieved ultrahigh detection sensitivity in vivo.¹⁵ Here we sought to address whether freshly isolated leukocytes could be labeled with GNRs to enhance scattering, and thus be detected in vitro and in vivo in the retina using OCT. We further determined the sensitivity for detection of GNRs inside retinal blood vessels using OCT in vivo. To our knowledge, this is a first demonstration of OCT-based cellular imaging at a high resolution and subnanomolar sensitivity, allowing single cell and blood capillary detection inside the retina in vivo.

OCT has long been used to image human eyes for clinical diagnosis^1,2,16 and several commercial OCT systems are FDA approved for ophthalmological use. GNRs were previously shown to be nontoxic^13,17 and have also been used for clinical trials in cancer immunotherapy¹⁸ and photothermal cancer therapy.^19,20 So, we expect our OCT imaging system paired with a nonsurgical minimally invasive procedure to be fully translatable to patients in the future. Additionally, depth and resolution offered by OCT, as well as the enhanced contrast of functionalized GNRs may complement existing clinical imaging techniques and help in better diagnosis and personalized treatments of disorders.

2.

Methods

2.2.

Labeling of Leukocytes with Gold Nanorod

Eight-week-old nu/nu mice (Charles River) were euthanized using ${\mathrm{CO}}_2$, and spleens and blood were harvested. The spleen was homogenized to get single cell suspensions and mixed with the blood [Fig. 1(a)]. The cells were then washed twice in DMEM, and red blood cells (RBCs) were lysed using $1\times$ lysis buffer (Biolegend) following manufacturer’s protocol. $2.5\mu \mathrm{g}$ of biotin-conjugated anti-mouse CD45 (Biolegend, Clone 30-F11) was incubated for 20 min at RT with $50\mu \mathrm{L}$ of OD 200 ${\mathrm{GNR}}^{\mathrm{Nav}}$ (see above) + diluted to 1 mL using ${\mathrm{diH}}_2\mathrm{O}$ to make GNR–anti-CD45 conjugates (${\mathrm{GNR}}^{\mathrm{CD}45}$). ${\mathrm{GNR}}^{\mathrm{CD}45}$ was washed twice in ${\mathrm{diH}}_2\mathrm{O}$ by centrifuging at $2250\times g$ for 10 min at RT, and finally resuspended in $50\mu \mathrm{L}$ $\mathrm{DMEM}+5\%$ FBS. The cells were resuspended in $200\mu \mathrm{L}$ $\mathrm{DMEM}+5\%$ FBS ($\sim 30\times {10}^6\text{cells}/100\mu \mathrm{L}$), mixed with the freshly prepared $50\mu \mathrm{L}$ ${\mathrm{GNR}}^{\mathrm{CD}45}$, and incubated for 2 h at RT. Cells were washed twice with $\mathrm{DMEM}+5\%$ FBS by centrifuging at $340\times g$ for 2 min at RT each time, and finally resuspended in 1 mL DMEM. A tube containing $200\mu \mathrm{m}$ of $\mathrm{DMEM}+5\%$ FBS and $50\mu \text{L}$ ${\mathrm{GNR}}^{\mathrm{CD}45}$ was subjected to the same staining and washing conditions as the labeled cells. Using this tube as a control, the amount of free GNRs post wash was measured to be $<0.01\mathrm{nM}$.

Fig. 1

Leukocyte labeling with ${\mathrm{GNR}}^{\mathrm{CD}45}$. (a) Schematic of leukocyte labeling with ${\mathrm{GNR}}^{\mathrm{CD}45}$. (b) From left to right OCT images of 0.5 nM GNR solution, 0.05 nM GNR solution, unlabeled leukocytes, and ${\mathrm{GNR}}^{\mathrm{CD}45}$-labeled leukocytes. All samples were loaded into $400\mu \mathrm{m}$ internal diameter glass capillaries for OCT imaging and imaged together. $\text{Scale bar}=100\mu \mathrm{m}$. (c) Mean of log of OCT intensities from contiguous regions of size 10 pixels or larger within the dotted boxes shown in (b). Data pooled over 150 B-scans from three independent experiments. * $p$ value $<0.001$ (unpaired $t$-test).

2.4.

Optical Coherence Tomography and Hyperspectral Imaging of Labeled Cells

Single cell suspensions (labeled and unlabeled, see above), or standard ${\mathrm{GNR}}^{\mathrm{mPEG}}$ solutions (0.05 and 0.5 nM) were drawn into $400\mu \mathrm{m}$ internal diameter glass capillaries and imaged with Ganymede at $2\mu \mathrm{m}/\text{pixel}$ in $X$ and $2\mu \mathrm{m}/\text{pixel}$ in $Z$ as described above. First, OCT intensities were calculated for individual pixels within regions inside the capillaries [Fig. 1(b)], which were devoid of reflection artifacts. Then, mean intensities were calculated from contiguous regions of 10 pixels or larger, which exhibited signal above background noise (measured from water-only capillaries). In addition, $\sim 20\mu \mathrm{L}$ of cells from each sample was mounted on glass slides with CytoSeal 60 (electron microscopy sciences) for dark-field imaging. Dark-field hyperspectral microscopy was performed using a Cytoviva™ system [Fig. 2(a)] equipped with a Fiber-Lite™ DC-950 light source, an Olympus UPlanFLN $100\times$ 1.30 N.A. oil objective, an Andor™ iXon3 hyperspectral camera, and a Dage-MTI™ XLMCT digital color camera. Hyperspectral images were normalized to the light source spectrum of the Cytoviva instrument.

Fig. 2

Hyperspectral detection of GNRs in leukocytes labeled with ${\mathrm{GNR}}^{\mathrm{CD}45}$. (a) Diagrammatic representation of the Cytoviva™ hyperspectral imaging setup. Dotted line and arrows indicate the light path. (b) Absorbance of GNRs measured by UV–Vis. (c) Hyperspectral image of GNRs (peak 830 nm) on a glass slide (left) and the entire spectrum (400 to 1000 nm) of randomly chosen GNRs indicated by red arrows (right). (d) Hyperspectral image of unlabeled leukocytes, post RBC lysis, washed and overlain on a glass slide (left) and spectra of randomly chosen points indicated by blue arrows (right). (e) Leukocytes, after removal of RBCs by lysis, and labeled with ${\mathrm{GNR}}^{\mathrm{CD}45}$ for 1 h, washed, and overlain on a glass slide (left) and spectra of representative pixels indicated by red and blue arrows in their corresponding colors (right). All images were acquired with a $100\times 1.3$ N.A. oil objective; (c) through (e) red, green, and blue highlights indicate bands that were used to represent the images on the left. Centers of bands are $\text{red}=887\mathrm{nm}$, $\text{green}=725\mathrm{nm}$, $\text{blue}=510\mathrm{nm}$; each band is Gaussian with an FWHM of 80 nm. $\text{Scale bars}=10\mu \mathrm{m}$.

2.5.

Imaging of Live Mouse Retina Using Optical Coherence Tomography

Nu/nu mice were anesthetized using 2.5% $\text{isofluorane}+{\mathrm{O}}_2$ (v/v). Once adequately anesthetized, the mice were mounted onto a platform and secured with stereotactic devices (Fig. 3). With the mouse secured, the stage was tilted so that the mouse was on the side and the eye was facing up, with the top of the cornea being approximately parallel to the table. In this position, a plastic O-ring was carefully placed surrounding the eye using vacuum grease (Dow Corning), so as not to touch the cornea and to form a fluid-tight seal. Then, pupillary dilation was achieved by applying one drop each of 1% tropicamide (Bausch & Lomb) and 2.5% phenylephrine hydrochloride (Paragon BioTeck) to the eyes, for 2 min each. If any fluid leakage was observed after application of the dilating solutions, additional vacuum grease was applied toward the outer edge of the base of the O-ring. 2.5% hypromellose solution (Gonak™, Akorn Inc.) was then placed over each eye as a contact solution. Enough hypromellose solution was applied to form an upward meniscus so that no air bubbles were formed when placing cover slips over the solution. Finally, 0.17-mm-thick glass coverslips were gently placed over the O-ring to complete a fluid-tight chamber over the dilated eye [Figs. 3(c) and 3(d)]. Anesthesia was continually maintained using a nose-cone delivering 1.5 to 2% $\text{isofluorane}+{\mathrm{O}}_2$. The level of anesthesia, hydration of the eye, and the body temperature of the mouse were continually monitored to prevent the formation of a cataract. Experiments where cataract formation was evident were not used for statistical analysis. OCT images of the live mouse retina were then acquired using Ganymede as described above.

Fig. 3

Setup for imaging of live mouse retina. (a) Inclined stage with an anesthetized nu/nu mouse restrained for retinal imaging. (b) Mouse is kept warm using a lamp, anesthesia is maintained by 1.5 to 2% isoflurane in ${\mathrm{O}}_2$ (v/v) delivered by a nose cone, and a tail vein catheter is inserted and secured for intravenous delivery of GNRs or cells. (c) Photograph and (d) diagram of a mouse eye encased using 2.5% hypromellose solution in a fluid-tight chamber.

3.

Results

3.2.

Optical Coherence Tomography Imaging of Living Mouse Retinae by Gold Nanorod Enhanced Detection Sensitivity

We next investigated the detection sensitivity of GNRs in vivo in mice retinae to deduce the degree of labeling necessary to allow detection of labeled cells in retinae. To adapt the Ganymede™ system for retinal imaging, we encased the eyes of anesthetized mice in a fluid-tight chamber forming a plano-convex lens system allowing the focal length of the Ganymede system to reach all the retinal layers (Fig. 3). Next, we progressively injected $200\mu \mathrm{L}$ total of a 10 nM solution ${\mathrm{GNR}}^{\mathrm{mPEG}}$ and measured OCT intensities inside retinal blood vessels [Fig. 4(a)]. OCT intensity increased within the retinal blood vessels following injection of ${\mathrm{GNR}}^{\mathrm{mPEG}}$ [Fig. 4(b)]. Changes in OCT intensities in retinal blood vessels could be distinctly differentiated from baseline intensities starting at an effective GNR concentration of 0.5 nM in blood [Fig. 4(c)]. At higher GNR concentrations in the blood, blood vessels with diameter $<20\mu \mathrm{m}$ could also be detected. ${\mathrm{GNR}}^{\mathrm{mPEG}}$ persisted in blood circulation for $\sim 4\mathrm{h}$ until they were cleared out, which was denoted by a return to preinjection scattering intensity levels [Fig. 4(d)]. It needs to be mentioned here that after GNR injection, when focus was established in the outer plexiform layer, we noticed lower intensities of backscattered light from the choroidal vasculature compared to the vessels within the plexiform layers. To confirm the presence of GNRs in the retinal blood vessels, we imaged unstained $5\text{-}\mu \mathrm{m}$-thick formalin-fixed paraffin embedded sections (without removing the paraffin) of retinae of mice injected with $200\mu \mathrm{L}$ of 10 nM ${\mathrm{GNR}}^{\mathrm{mPEG}}$ and euthanized 1 h postinjection [Fig. 4(e)]. Characteristic spectra of GNRs (887 nm, represented in red) were clearly visible inside the retinal vessels, confirming the presence of stable ${\mathrm{GNR}}^{\mathrm{mPEG}}$ within these vessels. These results show that using our OCT imaging technique, GNRs can be detected at a sensitivity of $\sim 0.5\mathrm{nM}$ inside retinal blood vessels following IV injection.

Fig. 4

Contrast-enhanced OCT imaging of retinal vessels after intravenous ${\mathrm{GNR}}^{\mathrm{mPEG}}$ injection. (a) Experimental design. Orange arrows indicate the time points for IV injection of ${\mathrm{GNR}}^{\mathrm{mPEG}}$. Black arrows indicate the times at which OCT imaging was performed. (b) Representative OCT images of mice retinae before and after ${\mathrm{GNR}}^{\mathrm{mPEG}}$ injection. Conditions corresponding to the images are indicated on the left. $\text{Scale bar}=500\mu \mathrm{m}$. (c) and (d) Measure of OCT intensity inside retinal blood vessels after ${\mathrm{GNR}}^{\mathrm{mPEG}}$ injection. (c) Sensitivity measurements and (d) measure of ${\mathrm{GNR}}^{\mathrm{mPEG}}$ signal decay from blood postinjection. $\text{Mean}\pm \mathrm{SEM}$; * $p$ value $<0.05$, $\mathrm{ns}=\text{not significant}$; all conditions in (c) and (d) are compared with preinjection images using one-way analysis of variance. Data represent three independent experiments. (e) Hyperspectral image of unstained $5\text{-}\mu \mathrm{m}$-thick formalin-fixed paraffin embedded section of a mouse retina 1 h after IV injection of ${\mathrm{GNR}}^{\mathrm{mPEG}}$. Gray—ambient scattering from tissue, red—pixels with characteristic spectra from GNRs. $\text{Scale bar}=50\mu \mathrm{m}$. Note the presence of GNRs inside a large vessel at the center.

3.3.

Migration of ${\mathrm{GNR}}^{\mathrm{CD}45}$-Labeled Leukocytes to Sites of Laser-Induced Retinal Injury

We expected labeled leukocytes with intensities greater than a 0.5 nM equivalent of GNRs to be detected in the retina by contrast-enhanced OCT, based on our GNR sensitivity measurements. We next imaged sites of laser-induced injury in nu/nu mice retinae to detect leukocyte migration within these areas. Following laser injury to the retina, we expect an increase in infiltrating leukocytes (predominantly neutrophils) to the site of injury.²¹ To this end, we injected ${\mathrm{GNR}}^{\mathrm{CD}45}$-labeled leukocytes in mice 30 min after laser injury. We were able to distinguish a few ${\mathrm{GNR}}^{\mathrm{CD}45}$-labeled leukocytes inside retinal blood vessels using enhanced OCT contrast [Fig. 5(a) and Video 1]. We measured the speed of the observed moving cells within the smaller capillaries to be $\sim 90\mu \mathrm{m}/\mathrm{s}$. These increased signals were more evident in outer plexiform layer, which exhibited low OCT intensity prior to injection of cells. As unlabeled leukocytes were poorly scattering (Fig. 1) and the amount of free GNR was at least 50-fold lower than OCT detection limits in vivo, these signals were unlikely from free GNRs or unlabeled leukocytes. Additionally, there was a small but significant increase in OCT intensity inside larger vessels after $60\times {10}^6$ and $100\times {10}^6$ GNR-labeled cells had been injected [Fig. 5(b)]. These results are indicative of the change in blood vessel intensity due to injected GNR-labeled cells. However, we were unable to distinguish ${\mathrm{GNR}}^{\mathrm{CD}45}$-labeled cells at the sites of injury, mainly because of speckle noise within the injured tissue that had intensities greater than our previously calculated detection limit of 0.5 nM GNR equivalent. Thus, we next investigated the presence of GNR-labeled cells in the retinae at the sites of injury by histological analysis using hyperspectral microscopy [Fig. 6(a)]. Two hours after laser injury and injection of GNR-labeled leukocytes, we observed characteristic GNR peaks (887 nm, represented in red) near the site of laser injury in mice retinae. These GNRs at the site of injury were closely associated with cells embedded in the choroidal region. GNR spectra were absent in the corresponding nonlasered eye obtained from the same mice [Fig. 6(b)]. We expected labeled leukocytes to constitutively migrate to the spleen. Thus, to further confirm the presence of injected GNR-labeled cells in these mice, we imaged histological spleen sections of the same mice and observed a large number of ${\mathrm{GNR}}^{\mathrm{CD}45}$-labeled cells inside the spleens [Fig. 6(b)]. We also imaged sections from eyes of mice that received laser thermotherapy but were not injected with GNR-labeled cells [Fig. 6(c)]. Expectedly, characteristic GNR spectra were absent in these control samples. From these results, we conclude that the hyperspectral GNR signals observed in the retina near sites of laser injury, but not near noninjured sites, are most likely due to ${\mathrm{GNR}}^{\mathrm{CD}45}$-labeled cells migrating preferentially to the sites of laser-induced injury in mice retinae and that these cells can be detected using hyperspectral histological analysis. While our current implementation of OCT can detect these cells inside vessels, they were not observable in the injured region due to excess speckle noise.

Fig. 5

${\mathrm{GNR}}^{\mathrm{CD}45}$-labeled leukocytes in retinae of nu/nu mice. (a) OCT image of a nu/nu mouse retina postinjection of ${\mathrm{GNR}}^{\mathrm{CD}45}$-labeled leukocytes (left, large blood vessels indicated by yellow arrows), and six sequential images of the boxed area inside the OCT image (each average of three B-scans) color-coded by time. Elapsed time is indicated inside each frame, with the first frame (blue) assigned time point 0. Superposition of the six frames (right) shows a multicolored path, indicative of a moving cell within a capillary. $\text{Scale bar}=200\mu \mathrm{m}$ (left) and $50\mu \mathrm{m}$ (right). (also refer to Video 1, MOV, 5.91 MB) [URL: http://dx.doi.org/10.1117/1.JBO.21.6.066002.1]. (b) OCT intensity in blood vessels shown in (a), with each data point representing intensity inside a single blood vessel summed over 100 B-scans before injection (pre) and after injection of $60\times {10}^6$ and $100\times {10}^6$ total ${\mathrm{GNR}}^{\mathrm{CD}45}$-labeled leukocytes. Red line and bars indicate $\text{Mean}\pm \mathrm{SD}$. * $p$ value $<0.05$ (unpaired $t$-test).

Fig. 6

Hyperspectral images of GNR-labeled leukocytes inside lasered eyes of mice. (a) Hyperspectral image of $5\text{-}\mu \mathrm{m}$-thick unstained section of lasered eye and (b) the corresponding nonlasered control eye of nu/nu mice that received ${\mathrm{GNR}}^{\mathrm{CD}45}$-labeled cells. The choroid, photoreceptor, and outer nuclear layer (ONL) regions of the retina have been marked inside the image. Red GNR-labeled cells are indicated by yellow arrowheads. (c) Hyperspectral image of $5\text{-}\mu \mathrm{m}$-thick unstained section of spleen of mice injected with GNR-labeled cells. Red GNR-labeled cells indicated by yellow arrowheads. (d) Hyperspectral image of $5\text{-}\mu \mathrm{m}$-thick unstained section of laser-treated eye of control mice without injected GNR-labeled cells. Center of bands are $\text{red}=887\mathrm{nm}$, $\text{green}=725\mathrm{nm}$, $\text{blue}=510\mathrm{nm}$; each band is Gaussian with an FWHM of 80 nm. $\text{Scale bars}=50\mu \mathrm{m}$.

4.

Discussion

In this report, we have demonstrated that contrast agents such as GNRs and the imaging depth and resolution achievable with OCT can provide an imaging technology capable of detecting signals at single cell level in a living mouse retina. This minimally invasive approach can achieve imaging depths of several millimeters and axial resolution of up to $2.1\mu \mathrm{m}$, thus providing superior detection capabilities than current noninvasive fluorescence imaging techniques. Moreover, the measured detection sensitivity for GNRs (0.5 nM) makes this OCT-based approach particularly attractive. Our custom synthesized GNRs¹⁵ provide better depth and resolution in OCT compared to smaller GNRs used in previous studies to enhance contrast in tissues and in vivo.^8,12,13 We further demonstrated that leukocytes obtained from mouse blood and spleen tissue can be successfully labeled with scattering contrast agents such as GNRs via a targeted labeling strategy. Such GNR-labeling can be utilized to enhance scattering intensity of these otherwise poorly scattering cells, allowing them to be detected by OCT. This, to our knowledge, is the first demonstration of targeted cell labeling using contrast agents that enhance scattering. This work demonstrates that GNRs can be used as multifaceted probes for contrast-enhanced OCT imaging and dark-field hyperspectral microscopy due to their unique scattering spectra. The ability to observe these nanoparticles in hyperspectral microscopy can corroborate the spectral images observed with OCT.

Migrating GNR-labeled leuokocytes were detected inside retinal blood vessels but were undetected at the site of tissue injury due to excess speckle noise. Improvements in the GNR labeling technique and utilization of instrumentation to reduce speckled noise may improve our OCT system to enable the detection of GNR-labeled objects within extravascular tissue. Currently, backscattered intensity after GNR injection in the choroid is comparable to preinjection speckle noise in the choroid, limiting the systems capability to detect GNR-labeled cells in the choroid. With our current imaging system, algorithmic removal of tissue speckle noise is insufficient to obtain the signal-to-noise ratio necessary for identification of GNR-labeled cells outside the blood vessels. In future studies, the ability of our GNRs to scatter optimally within specific wavelengths of incident light may be utilized to differentiate GNR spectra from ambient scattering from the retinal layers. After GNR injection, we also observed noticeably low signal intensity at the center of larger vessels, compared to the edges. This may be attributed to fringe washout commonly observed in large vessels in spectral domain OCT.²² In future studies, improved instrumentation and computational techniques can be used to remove washout artifacts.

We needed to make some adjustments for detecting GNRs in the retina by hyperspectral microscopy. First, we observed a shift in the GNR spectral peak ($\sim 830\mathrm{nm}$ in UV–Vis), which may be attributed to changes in plasmonic property of the GNRs due to high refractive index mounting media²³ and adsorbed proteins on their surface.²⁴ Even though H&E staining of histological section provides excellent anatomical references for the retinal layers, such staining interfered with the spectral detection of GNRs, leading us to image unstained sections. Additionally, to prevent loss of unbound ${\mathrm{GNR}}^{\mathrm{mPEG}}$ inside blood vessels, we imaged these sections without deparaffinization.

Our OCT imaging system can be extended in the future to image the spatiotemporal behavior of immune cells inside tissues and to detect microstructures or biomarkers within such tissues by specific labeling using functionalized GNRs. We envision that the methods described herein may be used in the future to shed light on the roles of immune cells^25–28 and biomarkers, such as amyloid beta,²⁹ VEGFR1,³⁰ and ${\alpha }_{\mathrm{v}}{\beta }_3$³¹ in atrophic age-related macular degeneration and other retinal disorders. These results will help us better understand the cellular and molecular basis of disease progression via longitudinal studies. OCT has been recently used in conjunction with imaging techniques such as scanning laser ophthalmoscopy,^32–34 fundus autofluorescence,³⁵ fluorescein angiography,^32,36,37 ultrasound,³⁸ and Indocyanine Green angiography,³⁹ helping in better diagnosis of diseased tissue. Multiphoton imaging has also been used to study fluorescent cells in the mouse retina.⁴⁰ While such fluorescence imaging can provide subcellular resolution, these techniques are limited by depth of penetration and the use of laser powers above the safety limits for humans.⁴¹ Thus, our strategies for leveraging the benefits of depth and resolution offered by OCT, as well as the enhanced scattering by functionalized GNRs may complement these existing clinical imaging techniques and help in better diagnosis and personalized treatments of disorders.