Single Source Fluorescence Imaging/Blue Optical Coherence Tomography in a GFP Mouse Model

Abstract

 

Purpose

 

Optical coherence tomography (OCT) is a commonly used in vivo three-dimensional imaging modality. However, conventional OCT systems cannot detect fluorescent markers that are commonly used in biological research. The purpose of this project was to develop a dual-modality imaging system combining anatomic imaging from OCT with fluorescent imaging.

 

Methods

 

A custom combined SDOCT and fluorescence system was developed utilizing a single blue illumination source (λ0=482nm, Δλ=30nm, NKT Photonics A/S) allowing for simultaneous detection of both signals (Fig. 1). The OCT signal consisted of backreflected blue light and was detected by a custom spectrometer at a rate of 20,000 A-scans per second. The OCT optical system was designed for isotropic 4.5µm^3 resolution. Fluorescence emitted from the sample was filtered (λ0=525nm, Δλ=39nm) from the blue light and detected via a photo-multiplier tube. Cx3CR1 (bone marrow derived cell [BMDC] receptor) GFP reporter knock-in mice and wild type C57BL/6 control mice were imaged in vivo using the blue OCT/fluorescence system under an IACUC approved protocol. A two-photon microscope (2P-M, λ = 910 nm) was used as reference to confirm presence or absence of GFP labeled BMDCs in the anterior segment.

 

Results

 

Figure 2 shows blue OCT/fluorescence images from a single volume acquisition in the anterior segment of a CX3CR1-GFP reporter mouse. The 700µm (W) x 700µm (L) x 1000µm (D) volume was acquired in 12.5 seconds. Striations in the fluorescence image (Fig. 2B) are due to bulk motion artifacts (i.e. mouse breathing, heartbeat). 2P-M verified the presence of BMDC GFP reporter in the cornea of this mouse. Wild type control mice did not show BMDC GFP using the blue OCT/fluorescence or 2P-M system.

 

Conclusions

 

We have developed a novel single source blue OCT/fluorescence imaging system that provides three-dimensional structural information as well as automatically co-registered en face molecular contrast information. This system has promise for rapidly screening and imaging model animals such as mice to simultaneously detect both anatomic changes and quantitate fluorescent signals.

 

 

Single Source OCT and Fluorescence System Diagram

 

 

All data from a single 700µm (W) x 700µm (L) x 1000µm (D) volume acquisition of GFP labeled mouse anterior segment A) Averaged OCT B-scan from central region B) En face fluorescence image showing GFP labeled macrophages within the cornea

 

Keywords: 552 imaging methods (CT, FA, ICG, MRI, OCT, RTA, SLO, ultrasound) • 599 microscopy: light/fluorescence/immunohistochemistry • 551 imaging/image analysis: non-clinical