3.2. Miniaturized 2-Color Fluorescence Microscopy (M-2CFM) System Development

The M-2CFM system (Doric Lenses Inc., Quebec, CA, Canada) consists of a broad band Ce:YAG light source and a blue LED source centered at 465 nm. The spectrum of the Ce:YAG source is filtered by a narrow bandpass optical filter centered at 561 nm. The two wavelengths or colors thus function as distinct excitation channels which can be controlled separately. The two colors are combined and coupled into a 200 μm core diameter multimode optical fiber. Light excitation for imaging is transmitted via an optical fiber coupled to a customized thin 1 cm long, 500 μm diameter cylindrical gradient index (GRIN) lens imaging probe, which fits coaxially into the LIT-IMD inner lumen. A metal sheath with 700 μm outer diameter and 100 μm wall thickness mechanically enhances and protects the probe. To enable side-viewing necessary for our application, the GRIN lens is coupled to a triangular prism at its distal end which redirects light 90 degrees (Figure 2b).

The probe both delivers light excitation and also collects the resultant fluorescent signal. The system operates in an epi-fluorescence manner, and the collected fluorescence images are received by two separate CCD cameras for both channels, one centered at 630 nm (red channel) and the other at 530 nm (green channel). The miniaturized optical imaging probe is mounted on a 4-axis stage system consisting of three linear stages (Thorlabs) for precise X-Y-Z movement and a fourth rotational stage (Figure 2a). The stages are controlled electronically using commercially available software (Thorlabs). Control of the M-2CFM system and subsequent imaging display from the CCD camera are also performed in real-time using commercially available software (Doric). There were two microscope cameras (Motic Instruments, Schertz, TX, USA) used to confirm satisfactory positioning and alignment of the imaging probe, and to ensure that the microdevice does not migrate within the live tumoral tissue during imaging.

A 10-μm thick section containing fluorescently stained macrophage cells was placed on a slide. The slide was moved in 25 μm increments away from the imaging probe using a linear stage (Thorlabs) until optimal focus and minimal blurring was qualitatively achieved; this distance was determined to be the optimal ‘working distance’ for the system. Images obtained using the M-2CFM system were compared to gold standard benchtop fluorescence imaging (Echo Revolve, San Diego, CA, USA) to determine the overall field of view and resolution of the M-2CFM system with the custom GRIN lens probe.