(27as) Development of a Supercontinuum Laser-Based Confocal Microscope System for Excitation-Scanning Hyperspectral Imaging

Widefield and confocal fluorescence microscopy are key tools that have been critical for the study of molecules, organelles, cells, tissues, and organisms. While these tools have been employed for many years in the biological and biomedical fields, there has also been continuous hardware and software innovation to improve the resolution, temporal sampling, sensitivity, and specificity of these tools. Spectral and hyperspectral imaging technologies have been employed by the fluorescence microscopy field for a variety of tasks, such as separating signals from similar fluorescent labels, discriminating fluorescent label signals from cellular or tissue autofluorescence, interrogating the molecular basis for autofluorescence, measuring intermolecular distances through Förstor resonance energy transfer (FRET), and others. The additional spectroscopic data provided by hyperspectral imaging has been invaluable for enabling these tasks. However, the measurement of image data at many wavelengths also invariably comes at a cost. In typical microscope systems, this cost is usually in the form of loss of the fluorescence emission signal due to filtering or dispersive optical elements needed to acquire spectral information. We have previously demonstrated that an alternate approach, called excitation-scanning hyperspectral imaging, which filters the fluorescence excitation light instead of fluorescence emission, may result in improved signal levels and enable faster imaging times. However, it has been difficult to translate the approach of excitation-scanning hyperspectral imaging to confocal microscope systems, due to the high collimation requirements. Here, we provide initial results of designing a system that combines a supercontinuum laser source, tunable filter, and spinning disk confocal microscope to provide excitation-scanning hyperspectral imaging confocal microscopy capabilities.

The system was implemented on an automated inverted microscope platform (Ti-U, Nikon Instruments). Broadband laser illumination was provided by a 15 W white light supercontinuum laser source (FIU-15, NKT). The supercontinuum source was coupled to an AOTF module (SuperK Select UV-VIS, NKT) to enable wavelength tuning in the range of 400-650 nm. Tunable laser excitation was then coupled into a beam-shaping and homogenization unit (Borealis, Andor) and into the spinning disk scanhead (CSU-W1, Yokogawa). A custom short-pass dichroic beam beamsplitter (Chroma) was used to enable an excitation scan range of 400-550 nm, while collecting all fluorescence emission above the 550 nm cutoff wavelength. Images were detected using a cooled EMCCD camera (iXon 897, Andor). Optical power transmission was assessed for various locations in the excitation lightpath using a laser powermeter (either PM100D, Thorlabs or ArgoPower, Argolight). Fixed slide test samples (FluoCells Prepared Slides, Invitrogen) were used to assess imaging performance.

System integration and testing results indicate that spectrally tunable illumination from the supercontinuum laser and AOTF module can be successfully coupled into the beam-shaping optics and confocal microscope scan head. Illumination powers of 5-80 uW were achievable at the sample stage, depending on wavelength. Unfortunately, wavelengths lower than 440 nm provided <10 μW of illumination power, which was insufficient for acquiring fluorescence images of appreciable signal-to-noise ratio. Further work will focus on optimizing the optical power transmission and excitation power available at the microscope stage in order to enable high-speed hyperspectral confocal imaging. This work was supported by NIH awards P01HL066299, R01HL58506, and R01HL137030, and NSF award MRI1725937. Drs. Leavesley and Rich disclose financial interest in a start-up company, SpectraCyte LLC, that was formed to commercialize spectral imaging technologies.