Selective plane illumination microscopy (SPIM) with time-domain fluorescence lifetime imaging microscopy (FLIM) for volumetric measurement of cleared mouse brain samples

• Published in: Review of scientific instruments Vol. 89; no. 5; pp. 053705 - 53712
• Main Authors: Funane, Tsukasa, Hou, Steven S., Zoltowska, Katarzyna Marta, van Veluw, Susanne J., Berezovska, Oksana, Kumar, Anand T. N., Bacskai, Brian J.
• Format: Journal Article
• Language: English
• Published: United States AIP Publishing LLC 01.05.2018
• Subjects: Animals; Brain - cytology; Brain - diagnostic imaging; Cell Culture Techniques; Dependovirus - genetics; Equipment Design; Fluorescence Resonance Energy Transfer; Genetic Vectors; Green Fluorescent Proteins - genetics; Green Fluorescent Proteins - metabolism; HEK293 Cells; Humans; Imaging, Three-Dimensional - instrumentation; Imaging, Three-Dimensional - methods; Lasers; Luminescent Proteins - genetics; Luminescent Proteins - metabolism; Male; Mice, Inbred C57BL; Microscopy - instrumentation; Microscopy - methods; Microspheres; Optical Fibers; Phantoms, Imaging; Red Fluorescent Protein; Tissue Scaffolds
• ISSN: 0034-6748; 1089-7623; 1089-7623
• DOI: 10.1063/1.5018846

We have developed an imaging technique which combines selective plane illumination microscopy with time-domain fluorescence lifetime imaging microscopy (SPIM-FLIM) for three-dimensional volumetric imaging of cleared mouse brains with micro- to mesoscopic resolution. The main features of the microscope include a wavelength-adjustable pulsed laser source (Ti:sapphire) (near-infrared) laser, a BiBO frequency-doubling photonic crystal, a liquid chamber, an electrically focus-tunable lens, a cuvette based sample holder, and an air (dry) objective lens. The performance of the system was evaluated with a lifetime reference dye and micro-bead phantom measurements. Intensity and lifetime maps of three-dimensional human embryonic kidney (HEK) cell culture samples and cleared mouse brain samples expressing green fluorescent protein (GFP) (donor only) and green and red fluorescent protein [positive Förster (fluorescence) resonance energy transfer] were acquired. The results show that the SPIM-FLIM system can be used for sample sizes ranging from single cells to whole mouse organs and can serve as a powerful tool for medical and biological research.