In vivo tracking of label-free resident cells in murine tissues via third harmonic generation microscopy

Authors

• D Malide

Abstract

We developed and validated a methodology for high resolution 3D harmonic imaging of intact murine tissues that requires no fluorescent labeling, based on epi- third-harmonic generation (THG) imaging by pulsed near-infrared light in the 1200–1300 nm range. We demonstrated intrinsic THG interface signals, outlining cell membranes and tissue inhomogeneities, revealing subcellular resolution of 3D-architecture in various murine tissues and organs. These included subcutaneous and perivisceral white and brown adipose tissue, blood vessels, and myelinated peripheral nerves. We also visually dissected intracellular lipid-rich granules in adrenal glands, mammary glands, sebaceous glands, pancreas, liver, and spleen. Characteristic patterns were observed in the skeletal muscle, heart, bone, skin, lung, brain, and spinal cord. Using this methodology, we examined the intracellular lipid deposits in healthy or diseased mouse tissues, including fatty liver, ischemic cardiomyocytes, and in the atherosclerotic aortic wall. Moreover, multiharmonic, combined THG and second harmonic generation (SHG) images can be assessed qualitatively and quantitatively to evaluate 3D-distribution of the cells in label-free tissues and organs. In addition, we demonstrated the feasibility of multimodal imaging by combining harmonic signals with fluorescence-excitation of red-shifted fluorescent proteins expressed in vivo. This methodology uses commercially available turn-key systems providing a versatile tool to track resident label-free cells in their undisturbed surroundings, as an alternative or complementary contrast mechanism to fluorescence-based techniques. THG imaging could especially facilitate development of noninvasive approaches for optical biopsies of various tissue-types in diagnostic medicine.

Scientific Focus Area: Cell Biology

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