Hyperspectral expansion microscopy

Abstract

Expansion microscopy (ExM) is a new tissue preparation technique that has the potential to revolutionize immunofluorescence imaging for histology [1, 2]. By physically expanding a biological specimen embedded in a dense swellable polymer, one can achieve an effective increase in resolution of «5X using an ordinary fluorescence microscope. The probes used for immunofluorescence in ExM are standard fluorescent conjugated antibodies and proteins. The photosensitivity of these fluorophores limit their usable lifetime and emission brightness. These limitations significantly reduce their effectiveness in ExM since the 5X lateral expansion results in an ≈125X reduction in signal per focal volume. In addition, traditional fluorophores have broad excitation and emission spectra that limit multiplex imaging to the number of spectra that can “fit” into the spectral bandwidth, largely comprising visible wavelengths.