We developed a new method, SUT (Scheme Update on tissue Transparency), to view cardiac microstructures and unveil the molecular changes underlying cardiac diseases. SUT is an effective method to clear whole-hearts from different species. Over the course of 4 - 6 days we obtained transparent whole-layer left ventricular tissues from mice with only an approximate 1% protein loss. In addition, EAL (Electrophoretic Antibody Labeling) was used to achieve fast antibody labeling by electric force, which significantly reduced antibody incubation time from days to hours. SUT, together with EAL and modern imaging techniques, were successfully used to visualize three-dimensional spatial distribution of various molecules in cardiac tissue. We also observed changes in the number and phenotypes of fibroblasts during post-myocardial infarction in a stereoscopic pattern. We believe that our technique opens a new avenue to explore the mechanisms underlying cardiac diseases.

A video-rate scanning two-photon excitation microscope (TPEM) has been successfully constructed and tested. The TPEM, based on a Nikon RCM-8000, incorporates a femtosecond pulsed laser, a pre-chirper, and a non-confocal detection box for ratio imaging. Fig. 1 shows the schematic layout of the main components of the instrument, each of which is briefly discussed below.Laser System: A Tsunami Ti: Sapphire laser (from Spectra-Physics) is optically pumped by a 5 W green laser (Millennia from Spectra-Physics) and is capable of generating 100 fs pulses at a repetition rate of 82 MHz and an average power of 0.8 W. The output wavelength is tunable from 690 to 1050 nm with three optical sets, each covering part of the spectrum with some overlapping.Pre-chirper: After leaving the Tsunami, the laser beam enters an optic unit known as a pre-chirper which pre-chirps laser pulses to compensate for the group velocity dispersion which will result when the laser beam goes through the microscope optics.