Second-harmonic generation and two-photon-excited autofluorescence microscopy of cardiomyocytes: quantification of cell volume and myosin filaments

Abstract

The ability to quantify changes in cardiomyocyte and myosin volume across gestation and in response to intrauterine insults will lead to a better understanding of the link between low birth weight and an increased risk of heart disease in adult life. We present the use of second-harmonic generation (SHG) and two-photon excitation autofluorescence (TPEF) microscopy to image unstained isolated fetal cardiomyocytes. The simultaneous collection of these two images provides a wealth of information on the morphology of cardiomyocytes. The SHG signal provides high-contrast images of myosin filaments and the TPEF signal can be used to clearly visualize cell morphology. A potential issue may arise if SHG microscopy is performed exclusively due to the lack of sensitivity to distinguish between mononucleated and binucleated cardiomyocytes. However, TPEF microscopy has the ability to efficiently separate the two types of cardiomyocytes. In addition, quantitative analysis of the SHG and TPEF images enables quantification of myosin filament level and accurate determination of cell volume. In short, we demonstrate that advanced nonlinear optical microscopy can be used to answer key physiological questions in the early origins of adult health with increased accuracy and speed compared to previously used methods.

Two‐photon microscopy of healthy, infarcted and stem‐cell treated regenerating heart

“…Scale bar ¼ 100 mm citation autofluorescence (TPEF) allows imaging of endogenous fluorescent structures such as extracellular matrix (elastin [1,2]) and myocytes [2,3]. Second harmonic generation (SHG), another non-linear optical process, is generated by non-centrosymmetric molecules, including collagen [1,2,4] and myosin [3,5,6].…”

Myofibrillogenesis in live neonatal cardiomyocytes observed with hybrid two-photon excitation fluorescence-second harmonic generation microscopy

“…5,6 Second harmonic generation (SHG) is intrinsic to a specific structure and thus enables the study of dynamical assembly of a myofibril without any protein labeling. [7][8][9][10] The combination of TPEF and SHG (TPEF-SHG) can provide more structural information in a cardiomyocyte, 11,12 which is ideal for tracking how specific sarcomeric proteins are assembled into the myofibrils during myofibrillgoenesis. Compared with corresponding single-photon excitation microscopy, the double wavelength requirement of TPEF and SHG can achieve a deeper penetration inside biological materials.…”

Automated cardiac sarcomere analysis from second harmonic generation images

“…[7][8][9][10][11][12][13] This unique contrast mechanism combined with recent technological advances in multiphoton microendoscopes/fibers [14][15][16] and ultrashort pulse laser sources 17 suggests that SHG might find clinical applications as an optical biopsy tool. 9 SHG has been studied in many applications related with collagen, 10,18,19 as it is a highly efficient frequency doubling biomaterial that can be detected by standard multiphoton microscopes.…”