Rapid Two-photon Holographic Scan Microscopy with Individual Quantum Dots and Its Applications in Biological Samples By

Two-photon (2P) excited fluorescence microscopy is particularly advantageous for imaging biological specimens since it offers reduced scattering, deep sample penetration, and intrinsic confocality when excited with point excitation. Here we report the first 2-photon microscopy of individual quantum dots in biological environment. Near-complete suppression of blinking and of photobleaching was achieved. Three schemes of 2-photon excitation are shown: 1) widefield, 2) diffraction-limited spot scanning with a single rastered spot, or 3) a multi-point excitation scheme employing a 9 × 9 matrix hologram that increases the scan rate by 80 fold. An array detector (EMCCD camera) was also used as a detector for 2-photon scanning microscopy, providing faster whole image acquisition and superior detection capabilities compared to a conventional single point detector (a PMT). Wide-field 2-photon excitation was used to resolve the step size of individual Myosin V motors in vitro, indicating the 2-photon field does not damage the ATPase of myosin. We validated the helical spatial arrangement of LamB receptors on live E. coli cells with the 2-photon fast hologram scan microscopy. The endocytosis of epidermal growth factor (EGF) receptors in breast cancer cells was verified by imaging quantum dot labeled fixed cell sample with the technique. The strong colocalization of EGFR and HGFR (c-Met) in the same clusters in EGF/HGF stimulated breast cancer cell was also revealed. Moreover, we obtained 3D localization at less than 3 nanometer accuracy and fast 3D imaging at confocal resolution, while doing no harm to the cell samples.