Video-Rate Scanning Two-Photon Excitation Fluorescence Microscopy and Ratio Imaging with Cameleons

Abstract

A video-rate (30 frames/s) scanning two-photon excitation microscope has been successfully tested. The microscope, based on a Nikon RCM 8000, incorporates a femtosecond pulsed laser with wavelength tunable from 690 to 1050 nm, prechirper optics for laser pulse-width compression, resonant galvanometer for video-rate point scanning, and a pair of nonconfocal detectors for fast emission ratioing. An increase in fluorescent emission of 1.75-fold is consistently obtained with the use of the prechirper optics. The nonconfocal detectors provide another 2.25-fold increase in detection efficiency. Ratio imaging and optical sectioning can therefore be performed more efficiently without confocal optics. Faster frame rates, at 60, 120, and 240 frames/s, can be achieved with proportionally reduced scan lines per frame. Useful two-photon images can be acquired at video rate with a laser power as low as 2.7 mW at specimen with the genetically modified green fluorescent proteins. Preliminary results obtained using this system confirm that the yellow "cameleons" exhibit similar optical properties as under one-photon excitation conditions. Dynamic two-photon images of cardiac myocytes and ratio images of yellow cameleon-2.1, -3.1, and -3.1nu are also presented.

On-Line Monitoring of Apoptosis in Insulin-Secreting Cells

“…We conclude in this study that TPLSM can be used to study FRET and induction of apoptosis using the C-DEVD-Y construct in insulin-producing cells. The ECFP-EYFP FRET pair was previously used with TPLSM to monitor cytoplasmic free Ca 2ϩ concentration (16). One important consideration in studies of living cells in general and apoptosis in particular is the degree of photo damage generated by the excitation light.…”

Measurements of the free luminal ER Ca 2+ concentration with targeted “cameleon” fluorescent proteins

“…Recent solid-state lasers, which can provide up to 25 mW at 430 nm, might be better suited for this application. YC can also be imaged with two-photon excitation in the wavelength range 770-810 nm [31,32]. Video-rate two-photon imaging of YC provides optimal spatial and temporal resolution, but is unfortunately not readily available to most laboratories.…”

Mitochondria Recycle Ca2+ to the Endoplasmic Reticulum and Prevent the Depletion of Neighboring Endoplasmic Reticulum Regions

“…The "cameleon" indicators based on green fluorescent proteins and calmodulin developed in the group of R. Y. Tsien (43, 44) appear better suited for calcium measurements in organelles. The bright fluorescence of the green fluorescent protein mutants, combined with selective targeting sequences, allows one to visualize the calcium signals in organelles by fluorescence ratio imaging (45)(46)(47). Furthermore, the calcium affinity of calmodulin can be adjusted by molecular engineering, enabling one to match the calcium concentration within the organelle of interest (43 ).…”