A new configuration of the Zeiss LSM 510 for simultaneous optical separation of green and red fluorescent protein pairs

Abstract

The power and simplicity of genetically encoded fluorophores (fluorescent proteins, FPs) have drawn many molecular biologists to light microscopy. First generation FPs suffered from overlapping excitation and emission spectra, which limited their use together in pairs (Patterson et al., J Cell Sci 2001;114 (Part 5):837-838). Image acquisition and processing techniques, collectively known as linear unmixing, have been developed to separate overlapping fluorescence signals encountered in the imaging of FP pairs and also in FRET. These specialized techniques are not without their potential drawbacks, including limitations on sensitivity and time-resolution for live cell imaging, and the risk of artifact in the hands of nonspecialists. With the advent of a new generation of red-shifted FPs (Shaner et al., Nat Biotechnol 2004;22:1567-1572 Verkhusha and Lukyanov, Nat Biotechnol 2004;22:289-296) careful selection of excitation sources and emission filters obviate the need for linear unmixing when simple two channel imaging of FPs is required. Here we introduce a new configuration of the Zeiss LSM 510 laser scanning confocal microscope, optimized for live cell imaging of green fluorescent protein (GFP) together with spectral variants such as mRFP1 and mCherry using standard photo-multipliers. A 2 mW, 594 nm HeNe laser was chosen as the excitation source for the red FP. This wavelength efficiently excites the aforementioned red variants without limiting the detection range of GFP emission during simultaneous two-channel imaging. Compared to excitation of GFP and mCherry at 488 and 543 nm, excitation at 488 and 594 nm approximately doubles the sensitivity of GFP detection and eliminates bleed-through of GFP into the mCherry channel. However, sensitivity of mCherry detection is decreased by 30%, suggesting the need for red FPs having longer emission peaks. Practical advantages to the simultaneous optical separation of FPs with nonoverlapping emission spectra include simplicity, robustness, reduced risk of artifact, and increased sensitivity during live cell imaging. q 2006 International Society for Analytical Cytology Key terms: laser scanning confocal microscopy; green fluorescent protein, GFP; red fluorescent protein, mRFP1; mCherry; HeNe laser 594 nm; linear unmixingIn the 1960s, development of epi-fluorescence microscopy was driven by the application of antibody labeling to localize proteins in fixed specimens. Techniques for live cell fluorescence microscopy followed in the 1970s and 80s; however, their utility was limited by the range of available fluorophores and substantially constrained by the available methods for introducing active, labeled biomolecules into living cells (1,2). The cloning of green fluorescent protein (GFP) (3,4) subsequently launched a revolution in cell and developmental biology based on the unparallel specificity and sensitivity for labeling structures within cells and tissues. Fluorescent protein (FP) constructs (5-7) have become widely used genetic tools to address fundamental questions ...

A practical protocol for particle bombardment-mediated transformation of Phyllostachys bamboo suspension cells

“…These FP expressing cells showed detailed clear cytological images as shown in Figures 6C-F. Since a useful configuration of the Zeiss LSM 510 for simultaneous optical separation of green and red FPs has been established (Anderson et al 2006), our findings show promise for further analytical works using these FP genes and LSM imaging in bamboo suspension cells. In the present study, a practical transformation protocol for P. nigra was successfully achieved using a cell suspension culture system.…”

“…Expression patterns of AcGFP1 and mCherry were observed under a fluorescence stereo microscope (SteREO Lumar.V12, Zeiss, Germany) and the LSM. The emission data and images of cells were acquired using lambda scan mode and plane mode, respectively according to Anderson et al (2006) and Lee et al (2008) with modification.…”

Fluid shear stress‐induced JNK activity leads to actin remodeling for cell alignment

“…The z‐slices shown are those with significant actin fibers, typically near the basal surface. To minimize cross‐talk between the FITC (Ex max : 495 nm, Em max : 520 nm) and TRITC (Ex max : 557 nm, Em max : 576 nm) fluorescence labels, the multi‐tracking setting of the Zeiss LSM 510 Meta AIM software was used, which sequentially illuminated and detected one fluorophore at a time (Anderson et al, 2006).…”

Evaluation of intensity‐based ratiometric FRET in image cytometry—Approaches and a software solution

“…In this case, 458 nm and 514 nm laser lines can be used for excitation. Simultaneous optical separation of fluorescent proteins is discussed in (21).…”