Figure 1
From: Imaging multiple phases of neurodegeneration: a novel approach to assessing cell death in vivo
Confocal scanning laser ophthalmoscopy (cSLO) and detection of fluorescent-labelled cells. Simultaneous detection of multiple markers was achieved by customising the cSLO as follows: for identification of annexin 488, an argon laser wavelength of 488 nm and a solid-state photodetector with a 521 nm cut-off filter and a 550 nm cut-on filter were used; for DiI, a diode-pumped crystal green laser with a wavelength of 532 nm was used with 550 nm cut-off and 600 nm cut-on filters; the same laser was used for PI visualisation but with a 650 nm cut-off. The inset graph shows the emission spectra of cellular marker fluorochromes used in this study. Alexa Fluor 488-labelled annexin V (green; annexin 488), DiI (blue; 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate) and propidium iodide (red; PI) have excitation/emission maxima of 495/519, 550/570 and 532/649 nm, respectively. (a–d) Monochrome images from each retinal area were acquired using the appropriate laser setup and colourised. Three-colour images were produced by combining each of the grey scale images from the cLSO into the green (a, annexin 488), blue (b, DiI) and red channels (c, PI) of an RGB colour image, and merged to form the image shown in (d) (scale bar 100 μm)
