Supplementary Fig. 1: How objective magnification affects the brightness of a CLSM image.

a, Schematic showing illumination and light collection for 40×/1.4 NA and 63×/1.4 NA objective lenses. The same objective is used for both illumination and collection in an epifluorescence geometry, but for clarity, the beampath has been unfolded. The incoming laser beam is focused to a small point in the specimen plane: the PSF. The shape of the PSF is determined by the NA of the objective. The intensity of the PSF depends on the size of the back aperture of the objective, which changes with the magnification of the lens. The incoming laser beam overfills the back aperture of the objective, with the result that the back aperture crops the outer rays of the laser beam, reducing the intensity of the incoming laser beam accordingly. For example, when switching from a 40×/1.4 NA objective to a 63×/1.4 NA objective, the aperture area is reduced by a factor of 40²/63² = 0.40. Hence, we would expect a CLSM image through the 63× objective to be ~40% of the intensity compared to using the 40× objective, if the NA and quality of the lenses are identical. Does this mean that a 40×/1.4 NA objective is more sensitive than a 63×/1.4 NA objective because of the increased brightness through the 40× lens? No. The increased brightness for the 40× lens stems from the fact that more of the laser beam passes through the aperture and hits the sample: this change in intensity by means of a physical aperture is no more helpful than changing the laser power in the software. On the other hand, a higher NA is always helpful for confocal microscopy since the collection efficiency of the objective increases with the NA² independent of magnification. b, CLSM image of a mouse kidney slide labeled with Alexa Fluor 488-WGA (Molecular Probes Prepared Slide #3) taken with a 40×/1.4 NA objective. c, CLSM image of the same field of view as b, taken with a 63×/1.4 NA objective using the same imaging parameters as b. The mean intensity of the image is reduced by ~36% in the 63× lens compared to the 40× lens. However, the power of the laser beam, which was measured using an oil immersion–compatible power meter (Thorlabs PM400 console with S170C sensor), was also reduced by 34%. This demonstrates that the dominant effect of changing magnification is to crop out a portion of the laser beam, for which one could easily compensate by increasing the laser power accordingly. The scale bar is 10 μm.