Evaluating different 3D fluorescence microscopy techniques

Saw this on the Confocal list… Several times in the last few years I and others in the lab have debated the advantages and disadvantages of different fluorescence microscopy techniques. As many of you know, fluorescence microscopy is becoming increasingly important for many cool neuroscience techniques. But equally important in knowing how to properly image fluorescence.

Here’s a really thorough 2007 article from J. Microscopy that does a nice job of comparing wide-field/deconvolution, spinning disk confocal, and laser scanning confocal microscopy. Punchline is after the jump.

A recommendation based on specimen contrast:

So that our work may achieve its goal of establishing guidelines for choosing between different modes of 3-D microscopy, we summarize here the main result of our comparisons. First, the magnitude of a simple specimen-dependent parameter that we have called the haziness index (H) provides a numerical criterion for choosing the most appropriate mode of microscopy. H is computed as a ratio of background in a WF image over signal: the intensity of background from out-of-focus fluorescence divided by the intensity of the fluorescence from a small in-focus object. ‘Small’ here means comparable in size to the Airy disk. In practice, the intensity of such small objects will be unmeasurable in a WF microscope for specimens with very large background, so it will typically be necessary to estimate H by some indirect means. For instance, it will often be possible to find a thin edge of the specimen where the background is not overwhelming and measurement of the intensity of some small object of interest is possible in a WF microscope. This number could then be combined with a WF measurement of the background in the thickest region of the specimen to give a good estimate of H. Alternatively, if both signal and background could be measured in a thin specimen, then knowing its thickness relative to a thicker specimen would be sufficient to calculate the value of H in the regions too thick for WF microscopy. In this regard, the graph in Fig. 2(B) will be useful, showing the decreasing contribution to the background from planes that are very far from focus.

Naturally, this sort of extrapolation will be somewhat imprecise, but fortunately all that is required is an order-of-magnitude estimate of H. For specimens with H less than ~20, WF microscopy with deconvolution can provide the best images. Over the range 20 < H < 200, spinning disk confocals are the best choice. From 200 < H < 1000, it will probably be necessary to use a spot scanning confocal, and beyond H = 1000, none of these methods is likely to be satisfactory.

I highly recommend taking a look at Figure 7 and the Results section on Contrast if you’re interested in the specifics. This paper seems very thorough.

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