What you see under the microscope · Fluorescence

What you see with a fluorescence microscope

A layer of endothelial cells stained with three fluorescent dyes, like the commercial FluoCells No. 1 slide: DAPI in the nuclei, phalloidin with Alexa Fluor 488 on the actin and MitoTracker Red CMXRos in the mitochondria. Each dye absorbs one colour of light and emits another, of longer wavelength.

Change the filter in the panel: UV, blue or green.

100×: the layer of cells

Endothelial cells form a layer of flat cells, side by side, like cobblestones. With the blue filter you can see their actin skeleton.

400×: one filter, one dye

Under 365 nm UV light the nuclei glow blue (DAPI). Under 485 nm blue light, the actin glows green (phalloidin). Under 525 nm green light, the mitochondria glow red (MitoTracker). The emission filter lets through the light emitted by the dye and blocks the light that illuminates it.

1000×: filaments and mitochondria

With the immersion objective you can make out the actin filaments and the mitochondria one by one. If you leave the light on, the fluorescence gradually fades: this is photobleaching.

A cell, up close

The nucleus, the actin skeleton and the mitochondria, each with its own dye. Under the microscope you see them separately, one with each filter.

3D view: under the microscope it looks flat, seen from above.

Which fluorescence microscope do you need?

You need an excitation light source and a filter set for each dye. The models that list fluorescence on their spec sheet have UV, blue and green LEDs with their filters.