Fluorescence microscopy

Fluorescence microscopy forms an image from the light emitted by fluorescent molecules in a sample: dyes, fluorescent proteins or quantum dots attached to the structures of interest. Each fluorophore absorbs at one wavelength and emits at a longer one, so filters can remove the excitation light and leave a dark background with only the labeled structures bright. The standard instrument is the widefield epifluorescence microscope, in which the same objective both illuminates the sample and collects the emission. With green emission near 520 nm and an NA 1.4 oil-immersion objective, the lateral resolution is about 230 nm and the axial resolution about 0.8 µm. The molecular process is covered under fluorescence; this entry covers the microscope.

The epifluorescence layout

Light from a lamp, LED or laser passes through an excitation filter that selects the absorption band of the fluorophore, reflects off a dichroic mirror set at 45°, and is focused by the objective onto the sample. The emission, collected by the same objective, has a longer wavelength and passes through the dichroic, then through an emission filter that rejects remaining excitation light, before the tube lens images it onto a camera or the eyepieces. The three elements are mounted together in a filter cube. A typical set for fluorescein or GFP, which absorb near 490 nm and emit near 515 nm, combines a 470/40 excitation filter (center 470 nm, width 40 nm), a dichroic with its edge near 495 nm and a 525/50 emission filter.

Because the emission is weaker than the excitation by many orders of magnitude, and travels back along the same path, the combined blocking of the cube at the excitation wavelength must be very high; the optical filter entry covers the specifications. Epi-illumination helps by sending most of the excitation away from the camera: only light reflected or scattered back into the objective must be rejected.

Resolution, collection and sampling

The lateral resolution follows the Rayleigh criterion of the diffraction limit,

$$d = 0.61\,\frac{\lambda_\text{em}}{\text{NA}},$$

about 227 nm at 520 nm and NA 1.4. A common estimate of the widefield axial resolution is $2n\lambda/\text{NA}^2$, about 0.81 µm for $n$ = 1.518.

The objective's numerical aperture also sets how much emission is collected. Fluorophores emit into all directions, and the collected fraction is $(1-\cos\theta)/2$ with $\sin\theta = \text{NA}/n$: 31% for NA 1.4 in oil, 17% for a dry NA 0.75 objective and 6.7% for NA 0.5. Because the excitation irradiance also grows with NA, the image brightness in epifluorescence scales approximately as NA⁴/M², with M the magnification; at the same NA, a 60× objective gives an image 2.8 times brighter than a 100× objective.

The camera pixel, projected back to the sample, should be no larger than about half the resolution. A 6.5 µm pixel behind a 60× objective samples 108 nm at the sample, just below half of the 227 nm resolution.

Variants

Widefield images include out-of-focus light from the whole illuminated depth, which reduces contrast in thick samples. Confocal microscopy rejects it with a pinhole; multiphoton microscopy excites only at the focus; light-sheet microscopy illuminates one plane from the side; and deconvolution removes part of the blur computationally using the measured point spread function. Total internal reflection fluorescence (TIRF) excites only a layer about 100 nm thick next to the coverslip with an evanescent wave, using objectives of NA 1.45 or more. Super-resolution methods such as STED, PALM and STORM reach tens of nanometers by switching fluorophores on and off.

Pitfalls

• Photobleaching: fluorophores are destroyed after a finite number of excitation cycles, so long exposures and repeated imaging dim the sample; phototoxicity damages live cells at the same time.
• Bleed-through: with several labels, the emission tail of one dye passes the filter of another, so single-label controls are needed before colocalization is claimed.
• Autofluorescence: cells, fixatives and some mounting media and immersion oils fluoresce, especially under blue and ultraviolet excitation.
• Chromatic shift: different colors focus at slightly different depths and positions unless the objective is well corrected, which can mimic or hide colocalization.
• Saturation and nonlinearity of the camera: quantitative comparisons need unsaturated images taken at fixed exposure and illumination.

Common questions

Why is the dichroic mirror needed?

It lets one objective serve as both condenser and collector: it reflects the shorter excitation wavelength toward the sample and transmits the longer emission toward the camera. A 50:50 beamsplitter would do the same with at least a 4× loss in signal and far poorer rejection.

What limits the resolution of a fluorescence microscope?

In widefield and confocal imaging, diffraction: about 0.61λ/NA laterally. Only super-resolution methods, which separate fluorophores in time or in excitation state, go below it.

Why do fluorescence images fade?

Photobleaching. Each excitation carries a small probability of an irreversible photochemical reaction, so the signal falls with cumulative exposure; lower irradiance, shorter exposures and antifade mounting media slow it.

References: J. R. Lakowicz, Principles of Fluorescence Spectroscopy, 3rd ed. (Springer, 2006); J. B. Pawley (ed.), Handbook of Biological Confocal Microscopy, 3rd ed. (Springer, 2006); D. B. Murphy and M. W. Davidson, Fundamentals of Light Microscopy and Electronic Imaging, 2nd ed. (Wiley-Blackwell, 2012); M. Born and E. Wolf, Principles of Optics, 7th ed. (Cambridge University Press, 1999).