Fluorescence microscopy

fluorescence microscopy forms images from spatially resolved fluorescence. Probe partitioning, optical resolution and photophysics constrain what a fluorescent pattern establishes about molecular composition or contact.

A fluorescent tracer is a marker detected through fluorescence whose distribution is used to infer transport, mixing or location. In a fluid experiment, a small amount of fluorescent dye can reveal where an injected portion of water travels. The marker's own motion and chemical interactions determine what it faithfully tracks.

A possible optical arrangement illuminates a sample at an excitation wavelength, rejects most excitation light with an emission filter, and records the remaining fluorescence with a detector. A fluorometer can turn this into a concentration measurement. For fixed optical conditions in a calibrated dilute regime, an illustrative model iswhere $c$ is dye mass concentration. A fluorescence calibration curve establishes the usable range and background. Assuming linearity at every concentration would hide effects of absorption, quenching or detector response.

The same marker can measure a volumetric flow rate. For steady injection at rate $q$ and stock concentration $c_s$ into an upstream flow $Q$ with background concentration $c_0$, complete downstream mixing givesFor $q=0.010\mathrm{L}{\mathrm{s}}^{-1}$, $c_s=1000\mathrm{mg}{\mathrm{L}}^{-1}$, $c_d=2\mathrm{mg}{\mathrm{L}}^{-1}$ and $c_0=0$, the inferred upstream flow is $4.99\mathrm{L}{\mathrm{s}}^{-1}$. The extra $0.01$ litre per second belongs to the injected stream.

The Bureau of Reclamation's manual describes this constant-injection method, requiring known injection rate, adequate downstream mixing and concentration measurement; fluorometers detect dye at levels too faint for visual comparison.^[ref] Adsorption, photobleaching or incomplete mixing can invalidate an ideal conserved-tracer inference. Observing a bright pattern alone does not establish that the dye is a quantitatively faithful measure of every constituent's motion.

Total internal reflection fluorescence microscopy excites labels close to a boundary using the evanescent wave accompanying total internal reflection. Illumination travels through the higher-refractive index side, typically a coverslip, at an angle above the critical angle for total internal reflection. The field penetrates the specimen side but decays with distance from the interface.

A prism or a sufficiently high-numerical aperture microscope objective supplies the required incidence. This confines excitation near the surface and reduces background from deeper fluorescent material, making it useful for surface-associated molecular events. Changing incidence angle changes the depth weighting; an intensity change can therefore reflect vertical motion as well as molecule number.^[ref]

Live-cell imaging follows living biological cells over time. Labels, illumination and immobilization must be checked for effects on the process being measured; an apparently sharp image is not sufficient evidence that the cell remains physiologically unperturbed.

Imaging of biological events in a living organism. Repeated cell tracking can establish event ordering but requires controls for phototoxicity, imaging access and sampling.