Chapter 5 Visualizing cells and the csk visible Light microscopy




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Fluorescence Instrumentation


Four essential elements of fluorescence detection systems can be identified from the preceding discussion: 1) an excitation source, 2) a fluorophore, 3) wavelength filters to isolate emission photons from excitation photons and 4) a detector that registers emission photons and produces a recordable output, usually as an electrical signal or a photographic image. Regardless of the application, compatibility of these four elements is essential for optimizing fluorescence detection. Fluorescence instruments are primarily of four types, each providing distinctly different information:

  • Spectrofluorometers and microplate readers measure the average properties of bulk (µL to mL) samples.

  • Fluorescence microscopes resolve fluorescence as a function of spatial coordinates in two or three dimensions for microscopic objects (less than ~0.1 mm diameter).

  • Fluorescence scanners, including microarray readers, resolve fluorescence as a function of spatial coordinates in two dimensions for macroscopic objects such as electrophoresis gels, blots and chromatograms.

  • Flow cytometers measure fluorescence per cell in a flowing stream, allowing subpopulations within a large sample to be identified and quantitated.

Other types of instrumentation that use fluorescence detection include capillary electrophoresis apparatus, DNA sequencers and microfluidic devices. Each type of instrument produces different measurement artifacts and makes different demands on the fluorescent probe. For example, although photobleaching is often a significant problem in fluorescence microscopy, it is not a major impediment in flow cytometry or DNA sequencers because the dwell time of individual cells or DNA molecules in the excitation beam is short.

Fluorescence Signals


Fluorescence intensity is quantitatively dependent on the same parameters as absorbance — defined by the Beer–Lambert law as the linear relationship between absorbance and concentration of an absorbing species. It is the product of the molar extinction coefficient, optical path length and solute concentration — as well as on the fluorescence quantum yield of the dye and the excitation source intensity and fluorescence collection efficiency of the instrument.


The general Beer-Lambert law applies to dilute solutions or suspensions as:
A = () * b * c
where A is the measured absorbance, () is the wavelength-dependent molar absorptivity coefficient with units of M-1 cm-1, b is the path length, and c is the analyte concentration. In, fluorescence intensity is linearly proportional to these parameters. When sample absorbance exceeds about 0.05 in a 1 cm pathlength, the relationship becomes nonlinear and measurements may be distorted by artifacts such as self-absorption and the inner-filter effect. Because fluorescence quantitation is dependent on the instrument, fluorescent reference standards are essential for calibrating measurements made at different times or using different instrument configurations. To meet these requirements, Molecular Probes offers high-precision fluorescent microsphere reference standards for fluorescence microscopy and flow cytometry and a set of ready-made fluorescent standard solutions for spectrofluorometry


A spectrofluorometer is extremely flexible, providing continuous ranges of excitation and emission wavelengths. Laser-scanning microscopes and flow cytometers, however, require probes that are excitable at a single fixed wavelength. In contemporary instruments, the excitation source is usually the 488 nm spectral line of the argon-ion laser. As shown in Figure 3, separation of the fluorescence emission signal (S1) from Rayleigh-scattered (diffraction) excitation light (EX) is facilitated by a large fluorescence Stokes shift (i.e., separation of A1 and E1). Biological samples labeled with fluorescent probes typically contain more than one fluorescent species, making signal-isolation issues more complex. Additional optical signals, represented in Figure 3 as S2, may be due to background fluorescence or to a second fluorescent probe.

Background Fluorescence


Fluorescence detection sensitivity is severely compromised by background signals, which may originate from endogenous sample constituents (referred to as autofluorescence) or from unbound or nonspecifically bound probes (referred to as reagent background). Detection of autofluorescence can be minimized either by selecting filters that reduce the transmission of E2 relative to E1 or by selecting probes that absorb and emit at longer wavelengths. Although narrowing the fluorescence detection bandwidth increases the resolution of E1 and E2, it also compromises the overall fluorescence intensity detected. Signal distortion caused by autofluorescence of cells, tissues and biological fluids is most readily minimized by using probes that can be excited at >500 nm. Furthermore, at longer wavelengths, light scattering by dense media such as tissues is much reduced, resulting in greater penetration of the excitation light.


Multicolor Labeling Experiments


A multicolor labeling experiment entails the deliberate introduction of two or more probes to simultaneously monitor different biochemical functions. This technique has major applications in flow cytometry, DNA sequencing, fluorescence in situ hybridization and fluorescence microscopy. Signal isolation and data analysis are facilitated by maximizing the spectral separation of the multiple emissions (E1 and E2 in Figure 3). Consequently, fluorophores with narrow spectral bandwidths, such as Molecular Probes' Alexa Fluor dyes and BODIPY dyes, are particularly useful in multicolor applications. An ideal combination of dyes for multicolor labeling would exhibit strong absorption at a coincident excitation wavelength and well-separated emission spectra (Figure 3). Unfortunately, it is not easy to find single dyes with the requisite combination of a large extinction coefficient for absorption and a large Stokes shift


Ratiometric Measurements

In some cases, for example the Ca2+ indicators fura-2 and indo-1 and the pH indicators BCECF and SNARF, the free and ion-bound forms of fluorescent ion indicators have different emission or excitation spectra. With this type of indicator, the ratio of the optical signals (S1 and S2 in Figure 3) can be used to monitor the association equilibrium and to calculate ion concentrations. Ratiometric measurements eliminate distortions of data caused by photobleaching and variations in probe loading and retention, as well as by instrumental factors such as illumination stability..

Comparing Different Dyes


Fluorophores currently used as fluorescent probes offer sufficient permutations of wavelength range, Stokes shift and spectral bandwidth to meet requirements imposed by instrumentation (e.g., 488 nm excitation), while allowing flexibility in the design of multicolor labeling experiments (Figure 4). The fluorescence output of a given dye depends on the efficiency with which it absorbs and emits photons, and its ability to undergo repeated excitation/emission cycles. Absorption and emission efficiencies are most usefully quantified in terms of the molar extinction coefficient () for absorption and the quantum yield (QY) for fluorescence. Both are constants under specific environmental conditions. The value of is specified at a single wavelength (usually the absorption maximum), whereas QY is a measure of the total photon emission over the entire fluorescence spectral profile. Fluorescence intensity per dye molecule is proportional to the product of and QY. The range of these parameters among fluorophores of current practical importance is approximately 5000 to 200,000 cm-1M-1 for and 0.05 to 1.0 for QY. Phycobiliproteins such as R-phycoerythrin have multiple fluorophores on each protein and consequently have much larger extinction coefficients (on the order of 2 106 cm-1M-1) than low molecular weight fluorophores.

Photobleaching


Under high-intensity illumination conditions, the irreversible destruction or photobleaching of the excited fluorophore becomes the factor limiting fluorescence detectability. The multiple photochemical reaction pathways responsible for photobleaching of fluorescein have been investigated and described in considerable detail. Some pathways include reactions between adjacent dye molecules, making the process considerably more complex in labeled biological specimens than in dilute solutions of free dye. In all cases, photobleaching originates from the triplet excited state, which is created from the singlet state (S1, Figure 1) via an excited-state process called intersystem crossing.


The most effective remedy for photobleaching is to maximize detection sensitivity, which allows the excitation intensity to be reduced. Detection sensitivity is enhanced by low-light detection devices such as CCD cameras, as well as by high–numerical aperture objectives and the widest bandpass emission filters compatible with satisfactory signal isolation. Alternatively, a less photolabile fluorophore may be substituted in the experiment. Molecular Probes' Alexa Fluor 488 dye is an important fluorescein substitute that provides significantly greater photostability than fluorescein , ), yet is compatible with standard fluorescein optical filters. Antifade reagents can also be applied to reduce photobleaching; however, they are usually incompatible with live cells. In general, it is difficult to predict the necessity for and effectiveness of such countermeasures because photobleaching rates are dependent to some extent on the fluorophore's environment.

Signal Amplification


The most straightforward way to enhance fluorescence signals is to increase the number of fluorophores available for detection. Fluorescent signals can be amplified using 1) avidin–biotin or antibody–hapten secondary detection techniques, 2) enzyme-labeled secondary detection reagents in conjunction with fluorogenic substrates or 3) probes that contain multiple fluorophores such as phycobiliproteins and fluorescent microspheres.


Simply increasing the probe concentration can be counterproductive and often produces marked changes in the probe's chemical and optical characteristics. It is important to note that the effective intracellular concentration of probes loaded by bulk permeabilization methods is usually much higher (>10-fold) than the extracellular incubation concentration. Also, increased labeling of proteins or membranes ultimately leads to precipitation of the protein or gross changes in membrane permeability. Antibodies labeled with more than four to six fluorophores per protein may exhibit reduced specificity and reduced binding affinity. Furthermore, at high degrees of substitution, the extra fluorescence obtained per added fluorophore typically decreases due to self-quenching.
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