Report
Following Single Cell Fluorescence This article includes t w o AVI movies as supporting information, which can be downloaded for free at http:// pubs.acs.org/ac.
rates, if not faster. This Report reviews how native fluorescence imaging can reveal details about cellular dynamics. Why native fluorescence microscopy?
The single most important piece of apparatus in the biology laboratory is the optical microscope (i). Even relatively inexpensive microscopes have launched budding grade-school students into careers in the life sciences. As we move up the scale in sophistication, spatial resolution, color correction, and special contrast modes are incorporated into the microscope. Binocular viewing, photography, and digital imaging are also added. The last feature is particularly beneficial for examining details, because various contrast-enhancing algorithms can be applied. It is also the key to recording dynamic (time-dependent) events in cells Optical images produced in sequences, or movies, have provided many fine details of the dynamics in cells. To gain even more insight, one would like to have chemical information as well. Among the various forms of optical spectroscopy, fluorescence is inherently the most sensitive. Fluorescence imaging opens the door for following dynamic chemical changes in cells at video Edward S. Yeung Ames Laboratory-USDOE and Iowa State University
Compared to MS, NMR, or vibrational spectroscopy, the amount of chemical information obtained from fluorescence spectrometry is relatively low. It is only slightly higher than that obtained from absorption spectrometry because of the existence of a second wavelength dimension. For this reason, selectivity is usually achieved by chemical derivatization of the target molecule. A host of biological stains have been developed to address this issue. With the advent of the laser, fluorophores that can be excited at common laser wavelengths particularly those from the continuouswave Ar+ laser have been svnthesized and are commercially available With fluoresefficiencies accroaching' detection sensitivity is often not a major concern. Fluorescence derivatization has several inherent limitations because a chemical reaction is involved. First, the measurement approach perturbs the biological system. Generally, the reagent can be modified to be noncytotoxic and can function in the actual cellular environment (pH and ionic strength). However, biological systems tend to respond efficiently to external perturbations. Even standard law-of-mass-
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action considerations will dictate a shift in the chemical equilibrium of the targeted molecule. Careful control experiments must be performed to avoid artifacts. Second, reactions take time. If the ratelimiting step is the chemical-labeling reaction, no useful dynamics can be deduced. The standard practice is to increase the concentration of the reagents to provide favorable diffusion-controlled kinetics, which comes at the expense of greater perturbation of the biological system. Third, the derivatization reaction rate also affects spatial resolution. There will be uncertainties about the point of origin of the dynamic event. For a typical small molecule, diffusion in water at room temperature is roughly one pixel (0.2 urn) every 01 ms (2) Fourth quantitative accuracy cannot be guaranteed While fluorescence spectrometry can boast many orders of magnitude in linearity the derivatization reaction is rarely 100% pffiripnt T h e cianal wiil al«n
be influenced by local variations in the concentrations of the tare-et and by the possi hie dpnlpfion of the rpao-ent
Ideally, fluorescence monitoring of biological processes should be based on the native (intrinsic) fluorescence of the biomolecules. With sensitive charged-coupled device (CCD) cameras, the photon flux in irradiation can be kept low to avoid heating or other cell damage. Moreover, the required photon flux is independent of the time scale of the biological event, which
Dynamics with Native
Microscopy determines the optimalframerate. Typically, tens offramesare needed in sequence to map out the temporal behavior, with eachframerequiring a fixed number of emitting photons to form an acceptable image. However, as long as the excitation source is turned off between exposures, the biological movies require very similar integrated photon fluxes to produce. Thus, the measurement is essentially nonintrusive. With native fluorescence monitoring, temporal uncertainties due to reaction kinetics, quantitative inaccuracies due to equilibrium and spatial spread due to delayed labeling 3TC 3.11 a v o i d e d Both the absorption coefficient and the fluorescence quantum efficiency of biomolecules are substantially less than those for suitably designed dye molecules. The decrease in response is 2-3 orders of magnitude, which makes laser excitation almost a requirement. UV laser lines are available at 275 nm and 305 nm for continuous-wave Ar+ lasers, and at 257 nm and 284 nm for frequency-doubled dr+ rnd Kr+ rasers, respectively. These are expensive commercial units that are rare in the laboratories of cell biologists. The power in each laser line however is sufficient to allow sharing among several independent setups (A variety of other UV wavelengths are available from less sophisticated but more ruffffed pulsed lasers However the beam from these is usuallv not imaging"
Fluorescence imaging opens
the door to following
dynamic chemical
changes in
cells at video rates, if not faster.
straightforward. The higher peak powers are also more damaging to cells.) The lower signal for native fluorescence means that stray-light reduction is a critical issue. As the excitation wavelength decreases, the number of fluorescent contaminants increases dramatically. Contaminants rangefromtrace organics in the buffer solutions to metal-ion centers in the optical components. Quartz objectives are necessary to reduce background fluorescence, although certain types of glass transmit down to 330 nm. The cost of a quartz objective can exceed that of the rest of the microscope hardware. The CCD element also needs to be UV-sensitive Back illumination of the chip and special coatings standard features in highgrade CCD elements The quantum efficiency can approach 30% at 300 nm Native fluorescence imaging comes at a price. Although all proteins and many peptides exhibit native fluorescence, many important biomolecules do not. An aromatic moiety must be present. Selectivity becomes a problem because chemical or biochemical recognition is not incorporated. The absorption and fluorescence spectra are rarely sufficient for discrimination. For example, although phenylalanine, tryptophan, and tyrosine have very different spectral characteristics, the absorption and fluorescence spectra of most proteins are remarkably similar. This similarity occurs because efficient energy transfer from
IQ tiot
Analytical Chernistry News & Features, August 1, 1999 5 2 3 A
Report
Figure 1 . Images of an astrocyte cell under different conditions. (a) Transmitted-light image, (b) UV-excited fluorescence image after serotonin uptake, (c) UV-excited fluorescence image after serotonin depletion. (d) Serotonin depleted from different locations of an astrocyte. A indicates cell regions showing a positive correlation between serotonin concentration and depletion rate. B indicates cell regions showing a high serotonin concentration but slow depletion rate. C indicates cell regions showing a low serotonin concentration but fast depletion rate, (e) Mapping of rates of serotonin depletion from the corresponding cell regions. Each frame is 80 x 135 urn. The reconstructed images in (d) and (e) were binned 2 x 2 . (Adapted with permission from Ref. 19.)
tyrosine to tryptophan favors emission from the latter ffuorophore. The only common exception is insulin, which lacks the tryptophan group and thus shows a characteristic short-wavelength fluorescence. DNA possesses little native fluorescence at physiological pHs (3). However, DNA is usually not the subject of biodynamic investigations except for cell division. Of substantial interest is cell signaling. For example catecholamines are an important subgroup involved in signal transduction. Fortunatelv many are naturally fluorescent (4) and can be followed wiih microscopy Because the selectivity associated with native fluorescence microscopy is poor, additional knowledge of the cellular components is needed to properly interpret the results. However, in a temporal sequence of images (a movie), subtracting each frame from the first frame (flat-fielding) can cancel the fluorescence emitted by stationary cellular components, such as the membrane proteins. Chemical changes in the cell (dynamics) can thus be revealed. Other confirmatory experiments can be performed. By using a large number of cells, the average dynamic behavior can be studied with other, more selective chemical probes. For example, biological tissues or cell cultures can be perfused in vivo or in vitro and sampled by microdialysis. Tech524 A
niques such as microelectrodes (5, 6)) LC (7), or CE (8-10), especially if followed by MS (11,12), can provide positive identification of the contents, even at the level of single cells. With a firm knowledge of the types and general quantities of the chemical species associated with each cell, dynamic measurements by native fluorescence microscopy can be highly informative. Blueprint of the setup
State-of-the-art CCD cameras have individual detector elements, or pixels, which are typically 20 um wide. With a lOOx microscope objective, the smallest dimension imaged is 0.2 um, right at the diffraction limit of light. So, there should be no loss of spatial resolution. To preserve picture quality and to faithfully reproduce rapidly changing quantitative information, the CCD element should have low noise, which is readily achieved by cooling, and a fast analog-to-digital converter with a large dynamic range Frame transfer has essentiallv eliminated all dead time between frames.n theframe-transfermode ,he image is first shifted raoidlv to a nonexposed area of the CCD T h e next imaffp
can then be acauired immediately while the first imacp is hpincy dicritiypH
At 1 MHz and at 5 MHz, a CCD camera's digitization resolution is presently
Analytical Chemistry News & Features, August 1, 1999
14 bits and 12 bits, respectively. So, a 512 x 512 image can be acquired at 4 Hz and 20 Hz, respectively. The latter is at a video rate approximating human visual perception. However, the increasedframerate implies shorter integration times, so more intense light sources are needed. Alternatively, image intensifiers have been added to the CCD element to boost the photon counts beyond the read noise, which is usually equivalent to 6-8 photoelectrons. However in this case both the linearity and the dynamic range of the compromised because of the inherent nature of microchannel plates Degraded spatial resolution and a distorted image can also be problematic depending on the coupling between the intensifier and the CCD element On the nr»
