Absorption and Emission Red Shift in Stained Cell and Tissue Samples

The design of fluorescence microscopes and selection of dyes are better served by the knowledge of environmental effects on dyes, whether they are in vitro or in vivo. All significant factors are related in the sense that the environment around the dye is being modified and, depending on the extent of environmental changes and type of changes, the shift in optical properties can be dramatic.1–3 This presents a major challenge when one attempts to image multiplexed signals to obtain quantitative information. In assays, it is critical to be able to understand and control these environmental influences in order to provide accurate analytical quantitation.4,5

In most studies, scientists rely on optical properties reported in the literature that were usually measured in a particular solvent. Little if any consideration is given to the above-mentioned environmental effects unless the study itself is being conducted to define the environment. For example, 4′-6-diamidino-2-phenylindole (DAPI) is used routinely to stain nuclei in cell-based assays or tissue samples. Usually, a UV laser at 355 nm or 375 nm is used to excite DAPI, although UV lasers are expensive and UV fiber couplers are not readily available. The filter set selection is generally based on in-solution spectra.

Photon collection efficiency

A high-level process map for photon collection efficiency (PCE) is depicted in Figure 1. The PCE transfer function can be defined as:

Figure 1 - Process map and main factors affecting photon collection efficiency. The study was focused on the environment-induced change in Stokes’ shift value (a.k.a. red shift) in biological samples.

PCE = ∫ [light source (λ)] * illumination optics (λ) * sample spectra (λ) * filters (λ) * detector ((λ) dλ~/∫
[light source (λ)] dλ

For fluorescence imaging systems in general, all of the above factors are significant and should be taken into account. Vendors of these systems usually provide spectral characteristics of the light source and detector. However, stained sample spectral properties are not readily available; thus engineers and scientists often rely on dye properties from a reagent catalog or a publicly available database (www.photochemcad.com). The latter is measured in solution. Ignoring spectral red shift in the samples may lead to suboptimal hardware design because exciting the fluorophore at a lower absorption value has a lower probability of producing a fluorescent photon than excitation near its peak absorption. Furthermore, collecting fluorescent photons with a bandpass filter outside of the dye emission peak exacerbates the problem. A filter wheel allows users to employ application-specific filters. Unfortunately, illuminator modification can be costly after an excitation source is selected.


Materials and methods

A variety of cell cultures stained with commonly used dyes were investigated. For example, HCT116 cell cultures on microscope slide substrates (75 × 25 × 1 mm) (Corning, Corning, NY) were investigated; the cells were grown on polylysine (PL)-coated coverslips and stained nonspecifically with a Cy5-coupled secondary antibody at three different dilutions of antibody (1:100, 1:300, and 1:1000) in a phosphate-buffered saline (PBS) buffer sealed with rubber cement. PL controls were used in the spectroscopic measurements.

Accurate analysis of histopathologic tissue samples is important in molecular imaging. A variety of stained tissue slides were interrogated for spectral red shift, e.g., breast and colon tissue samples (Figure 2) labeled with β-catenin and Cy dyes (GE Healthcare, Piscataway, NJ).

Figure 2 - Fluorescent image of Cy3-β-catenin-stained breast tissue section, original magnification 20×.

Images of Cy5- and Cy3-stained gels on polyvinylidene fluoride (PVDF) membrane were analyzed. Cy dye and Alexa Fluor®-labeled oligo microarrays (Invitrogen, Carlsbad, CA) (Figure 3) were studied earlier. Commercial fluorescence microscopes and scanners were used, including INCell and Typhoon (both from GE Healthcare), Axio (Carl Zeiss, Thornwood, NY), as well as a benchtop confocal scanning laser microscope.

Figure 3 - Image of a fragment of Cy3-labeled microarray (pseudocolor), 45-μm spot diameter.

Spectroscopic measurements

The fluorescence measurements were made to accommodate a wide range of light wavelengths and samples mounted on different substrates. Laser-induced fluorescence excited by R, G, and B lasers was analyzed first using a portable spectrophotometer. For greater accuracy, it was subsequently replaced with a model FS 900 spectrofluorometer (Edinburgh Instruments Ltd., Livingston, U.K.). The latter system corrects for the spectral responses of the xenon–arc lamp, monochromators, and photomultiplier tube (PMT), and has an excitation–emission mapping capability. Some data sets were replicated on a Fluorolog®-3 spectrofluorometer (Jobin Yvon, Edison, NJ) and were found to be in good agreement. 

Results and discussion

Figure 4 shows measured and catalog emission spectra for DAPI-stained sample. The data were corrected for variation of incident illumination power and normalized to a common vertical scale. The emission spectrum in the sample (curve 1) exhibits a significant shift to the red relative to the in-solution spectra (curve 2), as well as a noticeable change in peak shape. The red shift can be rationalized in terms of the large change in polarity and polarizability of the fluorophore environment. The peak shape change is most likely due to nonspecific attachment on the substrate surface and in the media.

Figure 4 - Normalized fluorescence emission spectra of DAPI dye in-solution (curve 1) and in a stained breast tissue (curve 2). Emission peak red-shifted by 29 nm.

It is worth noting that the Hoechst-stained sample exhibited different spectral behavior with a smaller emission red shift and a 50% lower quantum efficiency than DAPI, all other conditions being equal (see Table 1). Both dyes are used to stain nuclei, but it is expected that DAPI samples will look brighter under 405-nm excitation.

Table 1 Emission peak maximum: measured in the samples and catalog (in-solution) values

An emission peak red shift of 5–8 nm was typical for the enhanced green fluorescent protein (eGFP) samples the authors studied. This is good news, because a new 473-nm blue laser module can be used as well as a 488-nm standalone laser in an eGFP fluorescent channel.

Eosin-stained tissue spectra were analyzed, and a 15-nm emission red shift was present. While the value was consistent across all three slides, a larger sample size is needed to draw a conclusion due to a broad emission peak and known batch-to-batch variability.

A strikingly high value of emission peak shift was observed in tissue samples labeled with Cy-dye β-catenin (Figure 5). This shift was accompanied by considerable peak broadening, believed to be from a nonspecifically bound dye–conjugate in the tissue and on the substrate.

Figure 5 - Normalized fluorescence emission spectra of Cy3-β-catenin in-solution (curve 1) and in a stained breast tissue (curve 2). Emission peak red-shifted by 43 nm.

Red shift was encountered and analyzed in streptavidin–Cy dye-labeled microarrays during CodeLink BioChip (Applied Microarrays, Tempe, AZ) development. The effect was significant and exhibited the same magnitude for Cy dyes (Figure 6) and Alexa dyes (not shown), and varied depending on the substrate type, similar to the results demonstrated in Ref. 3. Smaller values of red shift were observed for Cy dye-stained gels on membrane.

Figure 6 - Normalized fluorescence emission spectra of streptavidin–Cy3 conjugate (curve 1) and on oligo microarray spotted on a glass substrate (curve 2). Emission peak red-shifted by 49 nm.

Table 1 summarizes the salient value of the emission spectra described above. Red-shift values in nm were converted to eV (E = h*c/λ) and the values varied from 0.01 to 0.2 eV. How much does it affect the transfer function (*)? Computing the integral (*) is a straightforward job, though quite tedious. The transfer function values were estimated using Microsoft® Excel (Redmond, WA), although the authors adopted high-throughput tools such as PhotochemCAD (Carnegie Mellon University [Pittsburgh, PA] and North Carolina State University [Raleigh, NC]) and Curve-O-matic (Omega Optical, Brattleboro, VT). The former is a spectral database with a built-in feature to search for peak maximum; the latter allows the plotting of dye spectra using a selected excitation source. The transfer function values with and without spectral red shift are summarized in Table 2. Quantum efficiency of the detector is factored in, while optical transmittance of the illuminator and objective assumed constant within the 40-nm bandpass. A 40-nm emission bandpass filter is centered on a wavelength corresponding to the peak emission in solution or in the sample (the adjusted values are in brackets).

Table 2 Calculated photon collection efficiency adjusted for absorption and emission red shift

Summary and conclusion

Figure 7 - Three-color image of a gut tissue section on glass substrate. Nuclei: Hoechst dye excited with 405-nm laser, 490/40 bandpass filter; laminin: Cy3 dye excited by 532-nm module, 580/40 bandpass filter; actin: Cy5 dye excited by 671-nm module, 700/40 bandpass filter.

Important improvements to fluorescence microscope hardware include:

  • A low-cost 405-nm laser that can replace more expensive UV lasers in the DAPI channel; the 405-nm laser is better suited for fiber optic-coupled illumination than UV lasers
  • In an eGFP channel, a 473-nm laser can replace a 488-nm laser without sacrificing efficiency
  • In a Cy3 channel, a 555-nm laser can increase PCE up to 50% compared to a 532-nm laser; a 671-nm laser can be used with Cy5 and Alexa 647 dyes with the same effectiveness as a 635-nm laser.

Figure 7 illustrates experimental results that support the following conclusions: Cell nuclei tagged with Hoechst (blue) were excited by the 405-nm laser, Cy3 dyes were excited by the green 532-nm laser, and Cy5 dyes (red) were excited by the 671-nm laser.


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Dr. Barash is a Physicist; Dr. Fomitchov is Staff Engineer; Dr. Filkins is Instrumentation Engineer; Mr. Mayers is Optical Engineer; Dr. Sood is a Chemist; Dr. Potyrailo is Principal Scientist; Dr. Xia is a Physicist; and Dr. Montalto is Manager, Molecular and Cell Biology Lab, GE Global Research, One Research Cir., KWC285, Niskayuna, NY 12309, U.S.A.; tel.: 518-387-7407; e-mail: [email protected]. The authors thank various GE colleagues for providing high-quality samples and for graciously allowing use of their equipment.