DAF-2 is one of the most commonly employed fluorescent probes for NO detection. To complicate NO measurements in biological samples using DAF-2, DHA reacts with DAF-2 under physiologically relevant conditions to form a unique series of fluorescent products, DAF-2-DHAs (Zhang et al., 2002
), which have almost the same fluorescence spectra as DAF-2-T, the fluorescent product of NO and DAF-2 (). DAR-4M is another frequently used fluorescent reagent for NO with several distinctive characteristics. For example, DAR-4M uses lower energy excitation at 560 nm, which results in less damage to biological samples.
Not surprisingly, given their similar reactive structures, DAR-4M also reacts with DHA, with the fluorescence profile of the DAR-4M-DHA product also showing high similarity to that of DAR-4M-T. In fact, the reaction of DAR-4M with DHA occurs to a greater extent than the reaction between DAF-2 and DHA. Semi-quantitative calculations based on the calibration curves of triazole fluorescence versus NO concentration indicate that 1 mM DHA with DAR-4M produced a similar fluorescence signal compared to ~100 μM NO (data not shown). In contrast, the fluorescence signal of the reaction mixture of DAF-2 with 1 mM DHA was similar to ~300 nM NO-donor NONOate (Zhang et al., 2002
). An interesting possibility is that the dyes themselves, by reacting with DHA, affect levels of DHA and AA in the cell; significant DHA and AA concentration changes would alter the redox state of the cell, modify NO production by NOS, and have an effect on NO lifetime. However, the concentrations of the probes are at much lower levels than DHA and so this is considered unlikely.
As DAFs and DARs both react with NO and DHA but to different extents, and the DAF derivatized products have different fluorescent profiles from DAR derivatized products, a ratiometric method has been created to produce images of NO and DHA levels. Because DHA is ubiquitously present in many cells at micromolar to millimolar levels (Kim et al., 2002
), in contrast to the nanomolar physiological levels of NO, even poor reactivity of these fluorescent reagents with DHA can generate significant fluorescence artifacts that lead to erroneous information on NO production. For a solution containing NO and DHA, their concentrations can be measured by using both DAF and DAR simultaneously. Therefore, the dual dye approach may eliminate the interference of DHA when measuring NO in samples, and allow DHA imaging without interference from NO.
We have shown that a linear regression model yields a linear calibration for the fluorescence response to different NO and DHA concentrations (R2
= 0.997). The scheme is capable of measuring DHA concentrations accurately, in our case, with a 3% error. The NO measurements deviate further from the true values and can be off several-fold at lower concentrations of NO. The issue relating to NO quantitation likely occurs for two reasons. First, DHA concentrations in the standard solutions, when chosen to be similar to physiological levels, are more than 100-fold higher than those of NO. More importantly, the absolute NO levels of standard solutions are not known precisely. While a saturated NO solution can be created and diluted to lower concentrations, NO is not stable, especially under dilute conditions. This makes accurate quantitation problematic. Our results indicate that the quantitative approach is well-suited for DHA measurements and eliminates the effects of DHA on NO measurements, but is not ideal for NO quantitation in the presence of high levels of DHA. If this occurs, then other approaches may be necessary (Kim et al., 2006
; Ye et al., 2004
The major goal of this work is not quantitation but is to develop protocols for imaging either or both NO and DHA in the presence of the other compound. By using both DAF-2 and DAR-4M, fluorescence images of NO and DHA can be created. One issue encountered with NO fluorescence imaging in biological samples using the DAF/DAR ratiometric method is the intracellular sequestration of the fluorescent dyes. With the incubation conditions commonly used such as 10 μM DAF-2 DA and 10 μM DAR-4M AM, the intracellular concentration of these dyes can reach the millimolar range (Rodriguez et al., 2005
). We observed strong fluorescence in PC12 cells. For example, the green fluorescence signal from DAF-2-T in PC12 cells is stronger than that from the mixture of 10 μM DAF-2 and a saturated 2 mM NO solution. The physiological range of NO production is from nanomolar to micromolar levels; thus it appears that the intracellular concentration of the dye is higher than its extracellular level in this case. Due to the accumulation of DAF-2 and DAR-4M inside the cell, calibration obtained with 10 μM DAF-2 and 10 μM DAR-4M reflects relative NO concentrations and not absolute levels.
The punctate nature of the fluorescence from PC12 cells suggests that the fluorescence imaging of NO using the DAF/DAR ratiometric method is capable of producing sufficient spatial resolution at the subcellular level. The observation of punctate fluorescence is in agreement with previous studies on NOS detection by immunohistochemistry and histochemistry (Gonzalez-Hernandez et al., 1996
; Hecker et al., 1994
). NOS is found within cells in association with subcellular organelles, especially with the endoplasmic reticulum and nuclear envelope and sometimes within the Golgi apparatus, and with the mitochondrial membrane (Faber-Zuschratter and Wolf, 1994
; Hecker et al., 1994
; Rothe et al., 1998
). NO detection using DAF-2-DA has demonstrated punctate fluorescence in cultured neurons (Chen et al., 2001
) and in brain slices (Brown et al., 1999
; Buskila et al., 2005
). L-NAME is an inhibitor of the constitutive isoforms of NOS (Moore and Handy, 1997
). In the presence of L-NAME, the average maximal fluorescence intensity of single cells greatly decreased. This result indicates that the NO observed is produced by constitutive NOS (e.g., nNOS). The punctate fluorescence seen within the cytoplasm of PC12 cells in the present study may therefore represent point sources of NO production. However, as the fluorescence does not diffuse away from the point sources even after a several-hour observation, it is more likely that these punctate sources represent sequestration of the probes rather than point sources of NO generation.
The improvement on NO fluorescence imaging using the DAF/DAR ratiometric method is noticeable in . The NO image obtained by using DAF-2 DA only shows a high level of background fluorescence from DHA, which is evenly distributed in the cells and is not suppressed by carboxy-PTIO (). The NO image acquired by using the DAF/DAR ratiometric method abolishes the fluorescence from DHA, thus the effects of the NO scavenger on NO concentration is obvious.
In differentiated PC 12 cells, individual punctate fluorescence observed at the fiber tips is brighter than the individual fluorescent puncta seen within the cell soma. This result is consistent with the immunofluorescence patterns of nNOS demonstrated in nerve growth factor differentiated PC12 cells, in which bright punctate regions of nNOS immunofluorescence was observed along the perimeter of cell soma and in the neurites (Arundine et al., 2003
). An increasing number of experimental studies have implicated NO as a positive regulator in neurite outgrowth (Bicker, 2005
; Poluha et al., 1997
; Rialas et al., 2000
). It has been shown that nNOS expression and NO production is required for the differentiation of PC12 cells (Hindley et al., 1997
; Peunova and Enikolopov, 1995
; Phung et al., 1999
). The DAF/DAR ratiometric method provides a new approach to directly study the modulatory role of NO in PC12 cell differentiation. The fluorescence images obtained using this methodology demonstrate NO production in PC12 cells with subcellular resolution, in good agreement with the immunofluorescence patterns of nNOS in PC12 cells.
We conclude that this accessible fluorescence imaging method effectively allows imaging of relative NO production in living cells without interference from DHA, is useful for identifying nitrergic cells in biological systems, and has application to a number of studies involving cellular NO production. Lastly, this ratiometric approach can be extended to detection and imaging of other compounds and fluorescence probes with similar interference problems.