BACKGROUND
cyanine family  of fluorescent dyes have been widely used in parallel microarray detection for array comparative genomic hybridization and gene expression analysis  <cit> .
the large molar extinction coefficients and ease of enzymatic incorporation of cy <dig> and cy <dig> allows both dyes to be combined for high sensitivity detection of low copy targets even when sample amounts are limited  <cit> .
however, a number of reports have been published documenting the instability of cy <dig> dye to elevated ozone levels in the environment causing distortion of gene expression  ratios  <cit> .
in summer months, when environmental ozone levels increase, microarray hybridization experiments can suffer disproportionately from cy <dig> signal loss over time impacting quality of data acquired from arrays.
unlike cy <dig>  the signal and brightness from cy <dig> remains stable during higher ozone periods.
to circumvent cy <dig> signal loss from ozone exposure, branham et.
al.
have proposed an engineering solution based on installation of a carbon-filtration system to eliminate ozone inside laboratories  <cit> .
while these systems can be effective in depleting ozone, they are expensive and difficult to engineer in open laboratory spaces allowing ozone levels to fluctuate posing continuous risk to data quality from often expensive arrays.
cy <dig> signal can also be impacted by dye photobleaching effects.
photobleaching can occur when arrays are exposed directly to light or when partially or even microscopically wet arrays are scanned for image acquisition.
like ozone, photobleaching of cy <dig> leads to reduction of absolute signals obtained from the arrays.
to circumvent these problems, our laboratory has been active in fluorescent dye research to find a suitable alternative to cy <dig> for microarray analysis with brightness and environmental stability matching cy <dig>  here, we report on the development of such a novel red fluorescent dye, known as hyper <dig>  with high ozone- and photo-stability that yields reproducible microarray performance all year round – irrespective of environmental conditions.
in this article, we describe the properties of hyper <dig> dye and compare its performance to cy <dig>  by using in-house fabricated arrays, we show resistance of hyper <dig> signal to repeat exposures of ozone pulses of  <dig> ppb over time.
data is presented on the marked 3– <dig> fold improvement in the photostability of hyper <dig> over cy <dig> following exposure of dyes to incandescent light source.
we functionally demonstrate the ability of hyper <dig> to detect chromosomal aberrations at multiple loci in array cgh experiments using patient samples and ability to perform array rescanning without loss of resolution.
furthermore, gene expression analysis using mouse arrays is demonstrated to show equivalent performance between hyper <dig> and cy <dig> dye in message detection.
microarray labeling data is also presented showing hyper <dig> incorporation rates matching cy <dig>
RESULTS
seasonal effects on microarray data quality have identified ozone to be the root cause of cy <dig> degradation.
an increase in ozone levels to between 5– <dig> ppb are known to severely impact cy <dig> signal  <cit> .
we compared the ozone resistance of new hyper <dig> against cy <dig> by repeatedly exposing in-house fabricated and hybridized mouse arrays to short pulses of  <dig> ppb ozone over time .
for each exposure, the slides were placed inside a box along with the ozone generator.
the  <dig> ppb ozone was delivered from an ozone generator for  <dig> minute intervals lasting up to  <dig> minutes  with immediate scanning after each treatment.
since ozone tends to be very unstable, high  doses of gas were necessary for each treatment.
as shown in figure 1a , a sudden reduction in cy <dig> signal  was observed after two ozone treatments .
yellow features, corresponding to near equivalent cy <dig>  and cy <dig>  signal, now appear to have mostly green cast from disappearance of cy <dig> signal.
in contrast, hyper <dig> signal was unchanged by ozone treatments.
the individual features  did not change their colour after repeated ozone treatments indicating resistance of hyper <dig>  to ozone .
quantification of signals before and after ozone treatments showed an immediate and dramatic drop in cy <dig> signal down to 50% within  <dig> minutes of exposure .
after  <dig> minutes, the detrimental effect of ozone on cy <dig> resulted in greater than 80% signal loss.
in contrast, hyper <dig> signal was found to be completely intact following ozone treatments demonstrating the resistance of hyper <dig> to ozone in the environment.
successive exposures of hyper <dig> arrays to  <dig> ppb ozone for up to  <dig> minutes did not drop its signal.
in fact, a slight increase in hyper <dig> signal was noted after the third ozone exposure .
this data shows that hyper <dig> is resistant to ozone and performs reliably on arrays even under elevated ozone conditions inside laboratories.
along with ozone sensitivity, cy <dig> arrays can also suffer signal loss from dye photobleaching.
the photobleaching properties of microarray dye can be characterized in two ways.
first, one can measure the relative stability of dye from continuous exposure to a light source.
second, in a microarray experiment, dye photostability can be monitored by multiple rescanning of hybridized array to determine if any loss in signal or resolution is seen .
both tests were used to measure the photobleaching property of hyper <dig> against cy <dig>  in the first test, duplicate samples of both dyes were exposed to an incandescent light source for seven days, during that period their absorbance were measured at regular intervals .
the data was normalized to control samples left in the dark for both dyes over the same time interval.
as seen in figure  <dig>  both samples of hyper <dig> dye were found to be completely photostable and resistant to photobleaching with no decline in absorbance readings over time.
in contrast, cy <dig> absorbance measurements showed steady decline from photobleaching with overall difference of more than  <dig> fold compared to hyper <dig> data.
hyper <dig> dye was tested functionally in array cgh assay by comparing its performance against cy <dig>  genomic dna isolated from three different patient samples  along with appropriate control samples were differentially labelled with cy3/cy <dig> and cy3/hyper <dig>  the dye-labelled microarray targets were then hybridized onto commercially available 3k bac arrays  and data analyzed for relative copy number changes using ratio analysis.
as seen in figure  <dig>  hyper <dig> dye labelled targets were able to detect known chromosomal aberration at 2p21- <dig>  and 15q <dig> - <dig>  comparable to cy <dig> targets, although initial signal intensity generated by the hyper <dig> targets were slightly diminished.
the lower brightness coefficient of hyper <dig> relative to cy <dig>  contributes to observed lower signal intensity from hyper <dig> array elements.
hyper <dig> dye was noticed to contribute slightly elevated noise to the data, although this had no impact on the ability to detect aberrations.
the source of higher background is not known but may be attributed to the more hydrophilic nature of hyper <dig> dye.
as mentioned above, dye photostability can also be measured by array rescanning to determine if loss of signal or aberration detection is observed from repeat scanning.
while cy <dig> dye is highly bright, precautions have to be taken to prevent signal loss from light due to photobleaching particularly when imaging is performed using laser-based scanners  <cit> .
we measured photo-stability of both dyes by the ability of arrays to be rescanned multiple times without suffering any distortion in dye ratios.
part of cgh arrays were rescanned multiple times over a period of  <dig> days .
from the data, it is obvious that hyper <dig> signals are as stable as cy <dig> signals whereas cy <dig> performance  dropped quickly in a period of  <dig> to  <dig> days, resulting in loss of aberration detection and an increase in standard deviation .
another important functional application of cy <dig> and cy <dig> lies in expression microarrays for analysis of gene expression  <cit> .
cy <dig> and hyper <dig> dyes were compared side-by-side in gene expression analysis using in-house fabricated mouse arrays.
due to the lower extinction coefficient of hyper <dig> , we found it necessary to use 30% more hyper <dig> labelled cdna onto each array for high sensitivity.
starting from mouse total rna, hyper <dig> was seen to perform similar to cy <dig> and cy <dig> dyes in parallel microarray hybridization experiments in the ability to detect similar number of genes from mouse array .
while hyper <dig> dye is an ozone and photostable version of cy <dig>  we wanted to determine whether labelling efficiency of hyper <dig> was comparable to cy <dig>  to test labelling, we synthesized hyper <dig> both as n-hydroxy-succinimide  reactive dye and as deoxycytidine triphosphate nucleotide  for microarray labelling experiments  <cit> .
starting from an input of  <dig> μg of mouse total rna, we observed more than 2-fold higher labelling yield from hyper <dig> using the nhs reactive dye  with post-labelling method.
with direct enzymatic  incorporation reaction, hyper5-dctp was found to give equivalent target yield to cy5-dctp using conventional first-strand cdna synthesis reactions .
the incorporation efficiency, as measured by nucleotide per dye ratio, of hyper <dig> dye was seen to be comparable to cy <dig>  higher labelling density was achieved with hyper <dig> nhs, although hyper5-dctp incorporation was slightly lower than cy5-dctp.
*nucleotide per dye ratios was determined by /.
data represented is average yield from n =  <dig> for hyper <dig> nhs, n =  <dig> for cy <dig> = nhs, and triplicates for hyper5- and cy5-dctp.
discussion
cy <dig> is a bright fluor with high extinction coefficient.
being in the red region of the spectrum, it offers the advantage of performing microarray assays with little or no background effects.
however, it is prone to oxidative damage caused by ozone in the environment.
here, we have described the properties of an alternative red dye, hyper <dig> that is stable to ozone and light which can generate reliable microarray data.
microarray experiments performed with hyper <dig> under elevated ozone conditions which rapidly degrade cy <dig> show no drop in hyper <dig> signal.
typically microarray laboratories experience cy <dig> signal loss during summer months when ozone levels rise to greater than  <dig> ppb.
we have demonstrated hyper <dig> performs functionally similar to cy <dig> making hyper <dig> a suitable replacement in microarray assays under challenging environmental conditions.
ozone experiments did show a modest increase in hyper <dig> signal intensity after repeated exposures .
the cause of this increase is unknown although rescanning experiments using cgh arrays, where ozone levels were being monitored and remained low, also exhibited a similar modest increase in hyper <dig> signal.
in cgh experiments, the signal increase leads to improved detection of aberrations benefiting from slightly higher signal-to-noise values .
in an attempt to understand the maximal effect of hyper <dig> signal increase, we accelerated this process by placing hyper <dig> arrays in desiccator overnight followed by image acquisition and data analysis.
these results showed an improvement in signal to noise in the range of 5–25% .
while this property of hyper <dig> improves cgh data, it may introduce slight variability in ratio analysis from arrays rescanned multiple times or if arrays were delayed for several hours prior to scanning.
however, the major property of hyper <dig> in providing ozone stability far exceeds the modest rise in hyper <dig> signal obtained from repeat scanning, ozone exposure, or slide storage.
furthermore, the slight increase in hyper <dig> signal did not alter the ratios significantly to impact aberration detection in array cgh experiments.
another difference between hyper <dig> and cy <dig> is exhibited in the photostability of new hyper <dig> dye.
constant exposure of arrays to light during different steps of the microarray experiment can lower cy <dig> signal intensities when arrays are finally imaged.
there is also a slight but significant loss of cy <dig> signal seen from repeat array scanning that is attributed to dye photobleaching.
hyper <dig> dye has been shown to be photostable and resistant to photobleaching.
the main benefit of photostability lies in the ability to perform multiple rescanning experiments without suffering from loss in resolution on array cgh slides.
additionally, stability of hyper <dig> signal may also allow the possibility of archiving cy <dig> and hyper <dig> arrays which were previously impossible due to cy <dig> instability.
functional data indicates that hyper <dig> can be substituted for cy <dig> in array analysis.
the protocols used for hybridization essentially allow hyper <dig> labelled samples to replace cy <dig>  rather than making direct dye swap on arrays, we find it necessary to use ~30% more hyper <dig> labelled targets for array hybridization than conventionally used with cy <dig>  this difference comes from the relatively lower brightness coefficient of hyper <dig> to cy <dig> which needs to be compensated by using more labelled targets for hybridization.
when using hyper5-dctp for labelling, increasing the amount of dye in the reaction will increase yield of labelled targets desired for hybridization.
for the hyper <dig> nhs reactive dye, labelling conditions can be kept identical due to higher coupling efficiency of hyper <dig> nhs compared to cy <dig>
CONCLUSIONS
the new hyper <dig> dye is a more ozone- and photo-stable version of cy <dig> for microarray applications.
we have demonstrated hyper <dig> to be resistant to repeated exposures of ozone at  <dig> ppb released from ozone generator inside a chamber, conditions at which cy <dig> shows dramatic loss of signal.
parallel photobleaching experiments performed on hyper <dig> and cy <dig> show hyper <dig> to be resistant to light-induced damage with 3-fold improvement in dye-stability over cy <dig>  the ozone- and photo-stability of hyper <dig> makes this dye highly suitable for array applications particularly during periods of high ozone and humidity levels in summer months, conditions at which cy <dig> data can be highly unpredictable.
additionally, the photostability of hyper <dig> is particularly advantageous in allowing multiple rescanning of arrays without loss of detection.
the functional use hyper <dig> is demonstrated in array cgh and gene expression experiments to acquire microarray data for copy number analysis.
the lower brightness coefficient of hyper <dig> may dictate the use of more labelled targets for hybridization to maximize array sensitivity.
with high labelling and coupling efficiency of hyper <dig>  it can be easily adapted to existing cy <dig> protocols for both array cgh and microarray gene expression analysis to perform array experiments under any environmental conditions.

 cyanine family of fluorescent dyes have been widely used in parallel microarray detection for array comparative genomic hybridization and gene expression analysis. the large molar extinction coefficients and ease of enzymatic incorporation of cy and cy allows both dyes to be combined for high sensitivity detection of low copy targets even when sample amounts are limited. however, a number of reports have been published documenting the instability of cy dye to elevated ozone levels in the environment causing distortion of gene expression ratios. in summer months, when environmental ozone levels increase, microarray hybridization experiments can suffer disproportionately from cy signal loss over time impacting quality of data acquired from arrays. unlike cy the signal and brightness from cy remains stable during higher ozone periods. to circumvent cy signal loss from ozone exposure, branham et. al. have proposed an engineering solution based on installation of a carbon-filtration system to eliminate ozone inside laboratories. while these systems can be effective in depleting ozone, they are expensive and difficult to engineer in open laboratory spaces allowing ozone levels to fluctuate posing continuous risk to data quality from often expensive arrays. cy signal can also be impacted by dye photobleaching effects. photobleaching can occur when arrays are exposed directly to light or when partially or even microscopically wet arrays are scanned for image acquisition. like ozone, photobleaching of cy leads to reduction of absolute signals obtained from the arrays. to circumvent these problems, our laboratory has been active in fluorescent dye research to find a suitable alternative to cy for microarray analysis with brightness and environmental stability matching cy here, we report on the development of such a novel red fluorescent dye, known as hyper with high ozone- and photo-stability that yields reproducible microarray performance all year round – irrespective of environmental conditions. in this article, we describe the properties of hyper dye and compare its performance to cy by using in-house fabricated arrays, we show resistance of hyper signal to repeat exposures of ozone pulses of ppb over time. data is presented on the marked 3– fold improvement in the photostability of hyper over cy following exposure of dyes to incandescent light source. we functionally demonstrate the ability of hyper to detect chromosomal aberrations at multiple loci in array cgh experiments using patient samples and ability to perform array rescanning without loss of resolution. furthermore, gene expression analysis using mouse arrays is demonstrated to show equivalent performance between hyper and cy dye in message detection. microarray labeling data is also presented showing hyper incorporation rates matching cy  seasonal effects on microarray data quality have identified ozone to be the root cause of cy degradation. an increase in ozone levels to between 5– ppb are known to severely impact cy signal. we compared the ozone resistance of new hyper against cy by repeatedly exposing in-house fabricated and hybridized mouse arrays to short pulses of ppb ozone over time. for each exposure, the slides were placed inside a box along with the ozone generator. the ppb ozone was delivered from an ozone generator for minute intervals lasting up to minutes with immediate scanning after each treatment. since ozone tends to be very unstable, high doses of gas were necessary for each treatment. as shown in figure 1a, a sudden reduction in cy signal was observed after two ozone treatments. yellow features, corresponding to near equivalent cy and cy signal, now appear to have mostly green cast from disappearance of cy signal. in contrast, hyper signal was unchanged by ozone treatments. the individual features did not change their colour after repeated ozone treatments indicating resistance of hyper to ozone. quantification of signals before and after ozone treatments showed an immediate and dramatic drop in cy signal down to 50% within minutes of exposure. after minutes, the detrimental effect of ozone on cy resulted in greater than 80% signal loss. in contrast, hyper signal was found to be completely intact following ozone treatments demonstrating the resistance of hyper to ozone in the environment. successive exposures of hyper arrays to ppb ozone for up to minutes did not drop its signal. in fact, a slight increase in hyper signal was noted after the third ozone exposure. this data shows that hyper is resistant to ozone and performs reliably on arrays even under elevated ozone conditions inside laboratories. along with ozone sensitivity, cy arrays can also suffer signal loss from dye photobleaching. the photobleaching properties of microarray dye can be characterized in two ways. first, one can measure the relative stability of dye from continuous exposure to a light source. second, in a microarray experiment, dye photostability can be monitored by multiple rescanning of hybridized array to determine if any loss in signal or resolution is seen. both tests were used to measure the photobleaching property of hyper against cy in the first test, duplicate samples of both dyes were exposed to an incandescent light source for seven days, during that period their absorbance were measured at regular intervals. the data was normalized to control samples left in the dark for both dyes over the same time interval. as seen in figure both samples of hyper dye were found to be completely photostable and resistant to photobleaching with no decline in absorbance readings over time. in contrast, cy absorbance measurements showed steady decline from photobleaching with overall difference of more than fold compared to hyper data. hyper dye was tested functionally in array cgh assay by comparing its performance against cy genomic dna isolated from three different patient samples along with appropriate control samples were differentially labelled with cy3/cy and cy3/hyper the dye-labelled microarray targets were then hybridized onto commercially available 3k bac arrays and data analyzed for relative copy number changes using ratio analysis. as seen in figure hyper dye labelled targets were able to detect known chromosomal aberration at 2p21- and 15q - comparable to cy targets, although initial signal intensity generated by the hyper targets were slightly diminished. the lower brightness coefficient of hyper relative to cy contributes to observed lower signal intensity from hyper array elements. hyper dye was noticed to contribute slightly elevated noise to the data, although this had no impact on the ability to detect aberrations. the source of higher background is not known but may be attributed to the more hydrophilic nature of hyper dye. as mentioned above, dye photostability can also be measured by array rescanning to determine if loss of signal or aberration detection is observed from repeat scanning. while cy dye is highly bright, precautions have to be taken to prevent signal loss from light due to photobleaching particularly when imaging is performed using laser-based scanners. we measured photo-stability of both dyes by the ability of arrays to be rescanned multiple times without suffering any distortion in dye ratios. part of cgh arrays were rescanned multiple times over a period of days. from the data, it is obvious that hyper signals are as stable as cy signals whereas cy performance dropped quickly in a period of to days, resulting in loss of aberration detection and an increase in standard deviation. another important functional application of cy and cy lies in expression microarrays for analysis of gene expression. cy and hyper dyes were compared side-by-side in gene expression analysis using in-house fabricated mouse arrays. due to the lower extinction coefficient of hyper, we found it necessary to use 30% more hyper labelled cdna onto each array for high sensitivity. starting from mouse total rna, hyper was seen to perform similar to cy and cy dyes in parallel microarray hybridization experiments in the ability to detect similar number of genes from mouse array. while hyper dye is an ozone and photostable version of cy we wanted to determine whether labelling efficiency of hyper was comparable to cy to test labelling, we synthesized hyper both as n-hydroxy-succinimide reactive dye and as deoxycytidine triphosphate nucleotide for microarray labelling experiments. starting from an input of μg of mouse total rna, we observed more than 2-fold higher labelling yield from hyper using the nhs reactive dye with post-labelling method. with direct enzymatic incorporation reaction, hyper5-dctp was found to give equivalent target yield to cy5-dctp using conventional first-strand cdna synthesis reactions. the incorporation efficiency, as measured by nucleotide per dye ratio, of hyper dye was seen to be comparable to cy higher labelling density was achieved with hyper nhs, although hyper5-dctp incorporation was slightly lower than cy5-dctp. *nucleotide per dye ratios was determined by /. data represented is average yield from n = for hyper nhs, n = for cy = nhs, and triplicates for hyper5- and cy5-dctp. discussion cy is a bright fluor with high extinction coefficient. being in the red region of the spectrum, it offers the advantage of performing microarray assays with little or no background effects. however, it is prone to oxidative damage caused by ozone in the environment. here, we have described the properties of an alternative red dye, hyper that is stable to ozone and light which can generate reliable microarray data. microarray experiments performed with hyper under elevated ozone conditions which rapidly degrade cy show no drop in hyper signal. typically microarray laboratories experience cy signal loss during summer months when ozone levels rise to greater than ppb. we have demonstrated hyper performs functionally similar to cy making hyper a suitable replacement in microarray assays under challenging environmental conditions. ozone experiments did show a modest increase in hyper signal intensity after repeated exposures. the cause of this increase is unknown although rescanning experiments using cgh arrays, where ozone levels were being monitored and remained low, also exhibited a similar modest increase in hyper signal. in cgh experiments, the signal increase leads to improved detection of aberrations benefiting from slightly higher signal-to-noise values. in an attempt to understand the maximal effect of hyper signal increase, we accelerated this process by placing hyper arrays in desiccator overnight followed by image acquisition and data analysis. these results showed an improvement in signal to noise in the range of 5–25%. while this property of hyper improves cgh data, it may introduce slight variability in ratio analysis from arrays rescanned multiple times or if arrays were delayed for several hours prior to scanning. however, the major property of hyper in providing ozone stability far exceeds the modest rise in hyper signal obtained from repeat scanning, ozone exposure, or slide storage. furthermore, the slight increase in hyper signal did not alter the ratios significantly to impact aberration detection in array cgh experiments. another difference between hyper and cy is exhibited in the photostability of new hyper dye. constant exposure of arrays to light during different steps of the microarray experiment can lower cy signal intensities when arrays are finally imaged. there is also a slight but significant loss of cy signal seen from repeat array scanning that is attributed to dye photobleaching. hyper dye has been shown to be photostable and resistant to photobleaching. the main benefit of photostability lies in the ability to perform multiple rescanning experiments without suffering from loss in resolution on array cgh slides. additionally, stability of hyper signal may also allow the possibility of archiving cy and hyper arrays which were previously impossible due to cy instability. functional data indicates that hyper can be substituted for cy in array analysis. the protocols used for hybridization essentially allow hyper labelled samples to replace cy rather than making direct dye swap on arrays, we find it necessary to use ~30% more hyper labelled targets for array hybridization than conventionally used with cy this difference comes from the relatively lower brightness coefficient of hyper to cy which needs to be compensated by using more labelled targets for hybridization. when using hyper5-dctp for labelling, increasing the amount of dye in the reaction will increase yield of labelled targets desired for hybridization. for the hyper nhs reactive dye, labelling conditions can be kept identical due to higher coupling efficiency of hyper nhs compared to cy  the new hyper dye is a more ozone- and photo-stable version of cy for microarray applications. we have demonstrated hyper to be resistant to repeated exposures of ozone at ppb released from ozone generator inside a chamber, conditions at which cy shows dramatic loss of signal. parallel photobleaching experiments performed on hyper and cy show hyper to be resistant to light-induced damage with 3-fold improvement in dye-stability over cy the ozone- and photo-stability of hyper makes this dye highly suitable for array applications particularly during periods of high ozone and humidity levels in summer months, conditions at which cy data can be highly unpredictable. additionally, the photostability of hyper is particularly advantageous in allowing multiple rescanning of arrays without loss of detection. the functional use hyper is demonstrated in array cgh and gene expression experiments to acquire microarray data for copy number analysis. the lower brightness coefficient of hyper may dictate the use of more labelled targets for hybridization to maximize array sensitivity. with high labelling and coupling efficiency of hyper it can be easily adapted to existing cy protocols for both array cgh and microarray gene expression analysis to perform array experiments under any environmental conditions. 
consequently, hyper dye can be used in parallel with cy under any environmental conditions in array experiments. our results show hyper signal to be stable to high ozone levels. additionally, hyper arrays are shown to preserve sensitivity and data quality from gene expression experiments. regardless of the microarray platform, cyanine dyes are one of the most widely used fluorescent dye pairs for microarray analysis owing to their brightness and ease of incorporation, enabling high level of assay sensitivity. our laboratory has been active in fluorescent dye research to find a suitable alternative to cy that is stable to ozone and resistant to photo-bleaching. photobleaching experiments show hyper to be resistant to light induced damage with 3- fold improvement in dye stability over cy in high resolution array cgh experiments, hyper is demonstrated to detect chromosomal aberrations at loci 2p21- and 15q - from three patient sample using bacterial artificial chromosome arrays. repeated exposure of mouse arrays hybridized with hyper5-labeled cdna to ppb ozone at and minute intervals resulted in no signal loss from the dye. cy can also suffer disproportionately from dye photobleaching resulting in distortion of ratios used in copy number analysis. the photostability of hyper is further documented by repeat array scanning without loss of detection. however, combining both dyes on arrays can become problematic during summer months when ozone levels rise to near parts per billion. hyper is demonstrated to be resistant to ozone at up to ppb, levels significantly higher than commonly observed during summer months.
 array-based comparative genomic hybridization and gene expression profiling have become vital techniques for identifying molecular defects underlying genetic diseases. regardless of the microarray platform, cyanine dyes are one of the most widely used fluorescent dye pairs for microarray analysis owing to their brightness and ease of incorporation, enabling high level of assay sensitivity. however, combining both dyes on arrays can become problematic during summer months when ozone levels rise to near parts per billion. under such conditions, cy is known to rapidly degrade leading to loss of signal from either "homebrew" or commercial arrays. cy can also suffer disproportionately from dye photobleaching resulting in distortion of ratios used in copy number analysis. our laboratory has been active in fluorescent dye research to find a suitable alternative to cy that is stable to ozone and resistant to photo-bleaching. here, we report on the development of such a dye, called hyper and describe its' exceptional ozone and photostable properties on microarrays.  our results show hyper signal to be stable to high ozone levels. repeated exposure of mouse arrays hybridized with hyper5-labeled cdna to ppb ozone at and minute intervals resulted in no signal loss from the dye. in comparison, cy arrays showed a dramatic 80% decrease in total signal during the same interval. photobleaching experiments show hyper to be resistant to light induced damage with 3- fold improvement in dye stability over cy in high resolution array cgh experiments, hyper is demonstrated to detect chromosomal aberrations at loci 2p21- and 15q - from three patient sample using bacterial artificial chromosome arrays. the photostability of hyper is further documented by repeat array scanning without loss of detection. additionally, hyper arrays are shown to preserve sensitivity and data quality from gene expression experiments.  hyper is a red fluorescent dye that behaves functionally similar to cy except in stability to ozone and light. hyper is demonstrated to be resistant to ozone at up to ppb, levels significantly higher than commonly observed during summer months. consequently, hyper dye can be used in parallel with cy under any environmental conditions in array experiments. 