BACKGROUND
over the past  <dig> years, confocal microscopy has become a centrally important technique for the analysis of biological samples.
by using a pinhole to exclude scattered light, confocal instruments can be used to optically section biological samples, producing 2- and 3-dimensional images with spatially resolved details at the sub-micron level.
beyond simply visualizing fluorescently labeled specimens, confocal microscopy has become a powerful tool for biologists in many disciplines for diverse applications, including establishing structure-function relationships at the cellular and tissue level, defining dynamic processes in living specimens, and for detection of close interactions between biological molecules at the subcellular level  <cit> .
most basic confocal microscopes are equipped with  <dig>   <dig>  or  <dig> lasers, and are generally configured to detect one fluorophore per laser, giving a maximum detection of four distinct fluorescent labels in a single sample.
there are several different factors that contribute to this limitation, including the fact that the most prevalent fluorescent probes are small organic molecules which have a small stoke's shift.
thus, with few exceptions, each fluorescent dye in an experiment requires a distinct laser for excitation, and the emission spectrum is slightly red-shifted, relative to the excitation wavelength.
as a result, the number of proteins or cell structures that can be imaged concurrently is quite restricted .
quantum dot -coupled detection reagents offer an opportunity to expand the capabilities of basic confocal instruments.
qdots are semi-conductor nanocrystals consisting of a cdse core and a surface chemistry treatment which allows the qdot to be coupled to proteins  <cit> .
a striking advantage of qdots over most organic fluorophores involves their long fluorescence half-life and high resistance to photobleaching, allowing them to be imaged extensively with minimal loss of signal  <cit> .
qdots have several additional properties which make them attractive for imaging applications, including a wide excitation spectrum, a narrow emission spectrum, and a long stoke's shift.
the physics governing qdots fluorescence is such that the emission wavelength is determined by the size of the qdot.
consequently, larger qdots have longer emission wavelengths.
importantly, all qdots share overlapping excitation spectra, with maximal excitation by ultraviolet  wavelengths, meaning that the stoke's shift for red and infrared qdots spans hundreds of nanometers, which clearly distinguishes these fluorophores from organic dyes  <cit> .
an additional consequence of this qdots property is that all qdots can be efficiently excited by a single laser in the uv to blue region of the spectrum  <cit> .
commercially produced qdots reagents are now available with defined emission wavelengths that extend from green to the near-infrared emission wavelengths.
the physical properties of qdots, predominantly their large size , dictate that numerous antibodies are coupled to a single qdot.
in contrast, when labeling with organic dyes , many dye molecules are coupled to an individual antibody.
thus, qdot coupled antibodies are both much larger and have many more ligand binding sites than organic dye coupled antibodies.
it is therefore reasonable to expect that these reagents may behave quite differently for applications such as the staining of detergent permeabilized cells.
in principle, qdot-labeled staining reagents can be combined with organic dye conjugates and/or fluorescent proteins to expand the number of parameters detected in a single fluorescently labeled sample.
given that quantum dot reagents with red and near-infrared  emissions are very bright and cover a region of the spectrum that is under-utilized on many basic confocal systems, staining reagents that are coupled to red and near-ir quantum dots represent a particularly attractive strategy to expand the multi-parameter capabilities of confocal microscopes.
however, due to the large size of quantum dot conjugates and their multivalency, these reagents perform poorly for one-to-one ligand-to-target binding  <cit> .
thus, qdot-labeled reagents can be expected to behave differently from organic dye-labeled conjugates in fluorescent labeling experiments.
we thus initiated a study to compare the quality of images obtained from qdot- and organic dye-labeled conjugates for the staining of fixed, detergent permeabilized cells, and to test the suitability of red and near-ir qdot reagents for performing multi-parameter fluorescence labeling experiments in combination with organic dye-based detection reagents.
together, our data demonstrate that, with simple and inexpensive modifications to a basic confocal microscope, red and near-ir qdot-labeled conjugates can readily be combined with conventional fluorophores, increasing the number of labeled structures that can be detected and cleanly discriminated in a single experiment.
RESULTS
to test the use of qdots in a basic confocal system, we employed a zeiss lsm <dig> pascal system equipped with a  <dig> mw  <dig> nm diode laser, a  <dig> mw argon laser tunable to  <dig> nm,  <dig> nm or  <dig> nm, a  <dig> mw  <dig> nm helium/neon laser, and two fluorescence detector channels, which utilize hamamatsu r <dig> photomultiplier tubes .
with this system, three-color detection of organic fluorophores in combinations such as dapi, alexa  <dig> and alexa  <dig>  or combinations of fluorescent proteins and organic probes, such as cfp, yfp, and alexa  <dig> are easily achieved.
however, acquisition of four or more fluorescence parameters from a single sample employing organic dyes and/or fluorescent proteins is quite difficult on this system, due to insufficient separation of excitation and/or emission spectra.
to expand the multi-parameter capabilities of our lsm <dig> pascal, we added additional emission filters to allow combinatorial detection of red- and near infrared-emitting qdots .
specifically, we added 655/ <dig> nm and 705/ <dig> nm band pass filters and a  <dig> nm long pass filter  to enable detection of qdots with fluorescence emissions at  <dig> nm,  <dig> nm and  <dig> nm, respectively .
details of the imaging parameters used during the collection of data for fig.
<dig>  amplifier offset and amplifier gain settings were constant throughout .
evaluation of the pascal's fluorescence detection hardware suggested that fluorescence emission from each of these qdots could be detected.
indeed, the pascal's hamamatsu r <dig> pmts maintain high quantum efficiency   through most of this spectral range .
although the pascal's pmts have <5% qe near  <dig> nm, we predicted that the extremely bright fluorescence from qdot  <dig> reagents could still be successfully detected, with adequate cell labeling.
the total cost of these filter upgrades was less than $ <dig>
to determine whether or not qdot conjugated antibodies behave comparably to organic dye conjugated antibodies for the staining of intracellular structures in fixed, detergent permeabilized cells, nih/3t <dig> fibroblasts were grown on glass coverslips, fixed with paraformaldehyde, permeabilized with a  <dig> % triton x- <dig> solution, and stained with a monoclonal antibody against α-tubulin.
duplicate coverslips were then stained with either an organic dye-labeled secondary antibody  or a qdot-labeled secondary antibody .
fluorescence data were acquired as z-stack images on our modified zeiss lsm <dig> pascal, and data were further processed by digital deconvolution and projection into a 2-dimensional image.
as shown in fig.
<dig>  the alexa 555-conjugated secondary antibody  and the qdot-conjugated secondary antibody  generally yielded images of similar quality, with individual microtubules labeled sharply and contiguously throughout the majority of the cell, both when comparing deconvolved z-stack maximal intensity projection images , and when comparing single xy planes, prior to deconvolution .
the one consistent difference in labeling that we noted was that the signal-to-noise ratio of the qdot  <dig> labeled samples was lower than observed with the alexa  <dig> labeled cells, which may account for the weak and variable staining of qdot 655-labeled microtubules at sites of adhesion of the fibroblasts to the coverslip .
however, it is important to note that this result is clearly due, at least in part, to the reduced quantum efficiency of the hamamatsu r <dig> pmt in the red and near ir region of the spectrum and the narrower band pass filter used for the collection of qdot  <dig> vs. qdot  <dig> fluorescence emissions  .
overall, the data in fig.
<dig> demonstrate that qdot-coupled and organic dye-coupled antibodies behave comparably for the staining of intracellular antigens.
however, bright organic dyes that fluoresce in the region of maximal pmt quantum efficiency may be more appropriate for the detection of low abundance antigens or for maximal resolution of fine structural details.
we next performed a multi-parameter labeling experiment to test the feasibility of separation of six distinct fluorescent signals on our modified lsm <dig> pascal system.
for this experiment, we used a d <dig> t cell line expressing pkcθ-gfp during interactions with a fibroblast cell line  engineered to specifically activate d <dig> through its t cell receptor .
we chose this particular combination of cell lines because we have previously characterized the antigen-stimulated redistribution of intracellular proteins  in the d <dig> line  <cit> , and because d <dig> t cells and fibroblasts have very different sizes and morphologies, allowing us to confirm the identity of each cell type independently of fluorescence data.
the 3t3-apc cells were grown overnight on sterile coverslips, and then labeled with the qtracker® q <dig> cell labeling kit, which results in accumulation of the qdot  <dig> label in cytosolic vesicles.
next, d <dig> t cells expressing a pkcθ-gfp fusion protein were added to the qtracker-labeled 3t3-apc cells.
the coverslips were incubated  <dig> min at 37°c to facilitate t cell interactions with 3t3-apc cells and consequent activation of the d <dig> t cells.
the cells were then fixed, permeabilized, and stained with biotin-xx-phalloidin for the detection of f-actin, and with primary antibodies to α-tubulin and rela, for the detection of microtubules and the nf-κb transcription factor, respectively.
we then added fluorescent secondary reagents: qdot  <dig> streptavidin for the detection of biotinylated phalloidin, qdot  <dig> anti-rabbit igg for the detection of the polyclonal anti-rela antibody, and alexa  <dig> anti-igg <dig> for the detection of the monoclonal anti-α-tubulin antibody.
immediately prior to mounting, nuclear dna was labeled with dapi.
slides were visualized using our modified zeiss lsm <dig> pascal confocal microscope, employing the imaging parameters outlined in fig.
<dig> and detailed in table  <dig>  fluorescent z-stack data were deconvolved and flattened as described in methods.
as shown in figs  <dig> and  <dig>  we were able to clearly separate the signals from the six fluorescent probes used in this experiment.
for each stain, the fluorescent signals were observed in the expected cell type and subcellular distributions, with minimal cross-talk between channels.
specifically, f-actin was found in a membrane-proximal region of the cytosol, with particular concentration in the t cells at the region of junction with the stimulatory 3t3-apc cells  <cit>  .
rela was found mostly in the cytosol of both the t cells  and the 3t3-apc fibroblasts .
dapi  and anti-α-tubulin  labeled the nuclei and microtubules of both cell types, respectively.
the qtracker  <dig> label was found exclusively in the fibroblasts , and the pkcθ-gfp signal was detected only in the d <dig> t cells , with the expected enrichment at the site of contact with the 3t3-apc cells  <cit> .
together, these data demonstrate that with the addition of three additional filters to the lsm <dig> pascal confocal and through the use of red and near-infrared qdot labeling reagents, six distinct fluorescent labels can be readily detected and cleanly discriminated from each other.
notably, due to the rather broad peak of the emission spectrum for qdot  <dig>  particularly in the near-infrared region of the spectrum, there is some signal bleed-through of the qdot  <dig> signal into the qdot  <dig> channel, with the specific filter design that we employed .
for our experiment, use of the qtracker  <dig> reagent produced a very intense qdot  <dig> signal, such that the qdot  <dig> signal bleed through was easily eliminated by raising the lower signal threshold in the qdot  <dig> channel.
as with any fluorescent staining protocol, it is imperative to empirically determine the degree of signal cross-talk and the intensity of staining in each channel, via analysis of singly-labeled samples imaged in all fluorescent channels, prior to attempting a combined labeling/imaging experiment.
although we found that the qdot-conjugated secondary antibodies yielded data of similar quality to organic dye labeled secondary reagents, we did note certain limitations of the qdot labels in the course of our study.
firstly, we found that good labeling with the qdot-conjugated antibodies was strictly dependent on use of the manufacturer's suggested blocking and staining buffers.
for example, qdot labeling was quite poor when serum was used as the blocking reagent in the secondary antibody labeling step, rather than albumin .
in contrast, excellent results were obtained with the organic dye labeled secondary reagents under either condition.
thus, for multi-parameter fluorescence microscopy experiments in which qdots- and organic dye-based staining reagents are combined, it is very important to use the blocking and staining buffers that are optimal for the qdots reagents.
additionally, the cost of qdot secondary reagents is considerably higher than organic dye labeled secondaries, when one considers the cost per staining reaction.
comparing alexa  <dig> anti-mouse to qdot  <dig> anti-mouse , the current list prices are $ <dig> for  <dig> μl and $ <dig> for  <dig> μl, respectively.
given that the respective working concentrations are 1: <dig> and 1: <dig> and assuming a staining reaction volume of  <dig> μl, the cost per stain is $172/ = $ <dig>  for alexa  <dig> anti-mouse vs. $191/ = $ <dig>  for qdot  <dig> anti-mouse.
thus, qdot-coupled secondary antibodies are approximately 27-fold more costly than comparable organic dye-labeled antibodies.
furthermore, we have observed that the useful shelf lives of the qdot secondary reagents are generally limited to their stated expiration dates , presumably reflecting a gradual uncoupling between the qdots and their antibody conjugates.
this limitation represents a further increase in the effective per stain cost of quantum dot reagents for those laboratories that use such reagents only occasionally.
in contrast, we have found that organic dye coupled secondaries are generally extremely stable for many years, with proper storage.
thus, qdot reagents have specific disadvantages relative to organic dye-based reagents for routine intracellular staining applications.
however, when considered in the context of the substantial potential benefits of the red and near ir quantum dots for multi-parameter confocal imaging, we view the above limitations as minor and manageable.
CONCLUSIONS
through the simple addition of appropriate emission filters, basic confocal microscopes can be modified for detection and discrimination of multiple red and near-infrared qdot reagents.
incorporating qdot-conjugated antibodies into staining protocols is a technically simple procedure that allows the utilization of existing lasers and pmt tubes , while dramatically expanding the multi-parameter imaging capabilities of this instrumentation at minimal expense.
in summary, we have shown that a generalizable and easily implemented upgrade can greatly augment the capabilities of pre-existing confocal instrumentation in many laboratories and core facilities.
we have also established staining conditions that yield optimal signal-to-noise and overall staining quality when qdot- and organic dye-based reagents are used in multi-parameter labeling experiments.

 over the past years, confocal microscopy has become a centrally important technique for the analysis of biological samples. by using a pinhole to exclude scattered light, confocal instruments can be used to optically section biological samples, producing 2- and 3-dimensional images with spatially resolved details at the sub-micron level. beyond simply visualizing fluorescently labeled specimens, confocal microscopy has become a powerful tool for biologists in many disciplines for diverse applications, including establishing structure-function relationships at the cellular and tissue level, defining dynamic processes in living specimens, and for detection of close interactions between biological molecules at the subcellular level. most basic confocal microscopes are equipped with or lasers, and are generally configured to detect one fluorophore per laser, giving a maximum detection of four distinct fluorescent labels in a single sample. there are several different factors that contribute to this limitation, including the fact that the most prevalent fluorescent probes are small organic molecules which have a small stoke's shift. thus, with few exceptions, each fluorescent dye in an experiment requires a distinct laser for excitation, and the emission spectrum is slightly red-shifted, relative to the excitation wavelength. as a result, the number of proteins or cell structures that can be imaged concurrently is quite restricted. quantum dot -coupled detection reagents offer an opportunity to expand the capabilities of basic confocal instruments. qdots are semi-conductor nanocrystals consisting of a cdse core and a surface chemistry treatment which allows the qdot to be coupled to proteins. a striking advantage of qdots over most organic fluorophores involves their long fluorescence half-life and high resistance to photobleaching, allowing them to be imaged extensively with minimal loss of signal. qdots have several additional properties which make them attractive for imaging applications, including a wide excitation spectrum, a narrow emission spectrum, and a long stoke's shift. the physics governing qdots fluorescence is such that the emission wavelength is determined by the size of the qdot. consequently, larger qdots have longer emission wavelengths. importantly, all qdots share overlapping excitation spectra, with maximal excitation by ultraviolet wavelengths, meaning that the stoke's shift for red and infrared qdots spans hundreds of nanometers, which clearly distinguishes these fluorophores from organic dyes. an additional consequence of this qdots property is that all qdots can be efficiently excited by a single laser in the uv to blue region of the spectrum. commercially produced qdots reagents are now available with defined emission wavelengths that extend from green to the near-infrared emission wavelengths. the physical properties of qdots, predominantly their large size, dictate that numerous antibodies are coupled to a single qdot. in contrast, when labeling with organic dyes, many dye molecules are coupled to an individual antibody. thus, qdot coupled antibodies are both much larger and have many more ligand binding sites than organic dye coupled antibodies. it is therefore reasonable to expect that these reagents may behave quite differently for applications such as the staining of detergent permeabilized cells. in principle, qdot-labeled staining reagents can be combined with organic dye conjugates and/or fluorescent proteins to expand the number of parameters detected in a single fluorescently labeled sample. given that quantum dot reagents with red and near-infrared emissions are very bright and cover a region of the spectrum that is under-utilized on many basic confocal systems, staining reagents that are coupled to red and near-ir quantum dots represent a particularly attractive strategy to expand the multi-parameter capabilities of confocal microscopes. however, due to the large size of quantum dot conjugates and their multivalency, these reagents perform poorly for one-to-one ligand-to-target binding. thus, qdot-labeled reagents can be expected to behave differently from organic dye-labeled conjugates in fluorescent labeling experiments. we thus initiated a study to compare the quality of images obtained from qdot- and organic dye-labeled conjugates for the staining of fixed, detergent permeabilized cells, and to test the suitability of red and near-ir qdot reagents for performing multi-parameter fluorescence labeling experiments in combination with organic dye-based detection reagents. together, our data demonstrate that, with simple and inexpensive modifications to a basic confocal microscope, red and near-ir qdot-labeled conjugates can readily be combined with conventional fluorophores, increasing the number of labeled structures that can be detected and cleanly discriminated in a single experiment.  to test the use of qdots in a basic confocal system, we employed a zeiss lsm pascal system equipped with a mw nm diode laser, a mw argon laser tunable to nm, nm or nm, a mw nm helium/neon laser, and two fluorescence detector channels, which utilize hamamatsu r photomultiplier tubes. with this system, three-color detection of organic fluorophores in combinations such as dapi, alexa and alexa or combinations of fluorescent proteins and organic probes, such as cfp, yfp, and alexa are easily achieved. however, acquisition of four or more fluorescence parameters from a single sample employing organic dyes and/or fluorescent proteins is quite difficult on this system, due to insufficient separation of excitation and/or emission spectra. to expand the multi-parameter capabilities of our lsm pascal, we added additional emission filters to allow combinatorial detection of red- and near infrared-emitting qdots. specifically, we added 655/ nm and 705/ nm band pass filters and a nm long pass filter to enable detection of qdots with fluorescence emissions at nm, nm and nm, respectively. details of the imaging parameters used during the collection of data for fig. amplifier offset and amplifier gain settings were constant throughout. evaluation of the pascal's fluorescence detection hardware suggested that fluorescence emission from each of these qdots could be detected. indeed, the pascal's hamamatsu r pmts maintain high quantum efficiency through most of this spectral range. although the pascal's pmts have <5% qe near nm, we predicted that the extremely bright fluorescence from qdot reagents could still be successfully detected, with adequate cell labeling. the total cost of these filter upgrades was less than $ to determine whether or not qdot conjugated antibodies behave comparably to organic dye conjugated antibodies for the staining of intracellular structures in fixed, detergent permeabilized cells, nih/3t fibroblasts were grown on glass coverslips, fixed with paraformaldehyde, permeabilized with a % triton x- solution, and stained with a monoclonal antibody against α-tubulin. duplicate coverslips were then stained with either an organic dye-labeled secondary antibody or a qdot-labeled secondary antibody. fluorescence data were acquired as z-stack images on our modified zeiss lsm pascal, and data were further processed by digital deconvolution and projection into a 2-dimensional image. as shown in fig. the alexa 555-conjugated secondary antibody and the qdot-conjugated secondary antibody generally yielded images of similar quality, with individual microtubules labeled sharply and contiguously throughout the majority of the cell, both when comparing deconvolved z-stack maximal intensity projection images, and when comparing single xy planes, prior to deconvolution. the one consistent difference in labeling that we noted was that the signal-to-noise ratio of the qdot labeled samples was lower than observed with the alexa labeled cells, which may account for the weak and variable staining of qdot 655-labeled microtubules at sites of adhesion of the fibroblasts to the coverslip. however, it is important to note that this result is clearly due, at least in part, to the reduced quantum efficiency of the hamamatsu r pmt in the red and near ir region of the spectrum and the narrower band pass filter used for the collection of qdot vs. qdot fluorescence emissions. overall, the data in fig. demonstrate that qdot-coupled and organic dye-coupled antibodies behave comparably for the staining of intracellular antigens. however, bright organic dyes that fluoresce in the region of maximal pmt quantum efficiency may be more appropriate for the detection of low abundance antigens or for maximal resolution of fine structural details. we next performed a multi-parameter labeling experiment to test the feasibility of separation of six distinct fluorescent signals on our modified lsm pascal system. for this experiment, we used a d t cell line expressing pkcθ-gfp during interactions with a fibroblast cell line engineered to specifically activate d through its t cell receptor. we chose this particular combination of cell lines because we have previously characterized the antigen-stimulated redistribution of intracellular proteins in the d line, and because d t cells and fibroblasts have very different sizes and morphologies, allowing us to confirm the identity of each cell type independently of fluorescence data. the 3t3-apc cells were grown overnight on sterile coverslips, and then labeled with the qtracker® q cell labeling kit, which results in accumulation of the qdot label in cytosolic vesicles. next, d t cells expressing a pkcθ-gfp fusion protein were added to the qtracker-labeled 3t3-apc cells. the coverslips were incubated min at 37°c to facilitate t cell interactions with 3t3-apc cells and consequent activation of the d t cells. the cells were then fixed, permeabilized, and stained with biotin-xx-phalloidin for the detection of f-actin, and with primary antibodies to α-tubulin and rela, for the detection of microtubules and the nf-κb transcription factor, respectively. we then added fluorescent secondary reagents: qdot streptavidin for the detection of biotinylated phalloidin, qdot anti-rabbit igg for the detection of the polyclonal anti-rela antibody, and alexa anti-igg for the detection of the monoclonal anti-α-tubulin antibody. immediately prior to mounting, nuclear dna was labeled with dapi. slides were visualized using our modified zeiss lsm pascal confocal microscope, employing the imaging parameters outlined in fig. and detailed in table fluorescent z-stack data were deconvolved and flattened as described in methods. as shown in figs and we were able to clearly separate the signals from the six fluorescent probes used in this experiment. for each stain, the fluorescent signals were observed in the expected cell type and subcellular distributions, with minimal cross-talk between channels. specifically, f-actin was found in a membrane-proximal region of the cytosol, with particular concentration in the t cells at the region of junction with the stimulatory 3t3-apc cells. rela was found mostly in the cytosol of both the t cells and the 3t3-apc fibroblasts. dapi and anti-α-tubulin labeled the nuclei and microtubules of both cell types, respectively. the qtracker label was found exclusively in the fibroblasts, and the pkcθ-gfp signal was detected only in the d t cells, with the expected enrichment at the site of contact with the 3t3-apc cells. together, these data demonstrate that with the addition of three additional filters to the lsm pascal confocal and through the use of red and near-infrared qdot labeling reagents, six distinct fluorescent labels can be readily detected and cleanly discriminated from each other. notably, due to the rather broad peak of the emission spectrum for qdot particularly in the near-infrared region of the spectrum, there is some signal bleed-through of the qdot signal into the qdot channel, with the specific filter design that we employed. for our experiment, use of the qtracker reagent produced a very intense qdot signal, such that the qdot signal bleed through was easily eliminated by raising the lower signal threshold in the qdot channel. as with any fluorescent staining protocol, it is imperative to empirically determine the degree of signal cross-talk and the intensity of staining in each channel, via analysis of singly-labeled samples imaged in all fluorescent channels, prior to attempting a combined labeling/imaging experiment. although we found that the qdot-conjugated secondary antibodies yielded data of similar quality to organic dye labeled secondary reagents, we did note certain limitations of the qdot labels in the course of our study. firstly, we found that good labeling with the qdot-conjugated antibodies was strictly dependent on use of the manufacturer's suggested blocking and staining buffers. for example, qdot labeling was quite poor when serum was used as the blocking reagent in the secondary antibody labeling step, rather than albumin. in contrast, excellent results were obtained with the organic dye labeled secondary reagents under either condition. thus, for multi-parameter fluorescence microscopy experiments in which qdots- and organic dye-based staining reagents are combined, it is very important to use the blocking and staining buffers that are optimal for the qdots reagents. additionally, the cost of qdot secondary reagents is considerably higher than organic dye labeled secondaries, when one considers the cost per staining reaction. comparing alexa anti-mouse to qdot anti-mouse, the current list prices are $ for μl and $ for μl, respectively. given that the respective working concentrations are 1: and 1: and assuming a staining reaction volume of μl, the cost per stain is $172/ = $ for alexa anti-mouse vs. $191/ = $ for qdot anti-mouse. thus, qdot-coupled secondary antibodies are approximately 27-fold more costly than comparable organic dye-labeled antibodies. furthermore, we have observed that the useful shelf lives of the qdot secondary reagents are generally limited to their stated expiration dates, presumably reflecting a gradual uncoupling between the qdots and their antibody conjugates. this limitation represents a further increase in the effective per stain cost of quantum dot reagents for those laboratories that use such reagents only occasionally. in contrast, we have found that organic dye coupled secondaries are generally extremely stable for many years, with proper storage. thus, qdot reagents have specific disadvantages relative to organic dye-based reagents for routine intracellular staining applications. however, when considered in the context of the substantial potential benefits of the red and near ir quantum dots for multi-parameter confocal imaging, we view the above limitations as minor and manageable.  through the simple addition of appropriate emission filters, basic confocal microscopes can be modified for detection and discrimination of multiple red and near-infrared qdot reagents. incorporating qdot-conjugated antibodies into staining protocols is a technically simple procedure that allows the utilization of existing lasers and pmt tubes, while dramatically expanding the multi-parameter imaging capabilities of this instrumentation at minimal expense. in summary, we have shown that a generalizable and easily implemented upgrade can greatly augment the capabilities of pre-existing confocal instrumentation in many laboratories and core facilities. we have also established staining conditions that yield optimal signal-to-noise and overall staining quality when qdot- and organic dye-based reagents are used in multi-parameter labeling experiments. 
most basic confocal microscopes are equipped to cleanly discriminate no more than four fluorophores in a given sample, limiting the utility of this method for co-localization, co-expression, and other multi-parameter analyses. in this study, we evaluated the use of red and near-infrared emitting quantum dot staining reagents to expand the multi-parameter capabilities of basic confocal microscopes. our data demonstrate that nearly any basic confocal microscope can be modified by the simple addition of appropriate emission filters, allowing the detection of red and near-infrared quantum dot conjugates. additionally, quantum dot- and organic dye-based secondary reagents can be successfully combined in complex intracellular staining experiments. we then performed direct comparisons between organic dye- and quantum dot-labeled detection reagents for the detection of subcellular structures. we found that the quality of staining was generally indistinguishable, although quantum dot reagents do have certain limitations, relative to organic dye conjugates. using the modified pascal system, three quantum dot conjugates, two organic dye conjugates, and one fluorescent protein, we demonstrated clean discrimination of six distinct fluorescent labels in a single sample. substantial expansion of the multi-parameter capabilities of basic confocal instruments can be achieved with a financial investment that is minimal in comparison to instrument replacement or upgrade with additional lasers. confocal microscopy is a widely employed methodology in cellular biology, commonly used for investigating biological organization at the cellular and sub-cellular level. we modified a three-laser zeiss pascal confocal microscope by the addition of two band-pass filters and one long-pass filter for the detection of three different red to near-infrared quantum dot conjugates.
 confocal microscopy is a widely employed methodology in cellular biology, commonly used for investigating biological organization at the cellular and sub-cellular level. most basic confocal microscopes are equipped to cleanly discriminate no more than four fluorophores in a given sample, limiting the utility of this method for co-localization, co-expression, and other multi-parameter analyses. in this study, we evaluated the use of red and near-infrared emitting quantum dot staining reagents to expand the multi-parameter capabilities of basic confocal microscopes.  we modified a three-laser zeiss pascal confocal microscope by the addition of two band-pass filters and one long-pass filter for the detection of three different red to near-infrared quantum dot conjugates. we then performed direct comparisons between organic dye- and quantum dot-labeled detection reagents for the detection of subcellular structures. we found that the quality of staining was generally indistinguishable, although quantum dot reagents do have certain limitations, relative to organic dye conjugates. using the modified pascal system, three quantum dot conjugates, two organic dye conjugates, and one fluorescent protein, we demonstrated clean discrimination of six distinct fluorescent labels in a single sample.  our data demonstrate that nearly any basic confocal microscope can be modified by the simple addition of appropriate emission filters, allowing the detection of red and near-infrared quantum dot conjugates. additionally, quantum dot- and organic dye-based secondary reagents can be successfully combined in complex intracellular staining experiments. substantial expansion of the multi-parameter capabilities of basic confocal instruments can be achieved with a financial investment that is minimal in comparison to instrument replacement or upgrade with additional lasers. 