TECHNICAL NOTE |
Correspondence to: Gian-Luca Ferri, Dept. of Cytomorphology, University of Cagliari at Monserrato, I-09042 Monserrato (Cagliari), Italy.
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Summary |
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Cyanine 5.18 (or Cy5) is a fluorochrome emitting in the long-red/far-red range, usually regarded as unsuitable for direct observation by the human eye. We describe here the optimization of a direct visualization approach to Cy5 labeling, based on a standard fluorescence microscope with mercury light excitation and applicable to both immunocytochemistry and fluorescent in situ hybridization. Crucial factors were (a) an excitation path in the microscope not absorbing light in the orange-red range, up to 640 nm, (b) a 588640-nm excitation filter range, distinctly below the excitation optimum for Cy5, (c) a 650700-nm emission filter range, transmitting the low-wavelength portion of Cy5 emission, and (d) high-efficiency filter set components allowing a narrow gap between excitation and emission ranges without visible cross-talk of excitation light in the emission path. (J Histochem Cytochem 48:437444, 2000)
Key Words: Cy5, fluorescence, immunohistochemistry, immunofluorescence, FISH, multiple staining, fluorescence filter, fluorochrome
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Introduction |
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In view of its emission in the long-red/far-red range (630 to over 700 nm), cyanine 5.18 (Cy5) provides a distinct fluorescent signal that can easily be separated from that of many other fluoro
chromes (
Although the human eye can detect light in the emission range of Cy5, its sensitivity in the long-red/far-red range is low (
The availability of advanced optical coating technology, suitable for fine-tuning of rejection and transmission regions in interference filters (
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Materials and Methods |
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Filter Set Specifications (Table 1)
Our first filter set for direct visualization of Cy5 (Set 1;
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Characterization of Filter Sets and Their Components
Complete excitation and emission pathways were assessed as described previously (
Spectral Tests with Cy5
A cuvette filled with Cy5 antibody or avidin conjugate (see below) diluted in PBS was used as sample. Monochromatic light (560645 nm) was projected through the excitation filter and the dichroic mirror onto the sample, and emission was collected after dichroic and emission filtering for photometry (at 660 nm: Fig 1D, Fluorolog-2 as above). Standard excitation and emission spectra of Cy5 conjugates were obtained for comparison.
Tissues and Immunofluorescence Experiments
Pituitaries and spinal ganglia (porcine and bovine, from a local abattoir) were fixed in either 4% paraformaldehyde or periodatelysineparaformaldehyde (
Fluorescent In Situ Hybridization
Lymphocytes were prepared from PHA-stimulated peripheral blood according to a standard cytogenetic protocol (-satellite pericentromeric probes for chromosome 1 (pUC177) and for chromosome 17 (p17H8) were labeled with Cy512-dUTP (Amersham; Poole, UK) and FITC12-dUTP (DuPont; Boston, MA), respectively, using standard nick-translation. Probes were denatured and hybridized in a standard hybridization mixture (
Fluorescence Microscopy and Photography
Slides were observed with BX-60 and BX-50 microscopes (Olympus Italia; Milan, Italy). These were equipped with 100-W high-pressure mercury lamps (HBO-103 from Osram, S. Giuliano Milanese, Italy; or 102D from Ushio, Tokyo, Japan), a collector lens with mirror [either achromatic-aplanatic "A" type or UV-transmissive "B" type, from Olympus; with or without their standard heat filter(s)], an accessory "conversion lens" (Olympus) aimed at maximizing excitation light, and a range of apochromatic objectives (x2 to x100). Standard or custom (
Direct Observation Tests
Signal intensity (of Cy5 and other fluorochromes) and excitationemission cross-talk (see below) visible through Sets 16 were assessed subjectively. The 010 subjective score used was intended to provide a relative within-test estimate of overall visual effectiveness. Therefore, score values for signal and for cross-talk were not comparable. To compensate for possible (although apparently low) fading during observation, the same fields were observed using the different filter sets in varying sequential order.
Depending on the degree to which excitation wavelengths are suppressed by the dichroic mirror and emission filter, unwanted excitation light may become visible through the emission path as excitationemission cross-talk. To maximize detection of such cross-talk, if any, a mirror was used in place of the preparation (Fig 1C). Whereas slide and coverslip surfaces reflect roughly 7% of the excitation light, the test mirror used had high reflectivity (>97%). Cross-talk was also assessed quantitatively using the exposure meter of the PM30 photographic system.
CCD Camera Tests
FISH and immunofluorescence preparations were captured using a high-sensitivity VarioCam CCD camera (PCO Computer Optics; Kelheim, Germany). Excitationemission cross-talk tests were also carried out using a mirror (as above) and further increasing sensitivity with a prolonged acquisition time (6 sec vs 0.6 sec, which was appropriate for imaging).
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Results |
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Filter Set Characterization
Excitation filter, emission filter, and dichroic used for each set are listed in Table 1. Differences in excitation path and emission path ranges (Fig 2, thick lines, vs excitation filters, emission filters, and dichroics, thin lines) can be summarized as follows: (a) progressively increasing upper limit of excitation, Sets 1, 2, 6, 3, 5, 4; (b) progressively increasing lower limit of emission, Sets 1, 3, 5, 2, 4, 6. In addition, for Set 6 excitation extended down to 577 nm (about 10 nm lower than other sets), and the emission filter showed a longpass profile extending to the infrared range. For the "very steep" dichroic 4, the rejection (reflection) region extended almost to the upper limit of the corresponding excitation filter range (Fig 2; approximately 8% transmittance at 638 nm).
Spectral Tests with Cy5
As expected, a steep ascending slope above 620 nm was apparent parallel to the ascending slope in Cy5 excitation (Fig 3). Significant differences in excitation efficiency were revealed, superimposable for avidin (Fig 3) and antibody conjugates (not shown). Sets 4, 3, and 5 (in decreasing order) proved most effective.
Microscope
Of the two collector lenses tested, the "A" type provided more consistent illumination of the microscopic field for excitation of Cy5 (orange-red light) and of the other fluorochromes tested (UV to green light). The accessory conversion lens significantly increased the amount of excitation light and was used for all tests. Removal of heat filters mounted along the collector lens resulted in significantly brighter signal. Further testing was therefore carried out with no heat filter, with no apparent problem. Although most excitation filters tested included an infrared blocking treatment, CCD camera capture and exposure metering revealed potential problems when such blocking was insufficient (Set 6; see below).
Direct Observation Tests
All filter sets tested permitted direct visualization of Cy5 labeling, which was seen as deep red signal over a very dark background for both immunostained and FISH preparations. Mediumhigh-power objectives (x20100) were most effective, lower magnification resulting in little visible signal. Brighter images were shown with higher numerical aperture objectives, such as Olympus PlanApo x40 (NA 0.95) vs UPlanApo x40 (NA 0.85). Naturally, in multiple staining visible signal intensity with Cy5 was well below that of Cy3, FITC, or Cy2. However, autofluorescence was exceedingly low with Cy5 labeling; therefore, the signal/noise ratio was distinctly high. Cy5 FISH labeling with both centromeric and chromosome painting probes resulted in bright visible signal against a virtually black background. Because of the low, virtually absent background, nuclei or interphase chromosomes were best located on the slide using a DAPI filter set (or an FITC set, where relevant) before switching to the Cy5 filter set.
For all preparations, distinct differences in visible signal intensity were observed, Sets 4, 3, and 5 (in decreasing order) being distinctly more effective than other sets (Table 2). Best results were seen when the upper limit of excitation was close to 640 nm and the lower limit of emission around 650 nm (Set 4). Even under the severe testing conditions used, visible excitationemission cross-talk was minimal for most sets (Table 2). Exposure metering revealed insufficient infrared blocking of Set 6 (see Table 2).
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Tests with Other Fluorochromes
In multiple- and/or single-staining preparations, AMCA, FITC, Cy2, Cy3, lissaminerhodamine, XRITC, and Texas Red labeling resulted in bright signal when viewed through the appropriate filter set. No signal was seen for AMCA, FITC, Cy2, or Cy3 through Cy5 Sets 1 to 5 (Table 2). These were designed with a low-wavelength excitation limit around 588590 nm. Conversely, Cy3 labeling was clearly visible through Set 6, in connection with its lower excitation limit at 577 nm. As expected, lissaminerhodamine, XRITC, and Texas Red showed visible signal through all Cy5 filter sets tested.
Photography
In preliminary tests (with filter Sets 1 and 2), blackwhite negative films required prolonged exposure with Cy5 (ISO 12 setting vs nominal ISO 400 for T-Max 400; ISO 100 vs nominal ISO 200 for the infrared-extended film SFX). Under the same conditions, color transparency films showed high sensitivity (exposure index around ISO 300 and 1000 vs nominal ISO 100 and 400 for E-100SW and Provia 400, respectively); therefore, these were used for all tests.
Photography showed differences that were less obvious but essentially similar to those resulting from direct observation tests. Sets 3, 5 and 4 resulted in bright photographic images. Longer emission ranges resulted in slightly (Set 4) and distinctly (Set 6) dimmer images, indicative of rapidly decreasing film sensitivity around the 640650-nm range and above.
CCD Camera Tests
Parallel direct and CCD camera observation through the same filters sets proved very effective, signal being bright with sets 4, 3, and 5 (in decreasing order; Fig 4). Even under demanding test conditions (mirror plus tenfold overexposure), images captured through Sets 15 revealed no excitationemission cross-talk. Set 6 showed a bright, diffuse background light (Fig 4, bottom right panel), which disappeared after addition of an infrared-blocking heat filter in the excitation path.
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Discussion |
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Specific optimization of excitation and emission ranges, as described here, resulted in a distinct increase in visible Cy5 signal over our previous filter design (
Cy5 conjugates show excitation and emission maxima around 655 and 670 nm, respectively (
Spectral transmission in the microscope's elements was considered, because excitation above 590 nm is not commonly used. Optics in the microscope illuminator can be expected to be transmissive up to the near-infrared range, Olympus optics (BX series) showing approximately 90% transmittance up to 700 nm (A. Hirohashi, personal communication). Differences in collector lens correction have been mentioned, lower chromatic aberration resulting in more even illumination. Because the commonly used mercury arc lamps show low intensity above 590 nm, any additional lens aimed at collecting more light for excitation, such as the additional conversion lens we used, provides a useful contribution. Alternative light sources, such as xenon lamps, may provide brighter excitation light in the orange-red range but are significantly more expensive. Heat filters well suited to many fluorochromes (e.g., AMCA, DAPI, FITC, Cy2, Cy3, Texas Red) may significantly block orange-red light. Therefore, all fluorescence work in our laboratory during the past several years has been carried out using microscopes devoid of heat filters, with no apparent shortcomings. Certain applications, such as the use of living cell preparations, polarizer filters, or infrared-absorbing elements, require properly designed heat filters (fully transmissive up to 640 nm). Finally, high numerical aperture planapochromatic objectives proved highly effective for direct visualization of both Cy5 fluorescence and multicolor fluorescence. Their multiple wavelength optical correction will be especially helpful for multiple-exposure photography with blue, green, yellow-orange, and long-red/far-red emitters.
In photography, the (slightly) lower signal with Set 4 vs Sets 3 and 5, and the poor outcome with Set 6, may be explained by a rapidly decreasing color film sensitivity in the long-red range, especially above 650 nm. Therefore, the 650700-nm emission range (Set 4) is probably a good compromise for parallel use in direct observation and photography. From a practical point of view, color transparency film proved well-suited to the present approach and is routinely used in our laboratory. When blackwhite photography is required, hypersensitized Technical Pan film may be an alternative (
Normal-intensity FISH (as for standard CCD camera imaging) and immunofluorescence labeled with Cy5 resulted in very bright CCD images with the direct observation filter sets described, suggesting that in many systems the same filters can be used for direct observation in parallel to CCD capture. Working concentrations of antibodies and conjugates for Cy5 fluorescence were optimized for direct eye observation. Therefore, it is reassuring that an excellent signal/noise ratio was also observed with CCD camera viewing. With more powerful light sources, such as lasers, it may be necessary to use higher dilutions. Most importantly, optimization for visible signal resulted in no excitationemission cross-talk, even under demanding test conditions. In FISH preparations, both centromeric and chromosome painting probes showed very clearly, to both the human eye and photographic film, against a virtually black background, and the usually problematic fixative-related autofluorescence was negligible. Detailed tests with intrisically dimmer and smaller locus-specific P1 and PAC probes were beyond the scope of the present study. In multicolor FISH, locus-specific probes are ideally revealed using FITC and/or Cy3 labeling, wheras Cy5 is of use for reference probes (usually centromere probes). For the highest possible detection sensitivity, different filter sets, optimized on Cy5 excitation and emission maxima, can be combined with CCD camera imaging.
In multiple immunofluoresence, Cy5 can be used as an additional visible label (
In conclusion, using the set-up described here, Cy5 can be used as an effective visible fluorochrome, alone or in combination with others in multiple labeling. The described filter sets are particularly useful because they work well for photography and CCD-based imaging and also allow direct examination of samples by eye. A distinct advantage lies in the (generally) very low cell and tissue autoflorescence in the Cy5 wavelength range, as well as in the better tissue penetration and limited scattering of the comparatively long-wavelength excitation light required (
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Acknowledgments |
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Supported in part by the Italian Ministry of University and Research (local Research Grants), Italian National Research Council (CNR), and Austrian Science Fund (P13652-GEN).
We thank the National Hormone and Pituitary Program, NIDDK, NICHHD, and USDA for providing antisera, and Olympus Italy for letting us use a VarioCam CCD camera. G-LF conceived and carried out the study, analyzed the data, and wrote the paper; JI provided FISH preparations; PB provided antibodies for staining and multiple staining; GG provided expertise and concrete cooperation with spectral testing.
Received for publication June 28, 1999; accepted October 21, 1999.
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Literature Cited |
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Berger P, Staindl B, Wick G (1988) Antigenic features of human protein hormones elucidated by monoclonal antibodies. In Paul JP, McCruden AB, Schuetz PW, eds. Strathclyde Bioengineering Seminars. The Influence of New Technology on Medical Practice. London, MacMillan Press, 212-219
Brelje TC, Wessendorf MW, Sorenson RL (1993) Multicolor laser scanning confocal immunofluorescence microscopy: practical applications and limitations. In Matsumoto B, ed. Methods in Cell Biology. Vol 38. New York, Academic Press, 97-181
Bridger J, Lampen S, Lichter P (1998) Fluorescence in situ hybridization to DNA. In Spector DL, Goldman RD, Leinwand LA, eds. Cells, a Laboratory Manual. Vol 3. Subcellular Localization of Genes and their Products. Cold Spring Harbor, NY, Cold Spring Harbor Laboratory Press, 1-45
Cullander C (1994) Imaging in the far-red with electronic light microscopy: requirements and limitations. J Microsc 176:281-286[Medline]
Ferri G-L, Gaudio RM, Castello IF, Berger P, Giro G (1997) Quadruple immunofluorescence: a direct visualization method. J Histochem Cytochem 45:155-158
Ferri G-L, Gaudio RM, Cossu M, Rinaldi AM, Polak JM, Berger P, Possenti R (1995a) The VGF protein in rat adenohypophysis: sex differences, changes during the estrous cycle and after gonadectomy. Endocrinology 136:2244-2251[Abstract]
Ferri G-L, Gaudio RM, Tirolo C, Anedda A, Corpino R (1995b) Simultaneous dual-fluorochrome visualization for co-localization studies. Cell Vis 2:413-419
Haugland RP (1996) Handbook of Fluorescent Probes and Research Chemicals. 6th ed Eugene, OR, Molecular Probes
Lennie P, Pokorny J, Smith VC (1993) Luminance. J Opt Soc Am 10:1283-1293. [A][Medline]
Marcus DA (1988) High-performance optical filters for fluorescence analysis. Cell Motil Cytoskel 10:62-70[Medline]
McLean IW, Nakane PK (1974) Periodate-lysine-paraformaldehyde fixative. A new fixative for immunoelectron microscopy. J Histochem Cytochem 22:1077-1083[Medline]
Mujumdar RB, Ernst LA, Mujumdar SR, Lewis CJ, Waggoner AS (1993) Cyanine dye labeling reagents: sulfoindocyanine succinimidyl esters. Bioconjug Chem 4:105-111[Medline]
Reichman J (1994) Handbook of Optical Filters for Fluorescence Microscopy. Brattleboro, VT, Chroma Technology
Smith AG (1986) Astronomical hypersensitization techniques applied to photomicrography: Kodak Technical Pan film 2415. J Microsc 144:39-44