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. 2018 Jun;127(2):235-245.
doi: 10.1007/s00412-017-0654-5. Epub 2017 Dec 12.

Photoconversion of DAPI and Hoechst Dyes to Green and Red-Emitting Forms After Exposure to UV Excitation

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Free PMC article

Photoconversion of DAPI and Hoechst Dyes to Green and Red-Emitting Forms After Exposure to UV Excitation

Travis J Karg et al. Chromosoma. .
Free PMC article

Abstract

The fluorescent dye 4'-6-Diamidino-2-phenylindole (DAPI) is frequently used in fluorescence microscopy as a chromosome and nuclear stain because of its high specificity for DNA. Normally, DAPI bound to DNA is maximally excited by ultraviolet (UV) light at 358 nm, and emits maximally in the blue range, at 461 nm. Hoechst dyes 33258 and 33342 have similar excitation and emission spectra and are also used to stain nuclei and chromosomes. It has been reported that exposure to UV can convert DAPI and Hoechst dyes to forms that are excited by blue light and emit green fluorescence, potentially confusing the interpretation of experiments that use more than one fluorochrome. The work reported here shows that these dyes can also be converted to forms that are excited by green light and emit red fluorescence. This was observed both in whole tissues and in mitotic chromosome spreads, and could be seen with less than 10-s exposure to UV. In most cases, the red form of fluorescence was more intense than the green form. Therefore, appropriate care should be exercised when examining tissues, capturing images, or interpreting images in experiments that use these dyes in combination with other fluorochromes.

Keywords: Chromosome; DAPI; Drosophila; Fluorescence; H33258; H33342; Photoconversion; Wavelength.

Conflict of interest statement

Conflict of Interest: The authors declare that they have no conflicts of interest.

Figures

Fig. 1
Fig. 1
Photoconversion of DAPI to a form with red fluorescence A Drosophila testis was stained with 2 μg/ml DAPI and mounted in Vectashield. Initially, a z stack was captured for UV excitation/blue emission at 50 ms exposure; green/red image at 500 ms. The testis was then exposed to UV for 3 min, and the z series acquisition was repeated. The images shown here are maximum projections of a series of six 0.2 μm z sections. Each image was individually optimized for display by scaling to the minimum and maximum intensity values of the original 16 bit image with gamma = 1.0. The displayed images were then exported from Slidebook as 8 bit grey scale images. The bottom row shows the colocalization of blue (x axis) and red (y axis) intensities at each time point. The points within the scatterplot are colored blue to red where dark blue represents single pixel events and red represents many pixels with identical intensity values. Axes are indicated as RFP (green excitation/red emission) and DAPI (UV/blue).
Fig. 2
Fig. 2
Photoconversion of DAPI to green and red fluorescent forms A Drosophila testis (T) and accessory gland (AG) were stained with 0.4 μg/ml DAPI and with an AlexaFluor488 antibody against a Drosophila telomere protein (punctate spots in green emission channel) and mounted in Vectashield. Single z section images for each channel were captured at the initial time point and after 1 min, 3 min and 7 min exposure to UV (cumulative values). Exposure lengths for image capture were 50 ms for the UV/Blue channel and 400 ms for Blue/Green and Green/Red channels. In this figure, images were scaled to reflect the change in intensity over time. The minimum and maximum intensity values were determined across the entire time course for each channel. The images from each time point within a channel were adjusted to the same scale, representing 0-99.5% of the full range of intensity values captured over the time course, with gamma = 1.0. Each image was then exported from Slidebook as an 8 bit gray scale image. The change in mean intensity over time across the entire image is plotted in the bottom panel. Values for the UV/Blue channel were multiplied by 8 to compensate for their shorter exposure.
Fig. 3
Fig. 3
Photoconversion of fluorescence in condensed sperm heads After staining with 2 μg/ml DAPI and mounting in Slowfade Gold, a Drosophila testis was visually scanned briefly in UV/blue with a neutral density filter of 1.5% transmittance in the excitation path to identify a region that had several mature sperm head bundles. A z series was acquired for each channel sequentially, with exposure lengths of 50 ms (UV exc.), 200 ms (blue) and 500 ms (green). The images shown here are maximum projections of those series. Image captures were repeated after 3 and 6 min cumulative exposure to UV. Display of each image was scaled separately to 0-99.5% intensity values of that image, and then exported from Slidebook as an 8 bit grey scale image. To quantitate the change in intensity over time, the UV/blue channel was used to create a mask over the sperm head bundle indicated by an arrow in the initial time point. The graphs below show the change for that sperm head bundle (UV/blue values multiplied by 10; blue/green values multiplied by 2.5, to account for different lengths of exposure). The UV/blue channel is plotted separately because of the extreme difference in scales.
Fig. 4
Fig. 4
Bleaching, photoconversion and recovery after DAPI staining A Drosophila accessory gland was stained with 2 μg/ml of DAPI, mounted in Vectashield and visually scanned briefly in UV/blue with a neutral density filter of 1.5% transmittance in the excitation path to identify DAPI stained nuclei. A z series was acquired for each channel sequentially, with exposure lengths of 500 ms (green or blue excitation), and 50 or 100 ms (UV excitation) using the 1.5% transmittance neutral density filter. After the initial capture the tissue was exposed to UV for 3 min, and the z series acquisition was repeated. After 7 days storage in the dark at room temperature, another z series was acquired. The images shown here are maximum projections of those series. The images from each time point within a channel were adjusted to the same scale, representing 0-99.5% of the full range of intensity values captured in that channel over the time course, with gamma = 1.0. Each image was then exported from Slidebook as an 8 bit gray scale image. The change in mean intensity over the entire tissue is plotted at the bottom. UV/blue values were multiplied by 10 or 5 to compensate for the shorter exposures. Inset figure for UV/Blue at 3 min (white box) is an image scaled to the 0-99.5% range of that image showing that, even though it is bleached, stained nuclei are still visible.
Fig. 5
Fig. 5
Bleaching, photoconversion and recovery after Hoechst 33258 staining A Drosophila testis was stained with 50 μg/ml of Hoechst 33258. After staining and mounting in Vectashield, a z series was acquired for each channel sequentially, with exposure lengths of 500 ms (green or blue excitation) or 100 ms (UV excitation) using the 1.5% transmittance neutral density filter. After initial capture the tissue was exposed to UV for 3 min and the z series acquisition was repeated. After 3 days storage in the dark at room temperature, the z series acquisition was repeated again. The images shown here are maximum projections of those series. The images from each time point within a channel were adjusted to the same scale, representing 0-99.5% of the full range of intensity values captured in that channel over the time course, with gamma = 1.0. Each image was then exported from Slidebook as an 8 bit gray scale image. Mean intensity was quantified over approximately the distal 1/4 of the portion of the testis imaged here, and is plotted at the bottom. UV/blue values were multiplied by 5 to compensate for the shorter exposures. Inset figure for UV/blue at 3 min (white box) is an image scaled to the 0-99.5% range of that image showing that nuclei are still visible.
Fig. 6
Fig. 6
Bleaching, photoconversion and recovery after Hoechst 33342 staining A Drosophila testis was stained with 50 μg/ml of Hoechst 33342. After staining and mounting in Vectashield, the testis was imaged as described in Fig. 5, except storage was for 7 days instead of 3 days. Captured images were treated and quantified as described in Fig. 5.
Fig. 7
Fig. 7
Photoconversion of fluorescence in mitotic chromosomes stained with DAPI Chromosomes from Drosophila larval brain mitoses were mounted in Vectashield with 10μM DAPI. Suitable chromosome spreads were identified with the UV/blue channel and a 1.5% transmittance neutral density filter in the excitation light path. After quickly identifying and focusing (estimated time 2-4 seconds), the shutter was closed and captures of all three channels were initiated. Exposure length was 1 ms for the UV/blue channel and 200 ms for the blue/green and green/red channels. After the initial capture, chromosomes were exposed to UV for 5s, 10s, 30s, 60s, 120s and 300s. (cumulative exposure lengths), and images reacquired after each interval. A subset of those images is shown here. The display of each image was scaled to the 0-99.5% intensity interval for that image, gamma = 1.0, and exported as an 8 bit grey scale image from Slidebook. A mask was generated in the UV/blue channel for the chromosome pair indicated by the arrow in the first image, and mean intensity over that chromosome pair was determined in each channel and for each time point. The change in intensity with UV exposure is graphed at the bottom.
Fig. 8
Fig. 8
Photoconversion of fluorescence in mitotic chromosomes stained with Hoechst 33258 Chromosomes from Drosophila larval brain mitoses were mounted in Vectashield with 50 μg/ml of Hoechst 33258. Chromosome spreads were identified and imaged as described in Fig. 7, except that cumulative UV exposure only extended to 120 seconds. A mask was generated over the chromosome pair indicated by the red arrow in the first image, and mean intensity over that chromosome pair was determined in each channel and for each time point. The change in intensity with UV exposure is graphed at the bottom.
Fig. 9
Fig. 9
Photoconversion of fluorescence in mitotic chromosomes stained with Hoechst 33342 Chromosomes from Drosophila larval brain mitoses were mounted in Vectashield with 50 μg/ml of Hoechst 33342. Chromosome spreads were identified and imaged as described in Fig. 7, except that cumulative UV exposure only extended to 120 seconds. A mask was generated over the chromosome indicated by the red arrow in the first image, and mean intensity over that chromosome was determined in each channel and for each time point. The change in intensity with UV exposure is graphed at the bottom.

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