Hoechst 33258 This page summarizes the optical absorption and emission data of Hoechst 33258 that is available in the PhotochemCAD package, version 2.1a (Du 1998, Dixon 2005). I reworked their data to produce these interactive graphs and to provide direct links to text files containing the raw and manipulated data. Although I have tried to be careful, I may have introduced some errors; the cautious user is advised to compare these results with the original sources.

You can resize any of the graphs by clicking and dragging a rectangle. If you hover the mouse over the graph, you will see a pop-up showing the coordinates. One of the icons in the upper right corner will let you export the graph in other formats.


This optical absorption measurement of Hoechst 33258 were made by R.-C. A. Fuh in the summer of 1995 using a Cary 3. The absorption values were collected using a spectral bandwidth of 1.0 nm, a signal averaging time of 0.133 sec, a data interval of 0.25 nm, and a scan rate of 112.5 nm/min.

These measurements were scaled to make the molar extinction coefficient match the value of 46,000cm-1/M at 345.5nm (Haugland, 1992).

Original Data | Extinction Data


The fluorescence emission spectrum of Hoechst 33258 dissolved in water. The excitation wavelength was 350nm. The quantum yield of this molecule is 0.034 (Härd, 1990). This spectrum was collected by on 06-24-1997 using a Spex FluoroMax. The excitation and emission monochromators were set at 1 mm, giving a spectral bandwidth of 4.25 nm. The data interval was 0.5 nm and the integration time was 2.0 sec.

Samples were prepared in 1cm pathlength quartz cells with absorbance less than 0.1 at the excitation and all emission wavelengths to uniformly illuminate across the sample, and to avoid the inner-filter effect. The dark counts were subtracted and the spectra were corrected for wavelength-dependent instrument sensitivity.

Original Data | Emission Data


The fluorescence yield of Hoechst 33258 increases dramatically in the presence of DNA (Weisblum, 1974) or in the solvent DMF.


Dixon, J. M., M. Taniguchi and J. S. Lindsey (2005), "PhotochemCAD 2. A Refined Program with Accompanying Spectral Databases for Photochemical Calculations, Photochem. Photobiol., 81, 212-213.

Du, H., R.-C. A. Fuh, J. Li, L. A. Corkan and J. S. Lindsey (1998) PhotochemCAD: A computer-aided design and research tool in photochemistry. Photochem. Photobiol. 68, 141-142.

Haugland, R. P. (1992-1994) Molecular Probes. Handbook of Fluorescent Probes and Research Chemicals.

Härd, T., P. Fan and D. R. Kearns (1990) A fluorescence study of the binding of Hoechst 33258 and DAPI to halogenated DNAs. Photochem. Photobiol. 51, 77-86.

Weisblum, B. and E. Haenssler (1974) Fluorometric properties of the bibenzimidazole derivative Hoechst 33258, a fluorescent probe specific for AT concentration in chromosomal DNA. Chromosoma (Berl.) 46, 255-260.