Hadamard transform spectral microscopy for single cell imaging using organic and quantum dot fluorescent probes.

We report the development of Hadamard transform fluorescence spectral imaging microscopy by using a movable one-dimensional Hadamard mask to encode the image signals and a linear charge-coupled device (CCD) to detect the encoded spectral image signal. The technique has the ability of multi-spectral imaging and images with four dimensions: location coordinate (X and Y), fluorescence intensity (Z) and wavelength (lambda) (or time (t)) can be obtained. The results show that the system has high spectral resolving power (0.3 nm) and imaging capability, where image resolution up to 511 x 512 can be easily obtained for analysis of tiny samples. Multicolor imaging of acridine orange/ethidium bromide double-stained fibroblast treated with glyoxal shows that 1.5 mM glyoxal obviously causes apoptosis, thus demonstrating that identification of apoptotic cells can be realized using the Hadamard system not only through morphologic changes but also by the spectral evidences of the fluorescently stained cells. In addition, based on immunostaining with quantum dots (QDs) emitting at 550 and 610 nm to tag and trace two breast cancer biomarkers human epidermal growth factor receptor 2 (HER-2) and estrogen receptor (ER) in human breast cancer tissue with in situ dual-color fluorescence imaging, sensitive spectra and images were obtained and show that the system can be applied to visualize and quantitatively measure the subcellular proteins inside the tumor tissues, especially when a single laser line is used as the illuminating source for multi-components with different emission peaks. The preliminary biomedical applications indicate the great potential of the Hadamard imaging system as a useful tool in biomedicine.