Nanometer-Resolution Imaging of Living Cells Using Soft X-ray Contact Microscopy

Soft X-ray microscopy is a powerful technique for imaging cells with nanometer resolution in their native state without chemical fixation, staining, or sectioning. The studies performed in several laboratories have demonstrated the potential of applying this technique for imaging the internal structures of intact cells. However, it is currently used mainly on synchrotrons with restricted access. Moreover, the operation of these instruments and the associated sample-preparation protocols require interdisciplinary and highly specialized personnel, limiting their wide application in practice. This is why soft X-ray microscopy is not commonly used in biological laboratories as an imaging tool. Thus, a laboratory-based and user-friendly soft X-ray contact microscope would facilitate the work of biologists. A compact, desk-top laboratory setup for soft X-ray contact microscopy (SXCM) based on a laser-plasma soft X-ray source, which can be used in any biological laboratory, together with several applications for biological imaging, are described. Moreover, the perspectives of the correlation of SXCM with other super-resolution imaging techniques based on the current literature are discussed

Cytotoxicity of PP(Arg)2- and Hp(Arg)2-mediated photodynamic therapy and early stage of apoptosis induction in prostate carcinoma in vitro

Porphyrin photosensitizers tend to localize in mitochondria. The depolarization of mitochondrial membrane is one of the early stages of apoptosis and Laser Scanning Fluorescence Microscopy allows to determine changes in transmembrane mitochondrial potential under influence of PDT depending on the kind of photosensitizer (PP(Arg)2, Hp(Arg)2), the energy dose (5, 10, 30 and 50 J/cm2) and time periods (24 and 48 hours after irradiation) in the LNCaP (lymphonodal metastasis of prostate carcinoma, the androgen dependent cell line). Cyototoxicity induced by PP(Arg)2- and Hp(Arg)2-based PDT depending on energy dose and time after irradiation in prostate carcinoma is determined with MTT. Generally, it was shown that lower energy doses induce greater changes in transmembrane mitochondrial potential. Hp(Arg)2-based PDT was more effective causing greater mitochondrial membrane depolarization and cell viability decrease in comparison to PP(Arg)2-mediated PDT (in the case of maximal nontoxic photosensitizer doses used)

Cytotoxicity of PP(Arg)2- and PP(Ala)2(Arg)2-based photodynamic therapy and early stage of apoptosis induction in human breast cancers in vitro

Mitochondria are cell energetic centers where ATP is produced. They also play a very important role in the PDT as intracellular sites of photosensitizer localization. Photosensitizers gathering in mitochondria (like porphyrin derivatives used in this work) are more effective in tumor cell destruction. Moreover, it was assumed that di-amino acid substituents attached to porphyrin ring will strengthen the effectivity of interaction with membrane receptors of examined cells. MTT assay was performed to investigate the influence of PP(Arg)2 and PP(Ala)2(Arg)2-based PDT on breast cancer cell viability for 24 h, 48 h and 120 h after cell irradiation. Then the influence of PP(Ala)2(Arg)2- and PP(Arg)2-mediated PDT on early mitochondrial apoptosis induction via measurements of the transmembrane mitochondrial potential changes was examined. Results showed that lower energy doses and maximal nontoxic photosensitizer doses of PP(Ala)2(Arg)2 and PP(Arg)2 applied in PDT can imply apoptotic cell death. It was confirmed that modification of the protoporphyrin IX by attaching two alanine substituents raised the efficiency of photodynamic therapy

Fabrication and geometric characterization of highly-ordered hexagonally arranged arrays of nanoporous anodic alumina

Anodic aluminum oxide (AAO) has been fabricated in the 0.3 M oxalic acid at voltage range 20-60 V and temperature range of 35-50oC. The resulting nanoporous alumina surfaces were characterized by high resolution scanning electron microscopy, and the images were quantitatively analysed by means of an innovative approach based on fast Fourier transform. The influence of operating anodization voltage and electrolyte temperature on nanopores geometry (pore diameter, interpore distance, porosity, pores density) and arrangement has been studied in details and compared to literature data and theoretical calculations. It was found that independently from the temperature, the best arrangement of the nanopores is for anodic aluminum oxide formed at voltages ranging from 40 to 50 V. Moreover, it was found that pore diameter and interpore distance increase linearly with voltage, what is in line with the literature data

Fabrication and geometric characterization of highly-ordered hexagonally arranged arrays of nanoporous anodic alumina

Anodic aluminum oxide (AAO) has been fabricated in the 0.3 M oxalic acid at voltage range 20-60 V and temperature range of 35-50oC. The resulting nanoporous alumina surfaces were characterized by high resolution scanning electron microscopy, and the images were quantitatively analysed by means of an innovative approach based on fast Fourier transform. The influence of operating anodization voltage and electrolyte temperature on nanopores geometry (pore diameter, interpore distance, porosity, pores density) and arrangement has been studied in details and compared to literature data and theoretical calculations. It was found that independently from the temperature, the best arrangement of the nanopores is for anodic aluminum oxide formed at voltages ranging from 40 to 50 V. Moreover, it was found that pore diameter and interpore distance increase linearly with voltage, what is in line with the literature data

Nanometer-Resolution Imaging of Living Cells Using Soft X-ray Contact Microscopy

Soft X-ray microscopy is a powerful technique for imaging cells with nanometer resolution in their native state without chemical fixation, staining, or sectioning. The studies performed in several laboratories have demonstrated the potential of applying this technique for imaging the internal structures of intact cells. However, it is currently used mainly on synchrotrons with restricted access. Moreover, the operation of these instruments and the associated sample-preparation protocols require interdisciplinary and highly specialized personnel, limiting their wide application in practice. This is why soft X-ray microscopy is not commonly used in biological laboratories as an imaging tool. Thus, a laboratory-based and user-friendly soft X-ray contact microscope would facilitate the work of biologists. A compact, desk-top laboratory setup for soft X-ray contact microscopy (SXCM) based on a laser-plasma soft X-ray source, which can be used in any biological laboratory, together with several applications for biological imaging, are described. Moreover, the perspectives of the correlation of SXCM with other super-resolution imaging techniques based on the current literature are discussed