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

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

Imaging of Cell Structures Using Optimized Soft X-ray Contact Microscopy

This work is to study the relationship between the exposure conditions and the quality of cell imaging with soft X-ray contact microscopy (SXCM). It is a crucial step in the efficient visualization of cell structures. Three different human cell lines: DU145 prostate carcinoma cells, HCC38 breast cancer cells, and Poietics mesenchymal stem cells were used to establish the optimal exposure conditions in SXCM. The image quality depended on the soft X-ray (SXR) absorbed energy and photoresist development conditions. At lower SXR energy (200 or 400 SXR pulses), sharp cell edges, membrane projections, and cell–cell connections were visible. In contrast, higher energy (600 or 800 SXR pulses) allowed observation of the cytoskeleton and the nucleus in a cell type-dependent manner (the influence of cell thickness and internal complexity was noted)