Development of a novel cryogenic microscope with numerical aperture of 0.9 and its application to photosynthesis research.

A novel cryogenic optical-microscope system was developed in which the objective lens is set inside of the cryostat adiabatic vacuum space. Being isolated from the sample when it was cooled, the objective lens was maintained at room temperature during the cryogenic measurement. Therefore, the authors were able to use a color-aberration corrected objective lens with a numerical aperture of 0.9. The lens is equipped with an air vent for compatibility to the vacuum. The theoretically expected spatial resolutions of 0.39µm along the lateral direction and 1.3µm along the axial direction were achieved by the developed system. The system was applied to the observations of non-uniform distributions of the photosystems in the cells of a green alga, Chlamydomonas reinhardtii, at 94K. Gaussian decomposition analysis of the fluorescence spectra at all the pixels clearly demonstrated a non-uniform distribution of the two photosystems, as reflected in the variable ratios of the fluorescence intensities assigned to photosystem II and to those assigned to photosystem I. The system was also applied to the fluorescence spectroscopy of single isolated photosystem I complexes at 90K. The fluorescence, assigned to be emitted from a single photosystem I trimer, showed an intermittent fluctuation called blinking, which is typical for a fluorescence signal from a single molecule. The vibronic fluorescence bands at around 790nm were observed for single photosystem I trimers, suggesting that the color aberration is not serious up to the 800nm spectral region.

Study of cell-differentiation and assembly of photosynthetic proteins during greening of etiolated Zea mays leaves using confocal fluorescence microspectroscopy at liquid-nitrogen temperature.

Fluorescence microspectroscopy observations were used to study the processes of cell differentiation and assemblies of photosynthesis proteins in Zea mays leaves under the greening process. The observations were done at 78K by setting the sample in a cryostat to avoid any undesired progress of the greening process during the measurements. The lateral and axial spatial resolutions of the system were 0.64µm and 4.4µm, respectively. The study revealed the spatial distributions of protochlorophyllide (PChld) in both the 632-nm-emitting and 655-nm-emitting forms within etiolated Zea mays leaves. The sizes of the fluorescence spots attributed to the former were larger than those of the latter, validating the assignment of the former and latter to the prothylakoid and prolamellar bodies, respectively. In vivo microspectroscopy observations of mature Zea mays leaves confirmed the different photosystem II (PS I)/photosystem I (PS II) ratio between the bundle sheath (BS) and mesophyll (MS) cells, which is specific for C4-plants. The BS cells in Zea mays leaves 1h after the initiation of the greening process tended to show fluorescence spectra at shorter wavelength side (at around 679nm) than the MS cells (at around 682nm). The 679-nm-emitting chlorophyll-a form observed mainly in the BS cells was attributed to putative precursor complexes to PS I. The BS cells under 3-h greening showed higher relative intensities of the PS I fluorescence band at around 735nm, suggesting the reduced PS II amount in the BS cells in this greening stage.

Less invasive surgical closure of patent ductus arteriosus in extremely low birth weight infants.

Minimally invasive surgery is widely used in pediatric surgery. Extremely low birth weight infants (ELBWI) are literally so fragile to surgical stress that the minimum invasive procedures should be required. We report 15 ELBWI cases with patent ductus arteriosus (PDA), who underwent surgical closure. All of them had failed treatment with indomethacin to close PDA or had contraindicated to its use. The mean gestational age at birth was 26.0+/-2.7 weeks (24-34 weeks) and birth weight 702+/-140 g (479-966 g). The mean age at operation was 23+/-11 days (2-48 days) and body weight at operation 679+/-151 g (428-969 g). The surgery-related mortality was none. No complications were also encountered. Our surgical procedures consist of 2 modalities, one is clipping PDA, not ligation. Clipping technique attributes to minimize the dissection of surrounding tissue of PDA. The other is posterolateral muscle sparing thoracotomy, which would reduce long-term physical impairment and deformity. We believe our surgical technique can be accomplished safely and would be an alternative approach for ELBWI with a lower probability of PDA closure with indomethacin or an increased risk of complications for medical treatment.