Fluorescent Western Protocol v2

Analysis of proteins using fluorescent immunoblot. Note: - The choice of secondary antibody depends on the choice of primary antibody, whether it is derived from a mouse (monoclonal) or a rabbit (polyclonal). - It is advisable to stick to the 800CW wavelength to avoid problems with chlorophyll autofluorescence encountered with the 680CW antibodies. Literature: Licor's "Fluorescent Western Blot Detection" Licor's "Good Westerns Gone Bad"

Phylogenetic position of Alternochelata lizardensis Kornicker, 1982 within Rutidermatidae (Ostracoda: Myodocopida), with an investigation into its green coloration

We collected the ostracod Alternochelata lizardensis Kornicker 1982 via sediment sampling and evening plankton tows at Lizard Island, Queensland, Australia. While only previously described from samples that lost their natural color, we report males of the species to have bright green bundles of pigment throughout the inner carapace membrane and at specific locations on the ostracod’s body and an unusually colorless and translucent carapace. Females have a heavier carapace and some green pigmentation. We found, in a morphological phylogenetic analysis of Rutidermatidae, that A. lizardensis is part of a paraphyletic grade with other species of Alternochelata Kornicker, 1958 and Scleraner Kornicker, 1975. The analysis also supports a monophyletic Rutiderma Brady & Norman, 1896. We also explored with microscopic and bioinformatic techniques the nature of the unusual green coloration of A. lizardensis and tested the hypothesis that it harbors photosynthetic symbionts. We first sequenced RNA extracted from the entire body of females and searched for genetic markers of possible photobionts. We found genetic matches for two species of cyanobacteria commonly found in seawater. Using fluorescent confocal microscopy to search for chlorophyll autofluorescence in the green patches, we nevertheless found no evidence for the presence of chlorophyll. From these analyses, we concluded there is no evidence that A. lizardensis harbors photosynthetic symbionts suggesting the green coloration is due to something besides photosynthetic symbionts. The framework we present here is nevertheless applicable for other taxa where photobionts are suspected.

chlorophyll autofluorescence in globular and heart-shaped embryos of some dicotyledons

The regions in early embryos of several species display chlorophyll autofluorescence in a certain order. First, autofluorescence in <em>Pisum sativum</em> appears in the basal part of globular embryos; in <em>Lathyrus vernus</em> in the basal part of early heart embryos; in <em>Cardamine pratensis</em> at the sides of the hypocotyl or in <em>Phaseolus vulgaris</em> in the hypocotyl of elongating heart-shaped embryos. Chlorophyll autofluorescence in an embryo proper of <em>Pisum</em> coincides with the development of a lamellar system in the plastids. The suspensorial plastids remain undifferentiated with one or two DNA positive nucleoids. <em>Cardamine</em>, <em>Lathyrus</em>, <em>Phaseolus</em> and <em>Pisum</em> suspensors give no chlorophyll autofluorescence.

Chlorophyll content of Plešné Lake phytoplankton cells studied with image analysis

Using image analysis, chlorophyll autofluorescence was measured in single cells of green alga Monoraphidium dybowskii and in filaments of cyanobacteria (Pseudanabaena sp. and Limnothrix sp.) in the vertical profile of small acidified mountain lake Plešné jezero (Plešné Lake) from May to November of 2003. Cell chlorophyll autofluorescence was converted to cell chlorophyll content using a conversion factor determined by comparing the total autofluorescence of phytoplankton in a microscope field with spectrophotometrically determined total chlorophyll concentration; the conversion factor did not differ between epilimnion (0.5 m depth) and hypolimnion (9 m depth). Vertical patterns of chlorophyll concentration and of cellular chlorophyll content depended on water column mixing: during the period of stable thermal stratification, a metalimnetic peak in total chlorophyll concentration was present and cellular chlorophyll contents in the metalimnion and hypolimnion were notably elevated compared to the surface. Monotonous vertical profiles of both total chlorophyll concentration and cell chlorophyll content were typical for the period of water column overturn. During the stratification period, hypolimnetic Monoraphidium cell chlorophyll content was on average twice as high (maximum difference 2.7-fold) compared to surface values (of 3.2–12.9 fg µm−3), while in filamentous cyanobacteria (surface cell chlorophyll content of 2.2–13.3 fg µm−3), the difference was much higher — six-fold on average, with an 11.6-fold maximum value. The values measured with image analysis in 2003 were compared to unpublished values of total phytoplankton biomass-specific chlorophyll concentrations obtained using manual phytoplankton biomass determination and spectrophotometric chlorophyll measurement in 1998 at the same locality. Good agreement was found in seasonal patterns and vertical profiles of chlorophyll between both seasons.

Photoinhibitory printing on leaves, visualised by chlorophyll fluorescence imaging and confocal microscopy, is due to diminished fluorescence from grana

By analogy with the starch printing technique, it was hypothesised that photoinhibition could be used to print images on leaves that would be invisible to the eye, but easily revealed by chlorophyll fluorescence imaging. We first illustrate the process of chlorophyll fluorescence printing on leaves of the shade plant, Cissus rhombifolia, using photographs of artefacts from starch printing experiments in the laboratory of Molisch. We then use portraits of current leaders in chlorophyll fluorescence research to demonstrate the stability of these images in living tissues. Text printing from microfilm of Ewart’s pioneering studies in photoinhibition shows the resolution of the method with the fixed-focus, portable, imaging system used here. The stability of images, as well as quenching analysis of images and of leaves, suggests that localised photoinactivation, rather than sustained photoprotection, is responsible for the detail displayed by fluorescence printing. Electron micrograph positives of stained thylakoids can be printed to create an illusion of what is imagined to be the source of chlorophyll fluorescence at the membrane level. Individual chloroplasts in adjacent cells under the grid pattern of granal stacks printed on leaves were also examined using a confocal microscope. Compared with chloroplasts in the shaded parts of the grid, those in the photoinactivated parts of the grid show greatly reduced chlorophyll autofluorescence. Moreover, these chloroplasts have lost the localised bright fluorescence from grana. Comparisons of fluorescence yields show that relative chlorophyll autofluorescence from grana observed in the confocal microscope parallels that determined in leaves. Our experiments provide direct visual evidence that fluorescence from grana is lost following photoinactivation of photosystem II in vivo.