Observe complex pigment mixtures in living cells

In a technological advance that allows researchers to observe active cells when performing photosynthesis, they have developed a method that can enhance the effect of biological images under the influence of light. Fluorescence is used to observe individual pigments within living bacterial cells.

This method provides a very clear picture of what is happening at the molecular level during photosynthesis. Promising to provide important information about the activities within the cell as they participate in the photosynthesis process, including the process of collecting light and turning light into chemical energy.

The above information is valuable in helping researchers control and regulate bacteria for different purposes, according to WU Vermaas, professor of ASU Life Sciences University and co-author The main pseudocode of the study: ' Multi-spectral fluorescence imaging helps identify location and pigment distribution in cyanobacterial bacterial cells '. The study report was published online during the week in the Early Edition version of the Proceedings of the National Academy of Sciences . ASU researchers have collaborated with scientists at Sandia National Laboratory and Albuquerque to find new ways.

Vermaas said: 'This is a new tool in our gadget box and is also a very useful tool'. The method is based on fluorescence imaging to distinguish different types of pigments in bacteria involved in photosynthesis. Fluorescence is a property that some compounds can emit specific light when excited by a specific light wavelength. Until now, modern fluorescence methods have had a hard time distinguishing compounds and pigments as well as fluorescence properties. That's why researchers are constrained to find out exactly what is going on inside the cell.

The focal fluorescence microscopy method has been shown to be an effective method to locate pigments in cells as long as there is a small spectral overlap between different fluorescence pigments. This multi-spectral fluorescence imaging method has extended the boundaries for engineering. It can separate each pigment based on similar fluorescence spectra.

Vermaas said the researchers applied a high-end imaging analysis developed at Sandia Laboratories.

Vermaas - a member of ASU's Center for Bioenergy and Photosynthesis - said: 'This is a superior analytical method compared to what the commercial analysis system can do. It tells you where the fluorescent material is located in the cell, what the fluorescence of each type of matter looks like and how much is emitted even if the fluorescence properties of the two different objects seem identical. together'.

Vermaas said the original study focused on pigmentation in the cyanobacterium bacterium - a bacterium that is interesting for the team. When applying this method, they demonstrated that the pigments involved in photosynthesis (chlorophyll, phycobilin and carotenoid) can be located in the living cells of cyanobacterium Synechocystis sp. PCC 6803 adopts the twisting process of individual fluorescence spectra emitted in very small volumes (0.03 mm ³ ) thanks to the multi-spectral focus fluorescence image.

Vermaas explains: 'The method allows us to extend the resolution limits of the confocal fluorescence microscopy approach especially when there is a mixture of different fluorescent compounds with quite similar spectra. Specifically in the case of cyanobacteria, the above method identifies the different pigments involved in the cell. Along with observing the pigment, we can locate two different photosynthetic systems in the cell. '

The researchers reported that the results showed a heterogeneous texture of thylakoid membrane of cyanobacteria cells: Phycobilin emissions are quite strong along the periphery of the cell while the chlorophyll fluorescence is relatively distributed. evenly in every position. This suggests that phycobilin fluorescence is more prominent on the periphery of thylakoid membranes. Carotenoid plant pigments are highly concentrated in cell walls and in thylakoid membranes as well.

Two elements of chlorophyll fluorescence component were also resolved: Short wavelength composition concentrated mainly in photosynthesis system II and was particularly strong in the area near the periphery of the cell; Long wavelength composition is characteristic of photosynthesis system I (it disappears in mutant forms lacking photosynthesis), has relatively strong intensity in the inner region of thylakoid membrane. The results show the structural heterogeneity between thylakoid membrane belts (the area inside the thylakoid membrane mainly occurs photosynthesis I).

Vermaas said that means that even a simple, small, cyanobacterium cell (less than 100 times smaller than the size visible to the naked eye), there is also a subtle functional division between lateral thylakoid membranes. in the cell take on different processes during photosynthesis in different areas of the membrane.

Vermaas said: "We found that the two photosynthetic systems are not located in the same position on the thylakoid membrane in the cell even when the thylakoid membranes look the same on the electron microscope image." On this basis, the cell's mode of action is: the area inside the thylakoid membrane mainly produces ATP (adenosine triphosphate) that supplies energy to the cell by transporting electrons in a cycle in the photosynthesis system I. ; The peripheral area that regulates the electron flow also produces ATP as well as nicotinamide adenine dinucleotide phosphate that transports the equivalents used to capture carbon dioxide.

'The important issue here is that even if the cell compartments cannot be identified (such as thylakoid membranes) by electron microscopy, there is also functional heterogeneity due to different protein groups in the regions. Different areas of thylakoid membranes. We have long questioned this chaos but have never been proven experimentally. '

Vermaas said: 'These results have demonstrated that a multi-spectral fluorescence image can provide new information about the organization and pigment location in tiny cells. It also provides a method for locating complex fluorescent compounds in high-resolution living cells. '

Multi-spectral fluorescence imaging is thought to be effective when it comes to understanding fluorescence multi-pigment cells. But according to Vermaas, these results have so far illustrated the power of technology in identifying the specific location of fluorescent compounds in cells. The study opens a new vision for locating some proteins, pigments and other fluorescent compounds inside cells. This method seems to be an indispensable tool in biology to provide a critical analysis of the interplay of proteins, metabolites, and energy states in cells to find out. How does the cell function?