Carol Hirschmugl

7. What is Infrared Spectroscopy?

9. Sources of Infrared Light

15. Why use a storage ring source?

17. Chemical Mapping and Chemical Image:

21. Perkin-Elmer Spotlight FTIR Microspectrometer

22. Multiple Source IR Microspectroscopy

23. Optics to Microscope (4 of 16 beams)

30. Experimental Conditions

32. The top image is a picture of an Euglena Gracilis algal cell that has been allowed to dry partially. (IR cannot measure through water of thickness higher than 10 microns) The items immediately below the titles Alga 1 and Alga 2 are dendograms. These come from the statistical analysis. They are basically trees, where the branches of the trees represent clusters of branches lower in the tree, and at the bottom of the tree there are enough branches for each branch to represent each individual spectrum used in the statistical analysis. Thus, human judgement is required to decide where to �slice� the tree, and therefore how many clusters of data are important. The dashed black lines represent the slice where 4 clusters have been chosen. From my analysis of these cells (and many other euglena gracilis cells) It is clear that the initial split (2 clusters) represents the difference between the spectra for inside the cell and those for outside and on the edge of the cell. I have chosen the 4 cluster example to emphasize how similar and different the data look for different samples. The difference between Alga 1 and Alga 2 is the nutrient condition (how much nitrogen in the form of Nitrate do they have available to ingest.) Below the title Average Spectra etc., I have included the color coded average spectra for the four clusters for each alga sample. The arrows indicate which alga the spectra come from, and the average spectra in the top of the graph originate from the right hand branch, while the average spectra in the bottom of the figure originate from the left hand branch. (Note that there are three average spectra "inside" the alga that is nutrient deficient, while there is only one inside the alga that is nutrient replete.) You can see that the signatures are similar, but are slightly varied, especially for the top two spectra - the carbohydrate (about 1000 cm-1) to protein (about 1600 cm-1) ratio is quite different between these two. For the top spectrum this ratio is greater than 1, while for the second it is approximately 1, which is indicative of the nutrient status of the algae. These similar patterns (for both the dendograms and the average spectra) have been observed for the 15 individual alga cells that we measured. The motivation for this work is two fold. The first is to understand the physiology inside the alga without destroying the alga cell, and ultimately to get higher spatial resolution images (which would be possible with the new Focal Plane array detectors coupled with the synchrotron facility - which I am trying to get funding for now - any ideas?). Then we can really associate the macromolecular pools with the �visible structure� in the cells. Here, the difference between the carbohydrate to protein ratios that I mention above, and ratios of other constituents are the most important results. The second aspect is to determine what exudates are released from the cells. This is why the spectra from the edges and around the cells are important, to isolate the chemical signatures for this, and ultimately determine how a mucaloge between cells forms since this can lead to algal blooms. In this sense, it is interesting that the starved algae have a larger variation between the spectra within the cell, while the the nutrient replete alga have a larger variation for the spectra on the edges and around the cell. We do not completely understand this yet, but could give some clues about the mucalage formation I have recommended a paper about the mucolage formation for other algal species that was published last year by one of my collaborators. I have also included a reference to a paper that describes the dendograms with a nice example for the algae ( section 7). (This is actually the basis for the other results which are very promising but need a bit more work). The top image is a picture of an Euglena Gracilis algal cell that has been allowed to dry partially. (IR cannot measure through water of thickness higher than 10 microns) The items immediately below the titles Alga 1 and Alga 2 are dendograms. These come from the statistical analysis. They are basically trees, where the branches of the trees represent clusters of branches lower in the tree, and at the bottom of the tree there are enough branches for each branch to represent each individual spectrum used in the statistical analysis. Thus, human judgement is required to decide where to �slice� the tree, and therefore how many clusters of data are important. The dashed black lines represent the slice where 4 clusters have been chosen. From my analysis of these cells (and many other euglena gracilis cells) It is clear that the initial split (2 clusters) represents the difference between the spectra for inside the cell and those for outside and on the edge of the cell. I have chosen the 4 cluster example to emphasize how similar and different the data look for different samples. The difference between Alga 1 and Alga 2 is the nutrient condition (how much nitrogen in the form of Nitrate do they have available to ingest.) Below the title Average Spectra etc., I have included the color coded average spectra for the four clusters for each alga sample. The arrows indicate which alga the spectra come from, and the average spectra in the top of the graph originate from the right hand branch, while the average spectra in the bottom of the figure originate from the left hand branch. (Note that there are three average spectra "inside" the alga that is nutrient deficient, while there is only one inside the alga that is nutrient replete.) You can see that the signatures are similar, but are slightly varied, especially for the top two spectra - the carbohydrate (about 1000 cm-1) to protein (about 1600 cm-1) ratio is quite different between these two. For the top spectrum this ratio is greater than 1, while for the second it is approximately 1, which is indicative of the nutrient status of the algae. These similar patterns (for both the dendograms and the average spectra) have been observed for the 15 individual alga cells that we measured. The motivation for this work is two fold. The first is to understand the physiology inside the alga without destroying the alga cell, and ultimately to get higher spatial resolution images (which would be possible with the new Focal Plane array detectors coupled with the synchrotron facility - which I am trying to get funding for now - any ideas?). Then we can really associate the macromolecular pools with the �visible structure� in the cells. Here, the difference between the carbohydrate to protein ratios that I mention above, and ratios of other constituents are the most important results. The second aspect is to determine what exudates are released from the cells. This is why the spectra from the edges and around the cells are important, to isolate the chemical signatures for this, and ultimately determine how a mucaloge between cells forms since this can lead to algal blooms. In this sense, it is interesting that the starved algae have a larger variation between the spectra within the cell, while the the nutrient replete alga have a larger variation for the spectra on the edges and around the cell. We do not completely understand this yet, but could give some clues about the mucalage formation I have recommended a paper about the mucolage formation for other algal species that was published last year by one of my collaborators. I have also included a reference to a paper that describes the dendograms with a nice example for the algae ( section 7). (This is actually the basis for the other results which are very promising but need a bit more work).

39. New Scientific Opportunities

43. Proof of Principle

44. First CSR Science:JPR in Bi2Sr2CaCu2O8

53. Signatures in Infrared Spectroscopy?