1. CMU Science and Technology Center NASA-ASTEP Detect and confirm sparse life on Mars Atacoma Desert Robot Study as model

2. CMU Science and Technology Center Science objectives To explore the limits of sparse microbe detection with minimal sample manipulation using a sensitive fluorescence instrument and innovative fluorescence reagents. To test the concept of multicolor biomarker correlation to minimize false positive identification of microbes.

3. CMU Science and Technology Center Fluorescence imaging

4. CMU Science and Technology Center Wavelength selection

5. CMU Science and Technology Center Chlorophyll emission spectra Figure 2: Absorption spectra of bacteriochlorophylls a, b, and c (upper panel) and eukaryotic chlorophylls a and b (lower panel).

6. CMU Science and Technology Center Detection of chlorophyll fluorescence Figure 5. Pennsylvania sandstone with photosynthetic biofilm. Biofilm is less than 100 micrometers thick at greatest depth. Left, visible light. Center and right images are fluorescence images, emission at 820nm (near-infrared fluorescence) for both. Center, 365 nm excitation;30sec exposure; right, 665nm excitation, 3sec exposure. The 820 nm emission captures the near-infrared tail of the chlorophyll emission. Notice the much-improved contrast between background signal and chlorophyll fluorescence obtained with longer wavelength emission. This figure illustrates the sensitivity of detection possible even with weak excitation and shows that wavelength selection capability is essential for optimal contrast.

7. CMU Science and Technology Center Properties of available dyes DyeAbsorption max (nm)Emission max (nm)Enhancement on binding DNA probes SYTO 9470530++++ SYTO BC480500++++ DRAQ56506700 Protein probes Fluorescamine380464++++ OPA/2ME334455+++ NDA/CN419493+++ CBQCA/CN465560+++ SYPRO Orange470570 +++ SYPRO Red550630++++ Lipid probes DPH353449+++ Nile Red559637+++ DiI(7)750780+ Carbohydrate probe Calcofluor White 360480+++

8. CMU Science and Technology Center Fluorescent biofilm indicator Figure 1: E. coli at edge of biofilm visualized using SYPRO Red dye.

9. CMU Science and Technology Center Relevant microorganisms ClassOrganism CyanobacteriaSynechocystis sp. Nostoc sp. EubacteriaEscherichia coli Bacillus megatherium Burkholderia cepacia Nitrosomonas europaea ExtremophilesThermus thermophilus Halobacterium salinarum Shewanella frigidimarina Acidovorax facilis

10. CMU Science and Technology Center Deliverable year 1 1: In Carnegie Mellon laboratories, completed testing of original fluorescent reagents on various soil samples and evaluation of reagents and instrumentation concepts. 2: Produce formal designs and construction of multicolor fluorescence detection system 3: Develop dye spritzing system.

11. CMU Science and Technology Center Deliverables year 2 1: Design and construct “drop box” containing multicolor fluorescence imaging unit. 2: Produce and evaluate the capabilities of the fluorescent reagents and drop-box imaging system placed manually on sites in Pennsylvania and the Southwest United states. Evaluation requires laboratory analysis of material from the sites. 3: Produce improvements and substitutes for reagents that do not function properly and make improvements to imaging system, excitation system and drop box. 4: Identify partner for robotic field study and produce machine plans for attachment of drop box imaging system to a rover (DONE!!).

12. CMU Science and Technology Center Deliverables year 3 1: Mount drop box imaging system on rover. 2: Produce results of field study for system on rover with laboratory analysis of material from sites visited. 3: Improved system instruments and reagents 4: Documentation and dissemination of results of studies.

13. CMU Science and Technology Center Personnel InvestigatorsInstitutionRoleWork Commitment FY03FY04FY05 Alan WaggonerCarnegie Mellon/STC PI[10] Victor WeednCarnegie Mellon/STC Co-I10 Byron BallouCarnegie Mellon/STC Co-I15 Lauren ErnstCarnegie Mellon/STC Co-I30 Ned MinkleyCarnegie Mellon/BHE Co-I12 Dave WettergreenCarnegie Mellon/RICollaborator Nathalie CabrolNASA Ames/SETICollaborator Jeff MoerschUniv of TennesseeCollaborator Rocco MancinelliNASA Ames/SETICollaborator

14. CMU Science and Technology Center Technological objectives To adapt currently existing fluorochromes (fluorescent biomarkers) for simultaneous multispectral detection of trace life. To design and synthesize improved fluorescent reagents. To define the most suitable spectral emission lifetime for detecting fluorescence due to traces of life in presence of background fluorescence from minerals. To construct an intermediate magnification fluorescence imaging instrument having both a high numerical aperture for effective light capture and an ability to survey 3-30 cm diameter areas with good light-gathering power. The instrument will be capable of multispectral fluorescence imaging at high sensitivity. To adapt image analysis software for identifying microorganisms in bio-films and colonies (perhaps individual microbes under ideal circumstances) and reporting quantitative measures and statistical evaluations of the concentrations of microorganisms in the context of their soil environments.