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NASA-ASTEPNASA-ASTEP

Detect and confirm sparse life on MarsAtacoma Desert Robot Study as model

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Science objectivesScience 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.

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Fluorescence imagingFluorescence imaging

camera

LED illuminators

light skirt

Xe flash illuminators

robot frame

black box

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Wavelength selectionWavelength selection

A1 A2 A3

B1 B2 B3

C1 C2 C3

push pull

push pull

push pull

autofocus element

system lens

protective sapphire plate

CCD

filter changers

shutter

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Chlorophyll emission spectraChlorophyll emission spectra

Bacteriochlorophylls

0.0

1.0

250 350 450 550 650 750 850 950

C B

A

Eukaryotic Chlorophylls

0.0

1.0

250 350 450 550 650 750 850 950

wavelength (nm)

A

B

Figure 2: Absorption spectra of bacteriochlorophylls a, b, and c (upper panel) and eukaryotic chlorophylls a and b (lower panel).

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Detection of chlorophyll fluorescenceDetection 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.

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Properties of available dyesProperties of available dyes

Dye Absorption max (nm) Emission max (nm) Enhancement on bindingDNA probesSYTO 9 470 530 ++++SYTO BC 480 500 ++++DRAQ56 506 700

Protein probesFluorescamine 380 464 ++++OPA/2ME 334 455 +++NDA/CN 419 493 +++CBQCA/CN 465 560 +++SYPRO Orange 470 570 +++SYPRO Red 550 630 ++++

Lipid probesDPH 353 449 +++Nile Red 559 637 +++DiI(7) 750 780 +

Carbohydrate probeCalcofluor White 360 480 +++

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Fluorescent biofilm indicatorFluorescent biofilm indicator

Figure 1: E. coli at edge of biofilm visualized using SYPRO Red dye.

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Relevant microorganismsRelevant microorganisms

Class Organism

Cyanobacteria Synechocystis sp.

  Nostoc sp.

Eubacteria Escherichia coli

  Bacillus megatherium

  Burkholderia cepacia

  Nitrosomonas europaea

Extremophiles Thermus thermophilus

  Halobacterium salinarum

  Shewanella frigidimarina

  Acidovorax facilis

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Deliverable year 1Deliverable 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.

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Deliverables year 2Deliverables 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!!).

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Deliverables year 3Deliverables 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.

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PersonnelPersonnel

Investigators Institution Role Work Commitment

FY03 FY04 FY05

Alan Waggoner Carnegie Mellon/STC

PI [10] [10] [10]

Victor Weedn Carnegie Mellon/STC

Co-I 10 10 10

Byron Ballou Carnegie Mellon/STC

Co-I 15 15 15

Lauren Ernst Carnegie Mellon/STC

Co-I 30 30 30

Ned Minkley Carnegie Mellon/BHE

Co-I 12 12 12

Dave Wettergreen Carnegie Mellon/RI Collaborator      

Nathalie Cabrol NASA Ames/SETI Collaborator      

Jeff Moersch Univ of Tennessee Collaborator      

Rocco Mancinelli NASA Ames/SETI Collaborator