Microfluorimetry for the Biosciences

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Micro-fluorimetry for the Biosciences
Diether J. Recktenwald PhD Contact email
diether_at_att.net
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Need of Biology Research
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Biological measurements

• Ideally measure all chemicals in an organism with
  spatial and temporal resolution
• Microscopy
• high spatial resolution to cell substructures,
  few cells,
• few parallel chemical measurements with
  subcellular resolution
• Flow cytometry
• spatial resolution to one cell, few to many
  cells, several parallel chemical measurements
  with one cell resolution
• Arrays
• low spatial resolution to macroscopic cell
  ensemble level, many cells, many parallel
  chemical measurements cell sample resolution

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Signal Transduction Network
Source DOE, genomes to life program
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System Components
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Detection Instrument Considerations

• Light source (wavelength, power, beam-shape)
• Excitation optics (direct beams vs. fiberoptics,
  multi-laser)
• Emission optics (collection efficiency NA,
  immersion optics)
• Spectral filtering (dispersive elements, filters,
  )
• Detectors (PMT, CCD, )
• Signal processing (analog vs. digital, data
  reduction)
• System background (electronic noise, photon
  statistics, )
• Fluidics (sheath flow vs undiluted sample, edge
  effects, focal depth)

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Excitation light sources

• High luminous density emitters
• (490nm a desirable wavelength for fluorescein)
• Arc lamps (Hg-arc, )
• Gas lasers (Ar, Kr, )
• Solid state lasers

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Fluorescence

• Absorbance
• Lifetime of excited state
• Stokes shift
• Quantum yield
• Saturation
• Intrinsic
• Extrinsic

Source www.olympusmicro.com
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Fluorescent Dyes

• Unsaturated organic molecules (photobleaching)
• Energy-transfer dyes
• Lanthanide derived pigments
• Quantum dots
• Dye combinations for multi-color fluorescence
• Single vs. multi-laser excitation
• Spectral overlap

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Fluorescence Measurements of Chemical Properties

• Intensity Concentration
• Lifetime Background reduction
• FRET (energy transfer) Proximity
• Polarization Rotational mobility
• Fluorescence correlation Lateral mobility

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Quantitative measurements

• Calibration with
• soluble fluorophors
• calibration particles
• photon statistics

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Fluorescence Quantitation by Volume Exclusion (1)
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Fluorescence Quantitation by Volume Exclusion (2)
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Assay considerations

• Reagent selection
• Selection of labels (spectral properties,
  overlap)
• Homogeneous vs. non-homogeneous
• Intrinsic fluorescence vs. use of labels
• Specimen and reagent auto-fluorescence
• Reference samples

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Systems, which use fluorescence
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Fluorescence Microscope
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Micro-arrays
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Micro-arrays
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Micro-arrays
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Cytometric Bead Array Assays
Quantum Dot Corp
Bead Array assays
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Flow Cytometers
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Flow Cytometer Fluidics
Cell Input
Injector Tip
Sheath fluid
Fluorescence
signals
Focused laser
beam
Cells after analysis, available for culture
CD-ROM Vol 3 Purdue University Cytometry
Laboratories
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Applications in research
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Applications of Microfluorimetry

• Measurements
• specific structures by immunofluorescence or
  in-situ hybridization
• protein content
• DNA and RNA with fluorogenic dyes
• gene expression with fluorescent proteins
• auto-fluorescencent components
• enzyme activity with fluorogenic substrates
• pH and other cations with ion-specific probes
• redox potential

Hela cells transfected with fluorescent protein
vectors for nuclei, mitochondria and tubulin.
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Applications of Microfluorimetry

• Cell sorting
• Multi-parameter cell subset analysis
• Organelle visualization
• Protein translocation
• Large scale cell composition changes
• Single fluorescent molecule detection
• (PCR vs. direct)
• Nucleic acid fragment sizing
• Protein-ligand interactions
• Virus counting

From Huang Z et al. in Cytometry 35 169-175
(1999)
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Single Molecule Detection
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Cellular Proteomics From Sample to Result
Transfect with Genes/GFP
Tissue
Blood
Cell Culture
Suspension of Single Cells
Genome Analysis
Purified Single Cells
Cell Disruption
Proteome Analysis
Cell Homogenate
Purified Cell Substructure Fractions
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Applications in Bio-defense
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Flow Cytometry for Microbiology Rapid TVC Assay
35 sec
10 sec
25 sec
60 sec
80 sec
90 sec
120 sec
Rana Alsharif, BD Biosciences
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Rapid Bioagent Identification
Fluorescence based detection and identification
of Bacillus anthracis within 10 minutes after
receiving a sample using widely available
instrumentation
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Simultaneous Measurements of Multiple Agents
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Applications in Clinical Diagnostics and
Monitoring
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Clinical Tests (CD34 counting)

• Large scale protein analysis in patient serum
  with arrays
• Cell subset analysis (special hematology)
• CD4
• CD34
• LL
• Histopathology

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Therapy
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Cell Sorting
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CD34 progenitor cell sorting

• Isolate human blood progenitor cells for cell
  transplantation
• CD34 at about 1 in mobilized peripheral blood
• for high purity several parameters are used
• 2-5106 CD34 cells needed for treatment
• analysis rate maximally at 105 cells sec-1
• sort rate at 2104 cells sec-1
• several hours of sorting required

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Outlook
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Challenges

• Better use of single molecule sensitivity
• Absolute quantitation
• Better multiplexing
• Efficient use of intrinsic fluorescence

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END Contact diether_at_att.net Presentation on web
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