CLARiTY Spectrophotometers

1. CLARiTY Spectrophotometers Scatter does not matter, only sample absorbance does! Unique to On-Line Instrument Systems (Olis) Illustrations by Dr. Paul Boxrud, Product Manager, Olis, Inc. February 21, 2012

2. Configuration of the Olis CLARiTY Sample detector Light Source Mono-chromator DSPC 1 DSPC 2 Reference detector Not to scale DSPC (DeSa Suspension Presentation Cavity): Quartz flask within a highly reflective chamber that holds the liquid, solid, frozen, or powder sample, causing perfect diffusion of the measurement beam into a “gas of photons”. DSPC 1 is the sample cavity while the DSPC 2 is the reference.

3. Absorbance in a Standard Cuvette Detector Measurement beam minus absorbance and scatter Focused incoming beam pathlength A normal 1 cm2 cuvette allows passage of the focused measurement beam to the awaiting detector. Pathlength is typically 1 cm or shorter.

4. Absorbance in a DSPC chamber Reflective coating (surrounds cell) Output is measurement beam (diffuse) minus absorbance Diffusing input port The CLARiTY’s DSPC ‘cuvette holder’ – because it is within a highly reflective chamber- causes the measurement beam to be fully scattered into a ‘gas of photons.’ The sample cannot further scatter the beam, and the effective pathlength is in the tens of centimeters because the photons bounce around extensively, traversing the flask trillions of times.

5. Standard UV/Vis Spectrophotometer Mono-chromator Light Source Scattered light Detector Standard cuvette • Focused light beam is scattered by sample • Scattered light is lost and not differentiated from absorbed light • Detector captures small percentage of light from the turbid sample, and reports an absorbance higher than actual value

6. Aminco DW 2000 Spectrophotometer Light Source Mono-chromator Detector • Spectrophotometer optimized for measurements of turbid samples • Most scattered light is collected by a large diameter detector • mounted close to sample • Largely removes the effect of scatter • Pathlength is typically 1 cm

7. CLARiTY Spectrophotometer Mono-chromator Light Source Detector DSPC cell • CLARiTY’s flask contains all the light (which is thus a ‘gas of photons’) • except that which passes through the 4 mm output aperture to the • immediate adjacent detector • Stirring and temperature control • Scatter does not matter, only sample absorbance does • Pathlength is enhanced up to 30-fold, depending on the volume • of the DSPC cell

8. CLARiTY Measurements to Date • Yeast characterization • Algae characterization • Bacterial characterization • Nanoparticle characterization • Oxygen binding to whole red blood cells • Mitochondria respiration

9. Chamber Does Not Need to be Filled Partially volume, leaving room for addition of reactants Very small volume, solid, or drop of suspension Solid, liquid, or powder sample within test tube that is lowered into DSPC Immobilized sample in solution

10. Technical Challenge: Absorbance Affects Path Length Low absorbance-> long pathlength High Absorbance-> shorter pathlength Solution: Absorbance is corrected for pathlength as described brilliantly by Fry et. al. Fry, E. S., Kattawar, G. W., Strycker, B. D., Zhai, P. W. (2010) Applied Optics 49, 575-577

11. Quantitating Absorbance in Partially-filled Cells • Modification of Fry 2010 algorithm used to correct absorbance for pathlength • Shorter pathlength and thus lower potential sensitivity than • when DSPCs are fully filled • Benefits: • Very convenient: common NMR tubes can be used • Tube from freezer to spectrophotometer • Simplifies cleaning between samples • Still perfect immunity to scatter

12. Technical Challenge: Differentiating Absorbance from Fluorescence Mono-chromator Light Source Detector Conventional Spectrophotometer Measurement beam is focused and reaches the detector. Fluorescence behaves like scattered light and does not reach the detector.

13. Technical Challenge: Differentiating Absorbance from Fluorescence CLARiTY Spectrophotometer Mono-chromator Light Source Detector Absorbance and fluorescence co-mingle in the flask and co-exit the detector Thus, to remove significant fluorescence from the detected signal, a bandpass filter or a monochromator needs to be used between the sample and detector, or a spectrometer such as the ArcOptix can be used for wavelength selectivity and detection.

14. Example Spectra: Oxygen binding to Whole Red blood cells Oxygen-Free Oxygen-Bound Sample: Human red blood cells suspended in water (1:4000 dilution) DSPC: filled (4 mL) Effective pathlength: 20 cm Captured: Oxygenated and Deoxygenated states of red blood cells using living, intact human red blood cells

15. Comparisons of Two Algae Strains

16. Hydrogel Before and After Addition of HCl -HCl +HCl

17. Polystyrene Baseline Comparison on Several Spectrophotometers Polystyrene beads added to water, and should result in zero baseline if scatter is corrected. The yellow trace was collected with a CLARiTY. Other traces include DW2000 dual mode (blue), DW2000 split beam (green), Cary 14 (purple), and HP Diode Array 8452 (white).

18. Difference Spectra of Yeast Deoxygenation over 300 Seconds Yeast suspension transitioning from aerobic to anaerobic environment. Data collected on a CLARiTY 1000 and reported as a series of difference spectra.