Wavelength (nm)

Taking the best two instruments — the DW-2000 and the CLARITY 1000A — and examining a solution of cytochrome C in water, we find the “gold standard” of yesterday to be poor competition for todayʼs new scatter-immune OLIS CLARITY system:

Absorbance Absorbance

CLARITY OF POSSIBILITIES

The OLIS CLARITY SERIES makes possible studies on living whole cells which are otherwise impossible.

The OLIS CLARITY 1000 and CLARITY 2000 can obtain high sensitivity scans on whole cells at rates to 100 scans per second. Mitochondria, photosynthetic suspensions, bacteria, and other whole cells can be studied accurately and kinetically as they change in response to their environment.

The electron transport chain – nominally along the 600-300 nm spectral range – can be monitored with sub-second time resolution.

CLARITY OF DESIGN

The OLIS CLARITY SERIES: Designed this way, because it has to be.

For a spectrophotometer to be useful with highly turbid samples which are undergoing metabolic change, five attributes are essential:

1. Dual beam detection to provide the required photometric stability, accuracy and sensitivity.

2. Monochromatic light to ensure against photobleaching.

3. Rapid-wavelength scanning to capture the entire 300 nm wide electron transport chain and thus all of the chromophores of interest in a single scan quickly

(ʻreal-timeʼ).

4. Photomultiplier tube detection to provide a vast range of speed and sensitivity settings. (Diode array,

CMOS, and CCDs are inadequate to the task.)

5. A diffuse light measurement system to eliminate the adverse affects of light scatter by the sample.

The CLARITY CHAMBER thus opens the world of studying living whole cells, aggregating proteins, and other light scatter samples. A host of sample chambers will support solution, powder, and even solid sample preparations. True immunity to scatter!

CLARITY OF INNOVATION

While all high performance spectrophotometers are dual beam, with monochromatic measurement beams, and photomultiplier tube detection, only the OLIS RSM 1000 has these

built around the patented DeSa Subtractive Double Grating Monochromator with Moving Intermediate Slit. The most popular niche market for this instrument before today was high speed kinetic studies such as stopped-flow and flash photolysis.

With the ability to collect up to 1000 scans per second, the Olis RSM 1000 has true one millisecond acquisition rates for full multiple wavelength scans. When working with clear sample preparations, the millisecond scan rate is entirely appropriate. When working with turbid suspensions, the millisecond scan rate is slowed to rates of 100 or 50 scans per second for enhanced sensitivity. (Should an occasion arise where the millisecond scan rate is required, it can be used.)

CLARITY OF FUNCTION

A diffuse light beam is achieved with our new isotropic homogeneous light field chamber. This Clarity Chamber includes one or two suspension presentation chambers for your sample (and optional reference). The suspension presentation chambers within the Clarity Chamber have inlet ports (up to five per sample), so you can add fluids and gasses to interact with your cells manually or in an automated fashion with the Olis

The Clarity Chamber eliminates the affect of scatter, which otherwise precludes study of living whole cells, aggregating proteins, and other light scattering samples. With the "Clarity Chamber" on the appropriate Olis spectrophotometer, recording the action of electron transport chromophores in living bacteria, yeast, mammalian and photosynthetic cell suspensions is possible, practical, and successful!

CLARITY OF DYNAMICS

The Clarity Chamber eliminates the affect of scatter at all wavelengths, including the UV, by creating diffuse light as the measurement beam.

Use of diffuse light makes a huge improvement over normal focused light (see Bateman and Monk, Science, 121, 441 (1955)) because highly diffuse light as the measurement beam cannot be diffused further by a sample which scatters as well as absorbs.

The Clarity Chamber also creates a huge increase in the effective pathlength and thus the sensitivity. The increase in sensitivity is related to the volume of sample and the character of the Clarity Chamber and may be as long as 20 centimeters using a vessel holding 3 mL of sample.

CLARITY OF CORRECTNESS

The OLIS CLARITY SERIES is an extraordinary advance over prior technology alternatives:

✓ Immunity to scatter

✓ Enhanced sensitivity

✓ High speed scan rates

✓ Photometric precision

✓ Low stray light

✓ The correct absorbance

using the method of Javorfi, et al (Jrl Photochem Photobiol. B, Biology 2006; 82(2):127-31).

Sample is a 5 mg/mL yeast suspension in water. The new OLIS CLARITY 1000A acquired this difference spectrum with a single sample preparation using two 5 second scans subtracted from each other.

Yeast Suspension Difference Spectrum, Reduced minus Oxidized

Dual Wavelength pairs commonly used to identify cytochromes in the electron transport chain, as described in Weiner, M.W., Lardy, H.A. J. Biol. Chem., 248(22), 7682 (1973). Imperfect wavelength correlation is attributed to the difference in samples: rat liver cells vs. yeast cells.

CLARITY OF PERFORMANCE

The test tube at right holds the yeast suspension which was used in this experiment.

The computer monitor at right shows the spectra (left) and kinetics (right) of the full reaction. The 3D presentation, below, plots the spectra captured during the 20 second active transition from oxidized to reduced, as available oxygen was consumed.

All data were acquired using the OLIS CLARITY 1000A, i.e., an Olis RSM 1000 absorbance spectrophotometer fitted with the Clarity Chamber.

Can you imagine working with a suspension this turbid?

Letʼs look more closely

now on the 20 second time frame of interest.

Notice that these data are “actual absorbance,” i.e., corrected to be the values returned if the measurement were made in a 1 cm pathlength cuvette.

Nine spectra during the 20 second transition period

First spectrum which will be used as the subtrahend

Difference Spectra: The classic reduced-oxidized spectra!

CLARITY OF NEED

Many components of cells and subcellular organelles --