MULTIPLE MEASUREMENTS

NOT MULTIPLE EFFORT

One makes a single sample preparation and puts it in the MULTISCAN. Within a few minutes, he will have CD spectra, emission spectra, and/or high quality second derivative spectra, all contributing to better understanding of the changes in the samples.

• A single instrument.

• A single software package.

• Less collection time.

• Less sample preparation time.

• Less sample volume.

• Fewer cuvettes.

• Small bench space.

• Less user involvement before, during, and after data acquisition.

• More emission information.

• The same or better quality spectra as one gets when doing a single measurement on other premium quality spectrophotometers.

This “extremely high throughput means of acquiring structure and stability data on a given sample” will save time, money, effort, error, and give you better results.

The more samples you analyze, the more savings a MULTISCAN will bring.

MULTIPLE DATA TYPES

All acquired data is stored automatically in a DATATREE. Using the DataExtractor, you can pull out (extract) exactly and only those data of immediate use to you from the full DATATREE. The remainder of the data might be of use to you today, tomorrow, or years from now; or, might be of use to a colleague or collaborator. Facilities in DataExtractor support quickly assembling the different measurements for further action.

See page 11 for more on the DATATREE, which is automatically created to sort and store the tens, hundreds, even thousands of data sets acquired during the course of a single session with your Olis MULTISCAN.

With an OLIS MULTISCAN, collect many optical measurements at once and eliminate “if only we had also looked at...”

Because when is one measurement ever sufficient?

MULTIPLE AQUISITION TYPES SUPPORTED

Absorbance • Fluorescence • Scatter* • Polarization of Fluorescence (POF)

etc.). What is distinctly new is the software which synchronizes activities and acquisition from multiple detectors.

Creating any given OLIS MULTISCAN involves selecting one of the two Peltier turrets (or other sample holder) and one of the four monochromators we offer. For a system which supports circular dichroism, the first monochromator must be a double monochromator. We offer three: the Olis Hummingbird, DeSa RSM 1000, and the Olis modernized CARY. On the emission side, most MULTISCANS will include an Olis RFL single monochromator. To support rapid-scanning emission (see pages 9-10), an RSM must be used, functioning as a rapid-scanning single.

*Scatter falls out of fluorescence spectra, so it will not be named individually in this document. Scatter, in this context, is the information when the excitation and emission wavelengths are the same. **POF and r use the same raw information, so when there is one, the other is just a mathematical conversion away.

MULTIPLE DIRECTIONS

With appropriate detectors and associated data acquisition channels, the MULTISCAN collects spectra in two (even three) directions simultaneously.

Optional reference detector

study in green

Looking straight across,

180º from the measurement beam, one detects absorbance signals. Absorbance includes UV/Vis, NIR, CD and LD.

Not all measurements can be made simultaneously. For instance, CD or LD will be collected; likewise, CD or CPL. The most likely combinations are CD and fluorescence (and absorbance), or absorbance and fluorescence (and POF).

MULTIPLE DETECTORS

Every MULTISCAN has at least two detectors: one or two for the absorbance (CD) measurement and one or two for the emission. The detectors will be photomultiplier tubes (PMT), InGaAs, or photon counting detectors. A fourth possibility is the optional CMOS array detector, added for high speed absorbance data to be used for second derivative absorbance (not shown).

PMTs are useful from the deep UV to 800 nm. They are the detectors for UV/Vis CD and LD and absorbance.

InGaAs detectors are useful in the NIR region. Like the PMTs, they can be very fast, allowing use in rapid-scanning applications, too.

Photon counters are used for all emission detection applications. They are useful from 280-630 nm or, with the compromise of higher dark counts, 200-850 nm.

MULTIPLE TEMPERATURES, MULTIPLE SAMPLES

One of the best justifications for an OLIS MULTISCAN is thermal denaturation studies which are traditionally very time consuming. Having a four or six position Peltier cell holder means that one can load the spectrophotometer with a reference and up to five samples and allow the instrument to run for hours, unattended, with no user intervention. At the end, up to five thermal denaturation data sets will have been collected.

[Scans] 39; Number of scans/assays to take

Data for each scan consists of a temperature (in degrees C) and an incubation time (in minutes) to wait before collecting data after the device has reached temp.

Temp	Incubation
10	1
12.5	1
15	1
17.5	1
20	1
22.5	.5
25	.5
27.5	.5
30	.5
32.5	.5
(Etc	etc)

The software follows a user edited ASCII (script) file which contains the temperature for each measurement. Along with the target temperature, the script includes a dwell time for the samples to reach equilibrium before data are acquired (a truncated example of a script is shown here). When the equilibrium is reached, the data is acquired for each of the samples and for each of the active measurements. When temperature stability is reached, the several measurements are made on each sample in turn and all data are placed in the DATATREE. This process is continued, unattended, until the end of the script.

The DATATREE, introduced on page 3, is explained in detail on page 11.

MULTIPLE SPECTRA

ARE BETTER THAN ONE

Why collect a single scan when you can collect multiple? Time is the only justifiable reason to limit your acquisition. The average of multiple scans results in better S/N than a single scan.

And, multiple scans which are collected as a function of something -- time, temperature, concentration, etc. -- return better answers than single dimension results.

-22 -23 -24 -25 -26 -27 -28 -29 -30 -31

40 50 60 70 80 90 Temperature, °C

Results from a CD thermal melt: multiple wavelength CD spectra at multiple temperatures, left graph; and single wavelength thermal profile (right graph). With the MultiScan, you can pull any single wavelength for closer examination but

Ellipticity

100

(recommended) analyze using all wavelengths.

On the following pages, you will see data acquired from an OLIS MULTISCAN 2626 (originally named “The Olis Protein Machine” by Prof CR Middaugh). We show the emission data acquired while the primary measurement -- CD -- was being collected as a function of temperature. Because the emission monochromator is a DeSa rapid-scanning monochromator (aka, Olis RSM 1000), hundreds of 150 nm wide emission scans per excitation wavelength were acquired. And, since the emission spectra are acquired using the CD measurement wavelengths as the excitation wavelengths, the emission data

are ʻfreeʼ on the time axis. The only added time is when excitation wavelength(s) in

addition to the CD wavelengths are used. And, no more time was spent collecting these dense and informative emission spectra than were assigned to the CD measurement!

MULTIPLE WAVELENGTHS

MULTIPLE INTEGRATION TIMES

Like temperature, the wavelengths of interest are scripted in a ASCII file, so that only and exactly those wavelengths of interest are acquired, each with potentially unique integration times.

As one example, let us consider collecting “CD, fluorescence, and second derivative absorbance” spectra. Such a script would have 260, 258, 256, etc., to 190 nm, so that secondary structure determination fits can be applied to the CD results. In addition to the CD specific 260-190 nm wavelengths, other wavelengths can be included in the script to produce fluorescence/scatter emission signals, such as 280-300 nm to record tryptophan emission changes; i.e., all the CD measurement wavelengths concurrently function as excitation wavelengths on the sample.

8000

7000

6000

5000

4000

3000

2000

1000

0

240 260 280 300 320 340 360 380 400 420 440

Emission Wavelength, nm

Count/sec

150 nm wide emission scans are being acquired at each given temperature at each of the CD (excitation) measurement wavelengths; that is, by a CD measurement wavelength and any additional excitation wavelengths in the script. All of these emission scans were acquired during the CD data point's acquisition time, which was 0.5 seconds. Recall, emission acquisition rates with a DeSa RSM are up to 1000 scans per second!

460

As with the Temperature Script (page 7), the Wavelength Script has a second column for setting the time axis, i.e., integration time, thus allowing unique times per wavelength. In this way, critical wavelengths, i.e., 222 and 280 nm, can be assigned long integration times, whereas wavelengths of lesser or no interest can be assigned short or zero integration times.

Emission caused by the low UV and the ʻappropriateʼ excitation wavelengths produce

two emission spectra, which will differ from each other if the protein has multiple tryptophans and/or as evidence of energy transfer. If the two results are different (and for that matter, if they are not) you know something more about your sample than if you

had limited your study to the presupposed ʻappropriateʼ excitation wavelength.

MULTIPLE DIMENSION ANALYSIS

Continuing with the emission data shown on the previous page, one has multiple choices for working with them. What we show here is applying SVD (singular value decomposition) and spectral fitting to find the emission spectrum of the protein using all of the excitation wavelengths at the particular temperature.

After invoking SVD, the above graphic is shown. The information, the data, are shown now separated from the non-information, the noise. In this case, a single spectrum is

visible in the first graph with an associated non-zero ʻkineticʼ curve. In this case, because of how the data were collected, the ʻkineticʼ is not a time axis but an excitation

wavelength. (Happily for all, SVD cares nothing about units, only numbers!)

Once the investigator accepts the computerʼs suggestion that there is a single colored

species in these raw data, the spectral fit is made and the emission spectrum (left graph, below) and the excitation spectrum (right) are returned.

The same mathematics can be used for any 3D extraction from the DATATREE, e.g., time, temperature, concentration, or other process being studied.

Post collection analysis of the CD data can employ SVD and appropriate fitting to calculate enthalpy and transition temperatures. In addition, any appropriate secondary structure determination algorithm can be invoked on the CD data.(Happily .....numbers, so data as a function of nearly anything (time, temperature, wavelength, etc.) can be analyzed in this modern global fashion.)

If a CMOS device is available on your MULTISCAN, a 340-240 nm absorbance spectrum will be collected at each temperature, supporting global absorbance data analysis plus second derivative presentation.

MULTIPLE FILES

ALL WITHIN A SINGLE ʻTREEʼ

In the course of collecting the data, the Olis software is automatically creating a DATATREE. This DATATREE has a TRUNK, BRANCHES, OFFSHOOTS, and LEAVES. Within the TRUNKS are the BRANCHES, as well as SCRIPTS and NOTES. All files with the Olis DATATREE are named

automatically.

The TRUNK is named for the date and time the experiment started. In this case, 09-06-23-1932 tells us June 23, 2009 at 7:32 pm.

The BRANCHES are named for the data type, e.g., ABS, EM, CD, etc.

The NOTES are any files you care to also associate with this experiment, such as who did it, details about the sample preparation, etc.

The SCRIPTS are for the wavelength and temperatures. In future, additional scripts might be offered too, including titration, pressure, and so forth.

PROCESSED DATA is where the analyzed results of SVD, second derivative fits, and so on are automatically stored for future use.

OFFSHOOTS are the folders created to match each of the up-to-six samples that were under study.

LEAVES are the individual scans within each OFFSHOOT folder. Here, we see the CD files collected at each of the (39) temperatures. The assigned file names are data type (e.g., CD), the sample (e.g., S1), and the temperature (e.g., T004).

Opening an individual LEAF brings the chosen data set immediately into GlobalWorks (See back cover).

MULTIPLE PEM AND POLARIZER POSITIONS

What makes the Olis MULTISCAN 400 series spectrometers so versatile is their sample compartment, which we refer to as the “Polarization Toolbox.” Within this spacious sample compartment are two positions for the photoelastic modulator and two positions for the two polarizers,

plus the four position Peltier cell holder.

Here, we show the PEM being lifted from the post sample position (as for CPL) to the pre-sample position (as for CD, see next photograph). Holding bolts position the modulator perfectly on either before or after the sample. When it is before the sample, the excitation light is being modulated, as for (r), POF, CD, LD, and FDCD. When the PEM is after the sample, the emitted light is analyzed, as for CPL and ORD. Absorbance and fluorescence do not employ either the polarizers or the PEM, so one might choose to remove these components from the light path during these non-polarization measurements.

Also noticeable in the upper picture is the excitation polarizer being in its storage position, out of the light path. When it is used, it is placed in front of the incoming light, just before the turret, as it is in the lower photograph.

CD and FDCD examine the chirality of your molecules in their ground state; CPL measures the chirality of your molecules in their excited state. The emission from the sample is analyzed for CPL. FDCD is the emission caused by the modulated measurement beam.

Here, we see the hardware in place for single beam CD and polarization of fluorescence (emission detector out of view) which is used on the 400 series MULTISCAN. This ToolBox can support all of the measurements listed on page 2, whereas the Olis 600 series (next page) is limited to those measurements which are possible with the polarizers and PEM in their default position and employing dual beam analysis.

OLIS MULTISCAN 600 SERIES

This configuration differs from the Olis MULTISCAN 417 in three ways. The 600 Series is fitted with a six position QNW Turret with Peltier and stirring. The polarizers and PEM are fixed in position, limiting measurement versatility. And, the 600 series employs two beams of light, so that the CD signal is twice as intense as in a single beam 400 series. With two beams to produce the CD signal, the intensity of the 622 is twice as bright, resulting in the square root of 2 better signal to noise. Thus, this model is the superior when CD and fluorescence (and absorbance) alone are required. For those laboratories wishing to do CPL and FDCD, the single beam detection system must be chosen. This is because dual beam CD sends both abs(R) and abs(L) light through the sample, which will cancel each other out during an emission reading.

WALKING AROUND AN OLIS MULTISCAN 417

We condense the components as tightly as possible, while leaving all hardware

available to access. The spectrophotometer is on the ʻtop floor;ʼ its computer, electronics, and power supply are in the ʻbasement.ʼ

From the front, starting at left, we have the lamp 1 lamp housing arrangement. Depending on the applications and monochromators used, the lamp will be a 75, 150, or ozone producing 150 watt model. Other choices can be substituted for special applications.

The taller object behind is the lamp cooling and nitrogen regulator box 2 Lamp cooling is used by 150 watt and larger lamps. Three independent valves allow regulation of nitrogen flow to the lamp housing, absorbance monochromator, sample compartment.

The absorbance monochromator 3 will be a DeSa RSM (as shown here), a Cary prism-grating, an Olis Hummingbird, or the single grating Olis RFL. For CD, the monochromator must be a double monochromator and thus any of the first three.

Beneath the monochromator are all of the electronics 4 If an electronic module fails, one disconnects this box and returns it to us. Simple.

The sample compartment 5 absorbance monochromator. The absorbance detectors 6 are mounted within this chamber and the emission detector is mounted within the emission monochromator 7 . From the rear, we can see the emission monochromator 7 heat exchange box 8 for use with the Peltier cell holder, the optional CMOS 9 by Avantes, and the xenon arc lamp power supply 10 .

MULTIPLE ACCESSORY CHOICES

This introduction to the OLIS MULTISCAN Series has focused on thermal studies. Just as appropriate to this multiple data acquisition mode product line are stopped-flow, titration, pressure, and other studies. For example, instead of a temperature script, you could use a titration script and acquire CD and fluorescence data simultaneously following a titration study. Or, you could acquire absorbance and polarization of fluorescence stopped-flow spectra simultaneously. If this document has sparked ideas for you, please speak with a sales representative at the company. The first three models were purchased by investigators at the University of Kansas, Wellesley College, and San Jose State University. Two of these support rapid-scanning emission, three support thermal studies, two include the Olis titrator, and one includes an Olis stopped-flow.

OLIS MULTISCAN 415

WITH OLIS STOPPED FLOW

An OLIS MULTISCAN being fitted with a stopped-flow mixing device. The Peltier cell holder has been removed and this rapid-mix device is used to produce freshly mixed solutions into an observation chamber, so that reactions of a few seconds or less can be captured.

OLIS MULTISCAN 415

WITH TITRATOR

Titration work presumes use of a single cell position, which can be one of the positions in the 4 or 6 position Peltier. All titration work is at a given temperature.

In todayʼs era of incredibly fast,

powerful computers with practically

limitless memory, the company that has

been exploiting computer technology for decades again finds a way to maximize their use to your benefit.

Megabytes of data are acquired with Olis MULTISCAN spectrophotometers during the course of any experiment.

The acquisition, storage, and

subsequent data analysis are as direct and easy as if handling a single data set from a competitive spectrophotometer. Our software

makes your work more efficacious, efficient, and effective.

Because of the intentional exploitation of computer technology that marks every Olis product, a

second and perhaps unexpected benefit occurs: Every Olis spectrophotometer is upgradeable for life. The high quality and well-housed optics have a lifespan of fifty years and longer. The computer control and associated electronics have a far shorter lifespan. We build our instruments so that the electronics and computerization are housed separate from the optical

bench. Thus, the ageless optical bench can be re-purposed with the latest computerization generation after generation. Clearly, the cost savings will be significant throughout oneʼs career!

Everyone benefits from more data, better time management, and cost savings, right? And who among us isn't grateful for the chance to upgrade rather than throw away! The era of “disposable” should be behind us. The era of ʻduplicate effortʼ should be behind us. The time to move to the performance and power of the is now. Join us!