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Spectrometers

Rapid-kinetics optical system - MOS-200

The MOS-200 is a simple and efficient optical system designed for Rapid-Kinetics experiments.

The MOS-200 uses a Xe or Xe(Hg) 150 Watts light source attached to a manual monochromator on an optical bench.
The connection to the Bio-Logic Stopped-Flow is done through a fiber optic specially designed to match the Stopped-Flow cuvette dimensions.
The signal detection is performed by a photomultiplier directly mounted on the Stopped-Flow and connected to its control unit. The photomultiplier can be attached at 180° of the light source or at 90° allowing absorbance or fluorescence measurements (both at the same time using an optional additional detection channel). For fluorescence measurements, standard filters can be installed in front the photomultiplier tube inside the holder. 
The photomultiplier control unit is connected to a 16-bit A/D board installed in the PC driven by our acquisition and analysis  Bio-Kine32 software.

MOS-200 diagram

• Light lamp*
  • 150 W Xe(Hg) or Xe arc lamp
  • Wavelength range (nm) : 220 to 700 for Xe(Hg), 200 to 700 for Xe
  • Stability : better than 1% for Xe(Hg), better than 0.3% for Xe
  • Nature of spectrum : "sharp lines" for Xe(Hg), "white" for Xe.
  • Standard air cooled with lens
• Light lamp power supply
  • Ripple (50 or 60 Hz) : Less than 0.15% peak to peak (0.1% rms)
  • Low frequency noise :  Less than 0.05 % peak to peak
  • Drift : Less than 0.1% per minute after one hour warm-up.
• Monochromator
  • Single grating
  • Large aperture (F/#=3.5) and short focal length (100 mm) to improve light throughput
  • Wavelength range : zero order and 200 to 800 nm
  • Linear dispersion (nm/mm) : 8 (delivered with 1 mm slits)
  • Accuracy (nm) : +/- 0.5
• Fiber optic
  • Material : Quartz
  • Dimensions
    • Monochromator end : 1 x 3 mm² (linear to match the slit image)
    • Stopped-Flow end : 1.9 mm diameter (round end to match cuvette image)
    • Length 1.5 m.
• Photomultiplier & control unit
• Data acquisition & data analysis
  • 16-bit digitization of data
  • Number of acquisition channels : up to 4. One for the main signal and the other three for external signals (optional additional detection channel, temp...)
  • Rate of data acquisition : 50 µs/sample to 1000 s/sample
  • Oversampling filtering
  • Linear or logarithmic time scale acquisition
  • Full integration of the Bio-Logic stopped-flow software

Xe(Hg) and Xe Lamp spectra

Options

1. Additional detection channel for simultaneous recordings of absorbance and fluorescence or of two different fluorescence wavelength.
2. Reference channel

*Note : Xe(Hg) lamps have a clear advantage in the UV below 300 nm where many bright lines provide a high light intensity. Strong lines are also present at higher wavelengths (313, 365, 405, 436, 546, and 577 nm for the most intense ones). In general, use of a Xe(Hg) lamp may prove to be very advantageous in cases where the highest possible light intensity is necessary as is the case for fast kinetics fluorescence

Fast UV/Vis Spectrometer - MOS-250

Multi-mode fast recording spectrometer for rapid reaction recordings and analysis

A unique instrument to fulfill most of the needs in rapid kinetics recordings

Can be interfaced with any rapid kinetics reactor via fiber optics

A view of MOS-250 associated with the SFM-400 Stopped-flow. Click to enlarge

General features

• Illumination spectral range : 220 to 1010 nm (by 1 nm steps)
• Emission spectral range : 220 to 1010 nm
• Wavelength steps : 1 nm
• Bandwidth : 5, 10 and 20 nm (+shutter)
• Fiber optics link to the stopped-flow or to an observation cuvette
• Built-in reference channel
• Full computer control (wavelength and bandwidth)
• 16-bit digitization of data (instruments delivered after mid-2001)
• One single software for fast kinetics and spectral recording
• Full integration of the Bio-Logic stopped-flow software

Fast kinetics specifications

• Rate of data acquisition : 50 µs/sample to 1000 s/sample
• Number of acquisition channels : up to 4
• Oversampling filtering
• Linear or logarithmic time scale acquisition

Spectral recordings specifications

• Two fast scanning direct drive monochromators for illumination and emission
• Rate of scanning : 0.1 to 130 nm/s

Options

1. Additional detection channel for simultaneous recordings of absorbance and fluorescence or of two different fluorescence wavelength.
2. Cuvette holder with programmable Peltier element for temperature regulation.

Performance test in kinetics and scanning modes

Fast modular UV-Vis spectrophotometer/polarimeter - MOS-450

For spectral recording in UV/Vis spectroscopic modes.

Fast recording modular spectrometer and spectropolarimeter for rapid reaction kinetics and analysis.

A unique instrument to fulfill all the needs in steady state and rapid kinetics recordings.

MOS-450/AF-CD associated with the steady state observation cuvette.
Click to enlarge

MOS-450/AF-CD associated with an SFM-300/400 Stopped-Flow
Click to enlarge

The modularity of the MOS-450 spectrometer makes it economical : a minimum of components to be rearranged for different modes of detection, and expandable.

The MOS-450 spectrometer comes as two basic configurations :

• MOS-450/AF for absorbance and fluorescence modes. This includes a dual illumination monochromator and single-channel detection.
• MOS-450/AF-CD for absorbance, fluorescence, fluorescence anisotropy and circular dichroism modes. This includes the same hardware as the /AF version, with in addition : polarizing optics, photoelastic modulator and synchronous signal detection.

Additional components can be added to upgrade the system for new developments or applications. Among these components : additional detection channel for simultaneous detection of two signals (e.g. CD + fluorescence), emission monochromator for measurement of emission spectra, diode array spectrometer, etc...

It's the optical system that grows with your research, not obsolete !

A unique Windows based software is used to control the instrument. It allows a true "single click" reconfiguration of the MOS-450 instrument between any of the operational modes. This instrument configuration is entirely computer controlled and does not require rewiring or realignment of the optics.

A few examples among many more:

• "Single click" conversion from full far-UV CD spectrometer into a transient kinetics recorder in fluorescence.
• "Single click" conversion from any of the available mode to a fluorescence anisotropy spectrometer.
• etc...

MOS-450/AF

• Double illumination monochromator at excitation for improved signal-to-noise ratio and stray light rejection in far UV
• Illumination spectral range : 180 to 800 nm
• Wavelength steps : 0.25 to 2 nm
• Bandwidth of illumination monochromator: 1 to 8 nm (by fixed exchangeable slits)
• 150 W Xe or Xe(Hg) arc lamp for illumination 
• All reflective achromatic optics
• Reference diode
• Emission spectral range : 200 to 999 nm (with the optional emission monochromator)
• Direct link to the Stopped-Flow or to the observation cuvette
• Full computer control with one single software for fast kinetics and spectral recording
• Full integration of the Bio-Logic Stopped-Flow software
• Rate of data acquisition : 50 µs/sample to 1000 s/sample
• Number of acquisition channels : up to 4
• 16-bit digitization of the signal (instruments delivered after mid-2001)
• Oversampling filtering
• Linear, logarithmic or free time scale data acquisition
• Wavelength reproducibility: +/- 0.1 nm
• Stray light at 222 nm: <3 10^-5
• Noise level in fluorescence mode: water Raman line rms signal to noise ration: >2000/1
• Noise level in absorbance mode: 5 10^-5 AU rms at 1 ms integration time constant

MOS-450/CD

• MgF₂ polarizing optics
• Unique single-pass excitation and emission fluorescence anisotropy spectral recordings (click here for more information)
• Noise level in CD mode:
  • Fast data acquisition: 1 to 2 mD° (rms noise with water in the cuvette, 222 nm wavelength, 4 nm bandwidth and 1 ms integration time constant)
  • Slow data acquisition: 0.04 mD° (in the same conditions as above but with 1 s integration time constant)
  • Baseline stability: < 10^-2 mD°/h after 1 h warm-up
• Noise level in anisotropy mode:
  • < 10^-3 rms anisotropy units at 1 ms integration time constant

MOS-450 Optional components

Schematic representation of MOS-450/AF-CD (including all options)

All examples below have been obtained with the MOS-450/AF-CD instrument associated either with a stopped-flow instrument or with a steady state cuvette.

Emission fluorescence of lysozyme (30µg/mL)
excitation : 280 nm
Xe(Hg) 150 W, integration time constant : 3 s
Rate of scan : 15 nm/mn
Bandpass : 4 nm at excitation, 8 nm at emission

Raman line of water
Excitation : 363 nm
Xe(Hg) 150 W, integration time constant : 3 s
Rate of scan : 15 nm/mn
Bandpass : 4 nm at excitation, 8 nm at emission

CD spectrum of lysozyme (100 µg/mL in a 1 mm cuvette)
Xe 150 W, integration time constant : 1 s
Rate of scan : 15 nm/mn

Fluorescence anisotropy of alpha-lactalbumin. 1 mg/mL in a 1 cm cuvette.
Emission > 360 nm.

Fluorescence detected refolding of lysozyme at low enzyme concentrations. Final concentration : 3 µg/ml ! This shows that kinetics with 10 times less enzyme (a few 100 ng/ml) may be recorded and exploited.
Excitation : 280 nm, emission : 330 nm, bandwidth : 6 nm, FC-15 cuvette

Refolding kinetics of Lysozyme
Followed by CD at 225 nm and simultaneous recording of fluorescence at >305 nm
Traces correspond to 5 accumulated shots.
Folding was initiated by 10 fold dilution of 3 mg/mL lysozyme denaturated in 6 M guanidine-cl (final concentration 0.3 mg/mL)
Cuvette light path = 1.5 mm (FC-15 model)
Experiment dead time = 2 ms

Fluorescence anisotropy changes recorded upon refolding of lysozyme (trace "A"). 
Reconstructed total protein fluorescence (trace "F") is shown below.

Kinetics of refolding of Bovine Alpha-lactalbumin followed by fluorescence anisotropy
Excitation : 297 nm
Emission : > 345 nm
Transition from molten globule to native state induced by a change from pH 2 to pH 7

Experiment shows reduction of a low concentration of DCIP (1.5 µM) by ascorbic acid. This low concentration was selected to demonstrate the sensibility of the system in absorbance mode.

It can be seen here that stopped-flow kinetics of amplitude in the range of 1 mAU are feasible.

This limit is, however, variable and is dependent on the rate constant that will have to be examined.

Titration of calcium induced conformational change of calmodulin
Observation of CD change at 222 nm induced by repeated injections of small concentrations of Ca²⁺ and EGTA.

High Speed Diode-Array Spectrophotometer - MOS-DA

The answer to spectral kinetics acquisition !

• Spectral ranges to suit your experimental needs
• Up to 1250 spectra/s (256 points/spectra)
• True 100% duty cycle
• Kinetics and steady-state modes
• Absorbance, transmittance, and photon counting modes

Spectral Kinetics acquisition

Data acquisition and analysis of whole spectra in the millisecond time range presents a formidable task in terms of electronics and computing power.

The MOS-DA diode array optical system meets this challenge by integrating the latest developments in transputer technology and parallel processing. This allows the MOS-DA to acquire as many as 1250 spectra/s (256 points/spectrum ; 0.8 ms/spectra). Use of the MOS-DA in conjunction with stopped-flow instrument creates a rapid kinetics system unmatched by any other with respect to experiment and detection versatility. Connection of the MOS-DA and stopped-flow systems is made with fiber optics allowing the MOS-DA to coexist with photomultiplier detectors and allow quick and easy exchange between the two detection systems.

MOS-DA Specifications

Number of diodes	256, or 1024 diodes
Spectral resolution	6nm (2nm/diode ; 256 diodes) 2nm (0.8nm/diode ; 1024 diodes)
Maximum Sampling rate (dependent on model)	1250 spectra/s ; 0.8 ms/spectra 250 spectra/s ; 4ms/spectra
A/D conversion	16 bit
Linear Signal range	0-0.8 a.u. (MMS module) 0-2.0 a.u. (MCS module)
Wavelength range/resolution	200-740 nm (256 diodes ; MMS)
	300-1100 nm (256 diodes ; MMS)
	200-1015nm (1024 diodes ; MCS)
Wavelength accuracy	Better than 0.1 nm
Wavelength reproducibility	Better than 0.07 nm
Noise	Better than ± 1x10-4 a.u. single scan Better than ± 1x10-5 a.u. average of 100 scans
Spectra	256 diodes - 3000 spectra 1024 diodes - 750 spectra

Data Acquisition and Analysis Software

A unique Windows based software is used to control the instrument.

The acquisition software allows acquisition of data over single or multiple time ranges. Acquisitions can be made using linear sampling, log-based sampling, or a mixture of both allowing adaptation of the acquisition to experimental needs.

Data files are fully compatible with SPECFIT/32 Global Analysis System for kinetics analysis.

The Diode Array Difference : Diode Array vs. Scanning Monochromators

Fast spectral recordings with the diode array.
Click here to enlarge 3D display.

MCS technology

MMS technology

Diode array spectrometers work by continuous and simultaneous detection of all the wavelength in the focal plane of a fixed monochromator (sometimes termed polychromator for this reason). The diode array is installed after the sample and the sample is illuminated by white light. In contrast, rapid scan systems record spectra using a rapid scanning monochromator coupled to a photomultiplier for light detection. Wavelengths are detected one by one. The monochromator can be installed before the sample which is illuminated by only one wavelength at a time.

The total overall rate of spectral acquisition of both techniques are comparable, but there is a difference in sensitivity and noise level.

Historically there are several misconceptions about the use of diode arrays vs. rapid scanning monochromator systems. Below are the most common misconceptions followed by detailed explanations.

• Statement: A PM tube is more sensitive than a diode.
• It is true that a PM tube is more sensitive than a diode, but this fact does not imply that a rapid scan system (which uses a PM tube) is more sensitive than a diode array (which uses diodes). 
  
  The best is to consider a practical case :
  Imagine a spectral recording from 300 to 556 nm with an accuracy of 1 nm. This corresponds to 256 pixels. Assume an acquisition of 1 spectrum/ms (1000 spectra per seconds). 
  
  A rapid scan system will scan from 300 to 556 in one millisecond in 256 steps, so the time of acquisition at each wavelength will be 1/256 ms or 3.9 µs. Once a wavelength has been scanned, there will be no more data acquisition during the next 1 ms. The ratio of acquisition time/sample period is called the Duty Cycle. In this example, the rapid scan system has a Duty Cycle of 3.9 µs/1 ms = 0.0039 or 0.39 %. This assumes an ideal rapid scan system that takes no time to return the monochromator from 556 to 300 nm.
  
  A diode array system will integrate the signal at all wavelengths for the entire 1 ms period. Here the duty cycle is 1 ms/1 ms = 1 or 100%! In other words, the PM tube in a rapid scan system will use only 0.39% of the photons while the diode array will use 100% !
  
  The Duty Cycle applies directly to the issue of sensitivity as follows : for the same light intensity, the signal acquired by the detection system is proportional to the duty cycle. Applying this to the example above shows that for the 256 nm scan range the signal recorded by the PM tube at each wavelength in the rapid scan system will be 256 times weaker than that recorded by the diode array system.
  
  Returning to the difference in sensitivity between a PM tube and a diode, even if a PM tube has a sensitivity ten times higher as compared to a diode (and this remains to be proved), the effective sensitivity of the diode array system will still be 25 times better as that of the rapid scan system (in a 256 pixel instrument). This difference in sensitivity in favor of the diode array can be is experimentally verified !

• Statement: A diode array system uses illumination of the sample by white light which causes sample bleaching.
• It is true that with a diode array the sample is illuminated with white light as opposed to only one wavelength at a time with a rapid scan system. Nevertheless, the intensity of light needed to be used with a diode array is much less than that needed for a rapid scan system. This is because of the efficient use of photons by the diode array system due to it's 100 % Duty Cycle. The diode array can also be used in conjunction with a filter to block a particular wavelength range in case there is a light sensitive chromophore in the system or with a computer-controlled shutter to block the illumination light when acquisition is not being made.

Separated optical components

These components may be used for MOS-250/450 upgrade as well as for most of the older versions of Bio-Logic Optical systems.
They may also be used for assembling an optical system under customer request and specifications.
Finally they may be purchased independently for upgrading or improving any other third party or home made optical system.

Light link

Contact us for more information.

Specialized adaptation for Jasco CD spectrometer J-600, J-700 & J-800 series

Click

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SFM-20 / J-810 detail.

We have designed in collaboration with Jasco a mechanical and optical adaptation between the Bio-Logic series of stopped-flow instruments and the Jasco CD spectrometers.

This creates a very efficient and performing instrument for fast kinectics CD recordings.

Features

• Supports SFM-20, SFM-300, SFM-400 instruments
• CD spectra may be recorded either by setting a standard cuvette in the stopped-flow head or by switching to the spectrometer built-in cuvette holder.
• Precise and reproducible optimization of the light transfer to the stopped-flow cuvette.
• Instant optical switch from the stopped-flow to the spectrometer cuvette without removing or misaligning the stopped-flow instrument.
• Two 90° ports on the stopped-flow for recordings of the fluorescence kinetics.

Stopped-flow software

The stopped-flow has its own driving software (the same for SFM-20 and SFM-300/400)

Data acquisition

First option : Control using Spectra Manager Software.

The system operates from the same computer having the J-810 and the stopped-flow software open independently. The stopped-flow is programmed to issue a trigger pulse which is sent to the trigger input of the J-810. Data acquisition is made by the J-810. Data manipulation can be performed by the Bio-Logic Bio-Kine software that reads the JASCO Spectra Manager *.jws files. This enables analysis of *.jws files using the power of Bio-Kine software.

Second option : use of the Bio-Kine software.

The data is acquired by the an A/D board in the computer. This board is driven by the Bio-Kine software. This requires a minor modification on the J-810 : soldering two wires to output the photomultiplier AC and DC signal. The stopped-flow is programmed to issue a trigger pulse which is sent to the trigger input of the J-810. Data acquisition is made by the Bio-Kine software. Data analysis is made with the Bio-Kine

The second option offers some features that are not available in the first :

• True real time data visualization and acquisition in the microcomputer at a maximum speed of 50 µs per data point (20 kHz)
• Autoscale feature active during data acquisition
• User controlled file averaging at each shot
• Automatization of multiple shots, synchronization with the recordings and file averaging (very useful for accumulation of many shots)
• Synchronization of the data acquisition with the titrator events (in the case of the use of the titrator accessory)
• Synchronization of the data acquisition with the Peltier temperature regulator events (in the case of the use of the Peltier element / titrator accessory)
• Recording stopped-flow parameters in the data file
• Single double or triple time base

Note: Direct control of the J-810 spectrometer by Bio-Kine software is also available.

Test experiment

see AN8 (PDF)

Data Acquisition and Analysis

Bio-Kine32 operates under the latest version of Windows environment (Win95, Win98, WinME, WinNT, Win2000, WinXP). It uses an A/D board from National Instruments, probably the best industrial standard in data acquisition systems.

The Bio-Kine32 software is designed to control the following optical systems:

• MOS-200
• MOS-200/M
• MOS-250
• MOS-450/AF & /AF-CD
• MOS-DA diode array
• Jasco J-810 CD spectrometer
• Accessories such as:
  • Thermostated bath
  • Peltier cells
  • Acquisition of any other analog signal (-10/+10V)

  It also provides a direct bridge to the Stopped-Flow driver software.

The Bio-Kine32 software can control these instruments and acquire data for transient kinetics, spectral scans, act as a virtual chart recorder and control external devices.
The use of similar controls for all modes makes Bio-Kine32 easy to use and eliminates the hassles of learning multiple programs for different types of experiments.

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Up four data channels of 8000 points each can be acquired and displayed simultaneously. Sampling rates from 50 µs/point (20kHz) to 100 s/point (0.1 Hz) can be used. Both linear and logarithmic sampling is available through the use of up to three times bases. Data can be acquired in two different spectroscopic modes simultaneously (Volt, Transmittance, Absorbance, CD or Anisotropy).

Scanning Spectrophotometer
The Scanning Spectrometer is used to control one or two monochromators to perform spectral scans.

Bio-Kine32 is capable of making spectral acquisitions in the same spectroscopic modes that are available for transient kinetics. In conjunction with the appropriate spectrometer system, Bio-Kine32 can act as a full feature absorbance, fluorescence or CD spectrometer.

Chart Recorder
The Chart Recorder is similar to the Transient Recorder, but specialized for slower acquisitions over long periods of time.

Up to four data channels and two spectroscopic modes can be recorded simultaneously. Data is displayed in real time, and scrolls along acquisition window. Files of several million points can be recorded with event markers being added at anytime.

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