Applied Photophysics Chirascan CCD User manual

Applied Photophysics Limited
21 Mole Business Park
Leatherhead
Surrey
KT22 7BA
United Kingdom
Tel: +44 1372 386 537
Fax: +44 1372 386 477
Email: info@photophysics.com
Applied Photophysics, Inc.
100 Cummings Center
Suite 440-C
Beverly
MA 01915
United States
Tel: +1 978 473 7477
Email: info.usa@photophysics.com
www.photophysics.com
User Manual
Chirascan CCD Emission Fluorometer

Document 4207Q277 V3.00 January 2018 Page 2 of 22
Chirascan CCD Emission Fluorometer
This document contains important safety information. Read this document, the
Chirascan V100 Spectrometer User Manual and the User Manual provided by
Ocean Optics before attempting to install or use the CCD Emission Fluorometer.
Failure to do so could result in death or serious injury.

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Chirascan CCD Emission Fluorometer
CONTENTS
CONTENTS........................................................................................................................................................3
USE OF THIS DOCUMENT ...............................................................................................................................4
ESSENTIAL SAFETY INFORMATION...............................................................................................................5
CHIRASCAN CCD EMISSION FLUOROMETER INSTALLATION AND OPERATIONAL REQUIREMENTS..6
GLOSSARY........................................................................................................................................................7
1 INTRODUCTION.............................................................................................................................................8
2 INSTALLATION ...............................................................................................................................................9
2.1 Single Cell Peltier Temperature Controller...........................................................................................9
2.2 6-cell Peltier Cell Holder .....................................................................................................................11
2.3 The CCD spectrometer.......................................................................................................................13
3 OPERATION..................................................................................................................................................14
3.1 Typical use..........................................................................................................................................14
3.2 Setting the CCD conditions.................................................................................................................14
3.2.1 CCD only mode .................................................................................................................................. 14
3.2.2 Operation with other signals............................................................................................................. 15
3.2.3 Acquiring a baseline .......................................................................................................................... 17
3.3 Pro-Data Viewer..................................................................................................................................17
3.3.1 Data files............................................................................................................................................ 17
3.3.2 Example: quinine sulfate reference .................................................................................................. 18
3.3.3 Example: anthracene reference ........................................................................................................ 19
3.3.4 Example: -chymotrypsin ................................................................................................................. 20
4 MAINTENANCE AND STORAGE .................................................................................................................22

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Chirascan CCD Emission Fluorometer
USE OF THIS DOCUMENT
This document is intended to inform the operator of Applied Photophysics Chirascan CCD Emission
Fluorometer on its design, installation and operation with the Chirascan V100 Spectrometer and the Single
Cell Peltier Temperature Controller, or the 6-cell Peltier Cell Holder. The CCD Emission Fluorometer is the QE
Pro Scientific-grade type supplied by Ocean Optics Inc., Dunedin, FL, USA, and this document should be used
in conjunction with the User Manuals supplied by Ocean Optics and those applicable to the spectrometer and
the Single Cell Peltier Temperature Controller, or the 6-cell Peltier Cell Holder. It is assumed that the user of
this document is familiar with the operation of the Chirascan spectrometer, and with Applied Photophysics Pro-
Data software. In particular it is assumed that the user is familiar with the hazards associated with the operation
of the spectrometer and the Single Cell Peltier Temperature Controller, or the 6-cell Peltier Cell Holder, and
has read the hazard and other safety information contained in their User Manuals and those supplied by Ocean
Optics.
The information in this document is subject to change without notice and should not be construed as a
commitment by Applied Photophysics, who accept no responsibility for errors that may appear herein. This
document is believed to be complete and accurate at the time of publication, and in no event shall Applied
Photophysics be held responsible for incidental or consequential damages with or arising from the use of this
document.
COPYRIGHT 2018 APPLIED PHOTOPHYSICS LIMITED. ALL RIGHTS RESERVED. THIS DOCUMENT
OR PARTS THEREOF SHALL NOT BE REPRODUCED IN ANY FORM WITHOUT THE WRITTEN
PERMISSION OF THE PUBLISHER.
THE SOFTWARE PROVIDED WITH THE CHIRASCAN AND ITS ACCESSORIES (PRO-DATA CHIRASCAN,
PRO-DATA VIEWER, ETC.) IS THE PROPERTY OF APPLIED PHOTOPHYSICS LIMITED AND IS
SUPPLIED UNDER LICENSE. APPLIED PHOTOPHYSICS IS WILLING TO LICENSE THE SOFTWARE
ONLY UPON THE CONDITION THAT THE LICENSEE ACCEPTS ALL THE TERMS CONTAINED IN THE
LICENSE AGREEMENT. THESE INCLUDE THAT THE LICENSEE MAY NOT SELL, RENT, LOAN OR
OTHERWISE ENCUMBER OR TRANSFER LICENSED SOFTWARE IN WHOLE OR IN PART TO ANY
THIRD PARTY. FOR A FULL COPY OF THE LICENSE PLEASE CONTACT APPLIED PHOTOPHYSICS OR
SEE THE SOFTWARE INSTALLATION DISK.
Chirascan is a trademark of Applied Photophysics Limited.
All other trademarks or registered trademarks are the sole property of their respective owners

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Chirascan CCD Emission Fluorometer
ESSENTIAL SAFETY INFORMATION
MAKE SURE THAT YOU HAVE READ AND UNDERSTOOD ALL THE SAFETY INFORMATION CONTAINED
IN THE MAIN CHIRASCAN USER MANUAL AND THE USER MANUAL PROVIDED BY OCEAN OPTICS
BEFORE ATTEMPTING TO INSTALL OR OPERATE THE CHIRASCAN CCD EMISSION FLUOROMETER.
IF YOU HAVE ANY QUESTIONS REGARDING THE OPERATION OF THE FLUOROMETER, PLEASE
CONTACT APPLIED PHOTOPHYSICS TECHNICAL SUPPORT AT THE ADDRESS SHOWN ON THE FIRST
PAGE OF THIS DOCUMENT.
OBSERVE ALL SAFETY LABELS AND NEVER ERASE OR REMOVE SAFETY LABELS.
PERFORMANCE OF INSTALLATION, OPERATION OR MAINTENANCE PROCEDURES OTHER THAN
THOSE DESCRIBED IN THIS USER MANUAL MAY RESULT IN A HAZARDOUS SITUATION AND WILL
VOID THE MANUFACTURERS WARRANTY.

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Chirascan CCD Emission Fluorometer
CHIRASCAN CCD EMISSION FLUOROMETER INSTALLATION AND OPERATIONAL
REQUIREMENTS
Environmental and electrical requirements
The Chirascan CCD Emission Fluorometer has no environmental requirements additional to those of the
Chirascan spectrometer, but will require a separate mains voltage supply.
Bench space
The Chirascan CCD Emission Fluorometer requires an additional bench space of about 150 mm depth and
200 mm width to the right of the Chirascan sample handling unit, but is low enough to fit below the Chirascan
detector.
Nitrogen purge gas
The Chirascan CCD Emission Fluorometer has no purge gas requirements additional to those of the Chirascan
spectrometer.
Circulating water
The Chirascan CCD Emission Fluorometer has no circulating water requirements additional to those of the
Chirascan spectrometer.
Servicing
Servicing of the Chirascan CCD Emission Fluorometer should only be undertaken by qualified personnel. If
you are in any doubt at all please contact the Applied Photophysics’Technical Support at the address given
on the front of this User Manual.

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Chirascan CCD Emission Fluorometer
GLOSSARY
The following abbreviations may be found in this User Manual
APL Applied Photophysics Limited
CCD charge-coupled device
CD circular dichroism
IR infrared
SHU sample handling unit
UV ultraviolet
HYPERLINKS
This document contains hyperlinks between references (for example the Contents tables, or references to
Sections or Figures in the text), and sources. To follow a link, place the cursor over the reference and use
CTRL+click. Hyperlinks in the text are indicated by underlined blue font.

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Chirascan CCD Emission Fluorometer
1 INTRODUCTION
The Chirascan CCD Emission Fluorometer is designed to be used with a Chirascan V100 Spectrometer and
the Single Cell Peltier Temperature Controller or the 6-cell Peltier Cell Holder. The detector is shown in Figure
1.1, with fitting for connecting to a Single Cell Peltier Temperature Controller.
Figure 1.1: the Chirascan CCD Emission Fluorometer
Fluorescence occurs when electromagnetic radiation absorbed by a sample is re-emitted at a longer
wavelength. With the Emission Fluorometer the emitted light is collected from the fluorescence window of the
Single Cell Peltier Temperature Controller or the 6-cell Peltier Cell Holder, and focused onto an optical fiber
light guide which takes it to a CCD spectrometer. The CCD spectrometer unit contains a grating that disperses
the incoming light and directs it onto a CCD detector with a 2-D arrangement of pixels (1044 horizontal x 64
vertical) that is responsive from 200 nm to 1000 nm. A full fluorescence spectrum can be acquired in just a few
seconds.
The CCD spectrometer is the QE Pro Scientific-grade mini-spectrometer type supplied by Ocean Optics Inc.,
Dunedin, FL, USA, and is supplied with a manual provided by Ocean Optics.

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Chirascan CCD Emission Fluorometer
2 INSTALLATION
2.1 Single Cell Peltier Temperature Controller
The fittings required for connecting the Emission Fluorometer to the Single Cell Peltier Temperature Controller
are shown in Figure 2.1.
Figure 2.1: the fittings required for connection to the single cell accessory,
left to right: dovetail insert, grub screw and nut, lens holder, light guide ferrule
Figure 2.2: the dovetail insert mounted on the Single Cell Peltier Controller
The dovetail insert mounts onto the Single Cell Peltier Controller fluorescence window as in Figure 2.2 (for
clarity, the Single Cell Peltier Controller is shown removed from the Chirascan sample handling unit). To hold
the insert in place, tighten the 16 mm grub screw that screws into the threaded hole in the center using a 1.5
mm hexagon key. Do not over tighten the grub screw, it is enough just to hold the dovetail in place.
16 mm grub screw
Hole for lens holder

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Chirascan CCD Emission Fluorometer
Figure 2.3: light guide collar with the light guide inserted.
Now insert the tip of the light guide through the grommet on the guide collar; the guide tip should protrude
about 3 cm through the grommet. Screw the light guide ferrule onto the tip of the guide (Figure 2.4).
Insert the ferrule into the lens holder, ensuring that it is fully seated home. Hold the lens holder in place by
tightening the two grub screws on the holder using a 1.5 mm hexagon key. Do not over tighten the grub screws;
they are only to hold the light guide in the lens holder.
The light guide collar mounts in the fluorescence port of the Chirascan sample handling unit as in Figure 2.4.
The lens holder inserts into the hole in the dovetail insert, with the 16 mm grub screw locating in the groove in
the lens holder. An M3 nut on the grub screw is used to hold the lens holder in place. For clarity, this
arrangement is shown with the Single Cell Peltier Holder removed from the Chirascan sample handling unit in
Figure 1.1.
Figure 2.4: the light guide mounted on the Chirascan sample handling unit,
Light guide
Light guide ferrule
Light guide collar
Grommet
Light guide
collar
CD detector
Lens holder

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Chirascan CCD Emission Fluorometer
Figure 2.5: the light guide mounted on the Single Cell Peltier Controller
2.2 6-cell Peltier Cell Holder
The lens holder for the 6-cell Peltier cell holder accessory is shown in Figure 2.6.
Figure 2.6: the lens holder for the 6-cell accessory
Mount the light guide collar on the light guide and screw the lens holder onto the end of the light guide, as in
Figure 2.7.
M3 nut
Alignment hole
Knurled ring

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Chirascan CCD Emission Fluorometer
Figure 2.7: light guide fitted with the lens holder and light guide collar
The light guide collar mounts in the fluorescence port of the Chirascan sample handling unit as in Figure 2.7.
The alignment hole on the lens holder (Figure 2.6) should be at the top, fitting over the protruding cap head
screw on the threaded ring on the 6-cell accessory. The knurled ring on the lens holder then screws onto the
corresponding threaded ring, as in Figure 2.8. Before tightening the knurled ring, make sure that the lens
holder is fully seated on the 6-cell ring.
Figure 2.8: the light guide mounted on the Chirascan sample handling unit
Light guide
Light guide collar
Lens holder

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Chirascan CCD Emission Fluorometer
2.3 The CCD spectrometer
The CCD spectrometer has connections for the light guide, a cable to the 5 volt DC power supply, and a USB
cable to the computer. These are shown in Figure 2.9.
Figure 2.9: connections to the CCD spectrometer
The CCD spectrometer can be positioned to the right of the Chirascan sample handling unit, as in Figure 2.10.
Figure 2.10: the CCD spectrometer positioned to the right of the Chirascan sample handling unit
USB cable
Power supply
Light guide

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Chirascan CCD Emission Fluorometer
3 OPERATION
3.1 Typical use
The CCD Emission Fluorometer is used to measure the fluorescence emission spectrum of a sample at a set
excitation wavelength or wavelengths. The total acquisition time for a reasonable spectrum is usually of the
order of seconds or less, so although the spectrum is acquired sequentially with the CD, ORD or other
spectrum, rather than simultaneously, only very little additional time is required.
A typical procedure would be to monitor the fluorescence spectrum during thermal unfolding of a protein. This
process could be followed by recording circular dichroism spectra at intervals of 1⁰C, for example, while
ramping the temperature continuously at 1⁰C per minute. The bandwidth required for the fluorescence
spectrum is usually larger than what would be used for a CD spectrum, so the Pro-Data software has the
facility for using different bandwidths for the CD spectra and the fluorescence spectra.
3.2 Setting the CCD conditions
3.2.1 CCD only mode
To run the Chirascan in CCD only mode, select CCD from the drop-down list on the Signal panel in the Pro-
Data Chirascan control software (Figure 3.1).
Figure 3.1: Pro-Data Chirascan control panel with CCD selected

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Chirascan CCD Emission Fluorometer
The conditions for the CCD are set on the Signal panel:
•Integration Time: sampling time for each wavelength scan; the longer the sampling time the better
the data quality, but note that there is a delay time of one second before the acquisition of the first
spectrum at each wavelength to prevent overspill between spectra, so if a single spectrum of 1000 ms
does not produce an acceptable spectrum, increase the number of scans using the Scans to average
function.
•Scans to average: number of scans to average for each spectrum; the more scans the better the data
quality. The total acquisition time for each fluorescence spectrum is the initial delay time + (integration
time x the scans to average).
•Boxcar width: the digital sampling bandwidth of the CCD spectrometer.
For a single wavelength scan, uncheck the Excitation scanning box. The Monochromator and Sequencer
panels will be disabled, with the exception of the Wavelength and Bandwidth boxes which can be entered as
usual. For fluorescence measurements, typical values for the bandwidth are 4 to 8 nm, depending on the
strength of the fluorescence signal.
For a wavelength scan, check the Excitation scanning box. The Monochromator panel will be enabled and
the Chirascan will scan as usual, with a CCD spectrum acquired at each wavelength. These will be stored in
the folder specified on the Mini-spectrometer dialog box (Figure 3.3), which can be opened by clicking the
Adv button on the Signal panel.
3.2.2 Operation with other signals
To run a CD spectrum with the CCD enabled, select Circular Dichroism or other signal from the drop-down
list on the Signal panel in the Pro-Data Chirascan control software, and check the Ccd box (Figure 3.2).
Figure 3.2: the Pro-Data Chirascan Signal panel
Click the Adv button next to the Ccd box to bring up the Mini-spectrometer dialog box (Figure 3.3). The
control parameters for the acquisition of the fluorescence spectra are set on the Settings panel.

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Chirascan CCD Emission Fluorometer
Figure 3.3: the Mini-spectrometer dialog box.
Integration Time, Scans to average and Boxcar width are described in Section 3.2.1.
•ExWavelength: the excitation wavelength set on the Chirascan monochromator for the fluorescence
measurements; more than one wavelength can be set, in which case the fluorescence spectra will be
acquired in the order in which the wavelengths are listed. Use the icon and the Append button to
wavelengths, and the icons to changeadd wavelengths, the icon to remove
the order in the list.
•Exbandwidth: the bandwidth set on the Chirascan monochromator for the fluorescence
measurements; this is usually set higher than the bandwidth for CD measurements, to allow more light
to reach the sample: typical values are 4 to 8 nm.
•Extra Delay time: the waiting time before each fluorescence measurement is made; this function may
be used, for example, to eliminate overspill between successive measurements.
•Default Directory: is the folder (directory) to which the fluorescence spectra will be saved; this may
be different form the folder to which the CD spectra are saved.

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Chirascan CCD Emission Fluorometer
3.2.3 Acquiring a baseline
When a fluorescence spectrum is measured on the CCD, the result will be offset from zero and will appear to
be a bit noisy. This can be corrected for by acquiring a baseline spectrum on the CCD with no light. This
provides a baseline which can be subtracted from the sample fluorescence spectrum. To run the baseline,
untick the Auto Shutter box in the panel on the top right of the Pro-Data Chirascan control software, ensuring
that the shutter is closed. The panel should appear as in Figure 3.4.
Figure 3.4: Auto Shutter box unticked with shutter closed
Examples of spectra before and after baseline correction are shown in Sections
3.3 Pro-Data Viewer
3.3.1 Data files
In CCD only mode, Pro-Data Viewer will generate a single file located in the folder specified as the Default
Directory on the Mini-spectrometer dialog box (Section 3.2.2).
In excitation scanning mode, Pro-Data Viewer will generate a separate file for each repeat scan. For example
a CD spectrum acquisition with three repeats, named Protein data with the numerical suffix 0001, will generate
a file of that name, located in the current home directory, as usual. There will three CD spectra in the file,
identified as 0, 1 and 2. It will also generate three Pro-Data Viewer files for each excitation wavelength specified
in the ExWavelength on the Mini-spectrometer dialog box (Section 3.2.2). These will be located in the folder
specified in the Default Directory on the Mini-spectrometer dialog box. If excitation wavelengths of 290 and
360 nm were specified in this example, there will be six a total of six CCD files, named as below:
Protein data 0001 290 repeat 0
Protein data 0001 290 repeat 1
Protein data 0001 290 repeat 2
Protein data 0001 360 repeat 0
Protein data 0001 360 repeat 1
Protein data 0001 360 repeat 2
These files are generated at the end of each CD scan.

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Chirascan CCD Emission Fluorometer
3.3.2 Example: quinine sulfate reference
The CCD fluorescence spectrum of a standard sample of 1 g/ml quinine sulfate from Starna Scientific Ltd.,
is shown in Figure 3.5, before and after baseline subtraction. The conditions used are shown in Table 3.1.
Figure 3.5: quinine sulfate before (left) and after (right) baseline subtraction
Sample
Quinine sulfate
Concentration
1.0 g/ml in 0.105 M perchloric acid
Baseline
No light (shutter closed)
Cell pathlength
10 mm
Excitation wavelength
347.5 nm
Excitation bandwidth
5 nm
CCD integration time
1000 ms
CCD extra delay time
0 ms
CCD spectra to average
5
CCD boxcar width
1 nm
Temperature
23.5⁰C
Table 3.1: conditions used for the fluorescence spectrum of quinine sulfate
Sample spectrum
Baseline spectrum
Corrected spectrum

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Chirascan CCD Emission Fluorometer
3.3.3 Example: anthracene reference
The fluorescence spectrum of a standard sample of anthracence from Starna Scientific Ltd., is shown in Figure
3.6 before and after baseline subtraction. The conditions used are shown in Table 3.2.
Figure 3.6: fluorescence spectrum of anthracene before (left) and after (right) baseline subtraction
Sample
Anthracene
Concentration
10-5 M in solid poly(methyl methacrylate)
Baseline
No light (shutter closed)
Pathlength
10 mm
Excitation wavelength
360 nm
Excitation bandwidth
2.5 nm
CCD integration time
1000 ms
CCD extra delay time
0 ms
CCD spectra to average
5
CCD boxcar width
1 nm
Temperature
23.5⁰C
Table 3.2: conditions used for the fluorescence spectrum of anthracene
Sample spectrum
Baseline spectrum
Corrected spectrum

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Chirascan CCD Emission Fluorometer
3.3.4 Example: -chymotrypsin
Figure 3.7 and Figure 3.8 show the temperature ramp CD and CCD fluorescence spectra respectively for -
chymotrypsin. The conditions used are given in Table 3.3.
Figure 3.7: CD temperature ramp data for -chymotrypsin; the arrow shows the direction of increasing temperature
Figure 3.8: CCD fluorescence temperature ramp data for -chymotrypsin after baseline correction; the arrow shows the
direction of increasing temperature
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