FLUOROMETER PERFORMANCE EVALUATION REPORT
9/2002
ABSTRACT
A series of performance evaluation tests were conducted at three tests sites to investigate the
overall performance of the Turner Designs’ SCUFA Submersible Fluorometer and the YSI
chlorophyll probe. Performance was determined through results designed to determine minimum
detection limit of chlorophyll a, dynamic range, accuracy of readings to actual chlorophyll a,
ambient light rejection, and response time. The results indicate that the SCUFA fluorometer
outperforms the YSI sensor but the performance of both sensors is adequate for most
environmental conditions. A performance limitation of the YSI is the response time. Using a
moving average calculation, it responds relatively slowly to changing concentrations and would
not be effective for vertical profiling applications. In some extreme conditions, such as
chlorophyll concentrations less than 1µg/L and high ambient light and/or high concentrations of
interfering compounds, the YSI may also not provide accurate measures of algal biomass.
INTRODUCTION
A series of performance evaluation tests were conducted to investigate the overall performance
of the Turner Designs’ SCUFA Submersible Fluorometer and the YSI chlorophyll probe. Tests
were conducted at three sites; Moss Landing Marine Laboratory, Turner Designs Testing
Laboratory and the USGS Menlo Park facility. Performance was evaluated through designing
tests to investigate the following areas; minimum detection limit, instrument range, response
time, light rejection and accuracy of fluorometer reading to actual chlorophyll a concentration.
Due to different specification standards, comparing instruments on published specifications is not
always straightforward. Below is the published performance data for each instrument. Through
our testing we have attempted to compare the instruments in a meaningful and informative way
for the customer. We have compared instruments identically and have attempted to present the
data in as an objective manner as possible. In most cases the raw fluorescence signal was
recorded in order to eliminate scaling issues related to calibration. The raw fluorescence was
recorded as %FS which indicates the % of a sample’s signal in relation to 100% full scale of the
particular instrument. Calibrated data is referred to as in vivo fluorescence or IVF.
Published Performance Specifications
YSI
0-400µg/L
Range
Minimum Detection Limit
N/A
0.1 µg/L, 0.1%FS
Resolution
Max Sampling Rate
NA
Depth
0-61m
SCUFA
4 orders of magnitude
0.02 µg/L
12 bit
5Hz
0-600m
Instrument Overview: While both instruments are chlorophyll fluorometers there are several
key distinctions between the two units. Firstly, the physical size and appearance is significantly
different. The SCUFA fluorometer is designed to operate as a stand-alone unit and can also be
interfaced to multi-parameter systems. Being a stand-alone unit, it incorporates many additional
features that are not required in the YSI probe. The following is a list of unique SCUFA features;
600m depth capability, temperature compensation, internal data logging, autoranging, turbidity
option, digital and analog output, configurable analog (0-5V) limits, and calibration software. The
presence of these features has resulted in a much different looking sensor. However, when
interfaced to a multi-parameter system, the SCUFA operates in a very similar manner to the YSI
probe. The YSI has been designed to interface directly onto YSI multi-parameter sondes. The
probe is also now available on an optical sonde that only offers fluorescence, conductivity,
1
FLUOROMETER COMPARISON REPORT
temperature and depth. The YSI probe provides an analog signal to the YSI sonde which
integrates the data from all of the sonde sensors into one digital data stream. The SCUFA
provides both digital and analog data continuously.
MATERIALS AND METHODS
All tests were conducted under controlled settings in a laboratory. In most cases, monocultures
on algae were used as the test media (see table below for algal culture details). In some cases
natural algal samples were used. Chlorophyll a extractions were conducted on all samples using
the Non-acidification fluorometric method (Welschmeyer, 1997) and the Acidification technique
(E.P.A. Method 445.0) in the case of the pheophytin testing.
Algal samples used:
Genus
Dunaliella sp.
Thallassiosira sp.
Phaeodactylum sp.
San Francisco Bay Delta
Algal group
chlorophyte
diatom
diatom
natural assemblage
1° Accessory Pigment
chlorophyll b
chlorophyll c
chlorophyll c
RESULTS
MINIMUM DETECTION LIMIT
The Minimum Detection Limit is the lowest concentration of algae in clean culture media than can
is distinguishable from a blank culture media solution. Three detection limit tests were run by
mixing a dilution series of algal cultures. The readings were allowed to stabilize and at least 10
datapoints were recorded per sample.
Dunaliella
Chl A
1.036
1.138
2.054
3.321
Phaeodactylum
SCUFA
Chl A
YSI
SCUFA
1.000
0.368
1.000
1.000
1.125
0.638
1.263
1.378
1.792
1.036
1.447
2.585
2.167
1.610
1.737
3.835
Thallassiosira
Chl A
YSI
SCUFA
0.000
0.140
1.460
0.340
0.930
5.920
0.700
0.890
8.330
2.060
4.760
15.420
Table 1: Dunaliella and Phaeodactylum test data from Moss Landing Marine Laboratory (MLML).
Data from Thallassiosira test from Turner Designs. The Chloro column is extracted chlorophyll a
data. The SCUFA and YSI column is the in vivo fluorescence data. From the data shown it is
clear the difference in detection limit between algal groups.
YSI
1.000
1.000
1.026
1.385
The data supports the fact that the YSI was unable to distinguish some algal cultures at
concentrations of 1µg/L chlorophyll a or less while the SCUFA was capable of distinguishing all
samples. The lowest concentration tested was 0.34µg/L of Thallassiosira culture. Because each
species of algae has varying in vivo fluorescence (IVF):extracted chlorophyll, the limits of
detection can vary between species. The YSI demonstrated the ability to detect concentration as
low as 0.79µg/L of Thallassiosira culture while not detecting other cultures that were above
1µg/L.
2
FLUOROMETER COMPARISON REPORT
Minimum Detection Limit Test #1
3.5
YSI
Minimum Detection Limit Test #2
SCUFA
IVF
20
2
R2 = 0.9203
1.5
15
1
10
0.5
5
0
R2 = 0.8504
0
0
1
2
3
4
5
0
1
chlorophyll a (ug/L)
2
3
4
chlorophyll a (ug/L)
Graph 1 & 2: MDL test data from Moss Landing Marine Lab showing that the YSI not
differentiating between 2.05µg/L and 1.1µg/L Dunaliella samples. MDL Test#2 shows data from
tests with Thallassiosira cultures indicate that the YSI did not distinguish the blank and the first
two culture samples.
RANGE
Both instruments provide an analog 0-5V output signal while the SCUFA has a digital output as
well. The 0-5V ranges are factory set for a given fluorescence range. The SCUFA comes standard
with a 0-100µg/L analog range while the YSI comes standard with a 0-400µg/L range. As the
analog range increases, the resolution of the data decreases and it is therefore advantageous to
set the 0-5V limits to the environments in which you work. The digital outputs from the YSI
Sonde and the SCUFA were used for all tests.
Both instruments demonstrated good linearity for typical chlorophyll a ranges found in natural
environments. The only difference of note for chlorophyll measurement is the ability to customize
the 0-5V range on the SCUFA. This abiltiy allows customers working in low chlorophyll
environments to decrease the 0-5V range and therefore improve the resolution of the analog
data.
Linearity Test
YSI
SCUFA
200
R 2 = 0.997
150
IVF
IVF
R2 = 0.9319
25
2.5
100
R 2 = 0.9941
50
0
0.00
-50
50.00
100.00
150.00
200.00
250.00
chlorophyll a (ug/L)
Graph 3: Data taken with calibrated values at MLML using Dunaliella cultures.
3
SCUFA
30
R 2 = 0.9737
3
YSI
FLUOROMETER COMPARISON REPORT
5
ACCURACY
In order to test the accuracy of measuring chlorophyll a fluorescence, the effects of various
interfering compounds were investigated. In an ideal situation, an in situ fluorometer would
measure chlorophyll a fluorescence only since chlorophyll a is the only photosynthetic pigment
that all algae and photosynthetic bacteria have in common. By measuring chlorophyll a only you
will obtain the best estimate of total, living algal biomass. Compounds that can interfere with
fluorescent signals optimized to detect chlorophyll a include accessory pigments such as
chlorophyll b and c, degraded chlorophyll a, pheophytin a, and dissolved organic matter. All of
these interfering substances have fluorescence spectra that can overlap with chlorophyll a given
the appropriate concentrations and fluorometer optics.
When discussing an instrument’s susceptibility to interfering compounds, the performance is
determined by the optics. The optics in a fluorometer encompass three components; a light
source, optical filters and a light detector. Both the SCUFA and the YSI instrument use solid-state
components, LED light source and photodiode light detector, which makes them equivalent in
this category except that the SCUFA utilizes two LED lamps while the YSI uses one.
The difference between the optics lies in the optical filters, specifically the emission filters. The
emission filter is placed in front of the light detector. It’s purpose is to block all light except for
the wavelengths of interest, in this case chlorophyll a fluorescence which peaks at 685nm. The
more specific the filter is to the chlorophyll peak, the more accurate the fluorometer will be at
measuring total algal biomass. The YSI uses long-pass emission filters which allows in all light
>600nm. This means that any material other than chlorophyll a that is excited by the blue LED
light source (peak at 460nm) and fluorescing above 600nm will be detected. Likely candidates for
this include pheophytin a, chlorophyll b, chlorophyll c and CDOM. The SCUFA, on the other hand,
utilizes a band-pass filter that allows in light from 660-700nm only, peaking at 685nm. Therefore,
the fluorescence from compounds other than chlorophyll a are much less likely to be detected.
The benefit of using a long-pass filter is the low material cost and higher transmission. The
downside is greater susceptibility to interfering compounds. The tests were effective in
demonstrating the effect of optical components in the pheophytin testing.
One of the most likely interference’s is pheophytin a. Pheophytin can accumulate in water column
at density gradients, close to the bottom, and be increased due to active zooplankton feeding. A
test was run to compare the fluorescence signal from a healthy algal culture and fluorescence
from the same culture after exposed to a weak acid that converted much of the chlorophyll in the
cells to pheophytin a. 1mL of 1N HCl was added to 350mL of Thallasisosira culture. The culture
was stirred for 1 minute and then the fluorescence was read using both instruments.
The results were as follows:
SCUFA
Before
After
FS% 0.45
0.163
Ratio 2.76
4
Before
2.91
1.36
YSI
After
2.14
FLUOROMETER COMPARISON REPORT
The Difference in the ratios indicates a difference in the fluorometers ability to detect pheophytin
fluorescence. The SCUFA ratio of 2.76 indicates that it is less sensitive to pheophytin fluorescence
than the YSI sensor that had a ratio of 1.36.
Regression Comprison
1.05
R2
1
0.95
TD
0.9
YSI
0.85
US
GS
-T
ha
l
TD
-T
ha
l
TD
-T
h
al2
M
LM
LPh
eo
M
LM
L-P
he
o
ML
ML
-D
un
ML
ML
-D
un
M
LM
L-D
un
TD
Fr
es
US
h
GS
-D
elt
a
0.8
Graph 4: The bar graph displays the correlations (r2) obtained from the minimum detection and
linearity tests conducted at all three testing sites. In most cases the SCUFA data correlated with
actual chlorophyll a more closely. All tests returned an r2 of greater than 0.85 or better. The
samples market TD Fresh and USGS-Delta are natural water samples with an assemblage of algal
species. All other samples were moncultures of algae.
AMBIENT LIGHT REJECTION
Ambient light rejection refers to the ability of the fluorometer to be unaffected by sunlight or
other ambient light sources. Light rejection is typically performed by flashing the LED light
source. When the light source is on, the instrument detects the fluorescence and any ambient
light, when the light source is off, the instrument detects the ambient light. The instrument
compares the two to determine the fluorescence.
Ambient light rejection test results indicate that there is a difference in how the two instruments
correct ambient light. The SCUFA displayed no significant difference in fluorescence signal while
the YSI showed a decrease in signal as ambient light increased and became more variable.
Ambient Light Rejection Test
Ambient Light Rejection Test #2
YSI
YSI
SCUFA
Scufa
70
1.50
60
% Full Scale
50
IVF
40
30
20
10
0
-10
no light
light at 90 degrees
1.00
0.50
0.00
Lit Lab
light at 180 degrees
Position of Light Source
-0.50
Dark Lab
Light 12"
Direct
Light 12"
Variable
Graph 5 & 6: Graph 5 displays data from Moss Landing Marine Lab. The instruments were
placed in a solution of rhodamine WT dye. A halogen light source was used to test ambient light.
Graph 6 displays data from tests run at Turner Designs with a halogen light source. The units
were not placed in a solution.
5
FLUOROMETER COMPARISON REPORT
RESPONSE TIME
The graph below shows the response time of the two instruments as they were alternately placed
into samples of natural water containing algae and blank deionized water. The readings were
taken at one-second intervals. The difference in the graphs displays a significant difference
between the two instruments. The YSI system uses a moving average as opposed to the ‘realtime’ sampling of the SCUFA. The moving average allows the YSI system to smooth the data as
well as enables it to improve the limit of detection. The SCUFA, designed for high-speed vertical
profiling applications, samples at a rate of 10Hz and outputs data at 1Hz or 5Hz. The spikes at
the beginning of the SCUFA readings of the algal cultures is due to removing the instrument from
the blank water and then placing it into an algal culture. The change from air to liquid creates
some bubbles that cause spikes in the readings. The YSI sensor is less sensitive to these changes
due to the slow moving average (~10sec) of the data which smoothes the data but does not
supply instantaneous data.
IN VIVO FLUORESCENCE
RESPONSE TIME TEST
45
40
35
30
25
20
15
10
5
0
13:55:55
-5
YSI
SCUFA
13:56:38
13:57:22
13:58:05
13:58:48
13:59:31
14:00:14
14:00:58
14:01:41
TIME
Graph 7
CONCLUSIONS
The results of the performance tests indicate that both instruments are effective at estimating
chlorophyll a concentrations in typical environmental conditions. Although accuracy tests favored
the SCUFA fluorometer, most correlations between the in vivo fluorescence and the actual
chlorophyll concentration exceeded 0.9 for both instruments. It is in the presence of interfering
compounds where the data from the two units may differ significantly. Results from testing with
degraded chlorophyll, pheophytin, support this conclusion, with the YSI not differentiating
between chlorophyll and pheophytin as well as the SCUFA.
Other extreme environmental conditions could limit the effectiveness of the YSI probe.
Conditions such as high ambient light ,from shallow waters with reflective sediment, and low
productivity waters could be problematic. Also, due to the slow response time of the YSI probe,
vertical profiling would be a challenge. On the other hand, the YSI probe seems well suited for
deployed use in coastal or freshwater environments where chlorophyll levels exceed 1µg/L and
ambient light and interfering compounds are not a significant issue. The SCUFA performed well
in all categories. It is a more versatile instrument because of ability to be used in self-contained
mode.
6
FLUOROMETER COMPARISON REPORT
REFERENCES:
Arar, E.J. and G.B. Collins 1996. In vitro determination of chlorophyll a and pheophytin a in
marine and freshwater algae by fluorescence. E.P.A. Method 445.0, Revision 1.2
Welschmeyer, N.A. 1994. Fluorometeric analysis of chlorophyll a in the presence of chlorophyll b
and pheopigments. Limnol. Oceanogr. 39: 1985-1992
TESTING FACILITIES:
MOSS LANDING MARINE LABORATORY, MOSS LANDING, CA. Tests run under the supervision of
Dr. Nick Welschmeyer.
TURNER DESIGNS TESTING FACILITY, SUNNYVALE, CA.
UNITED STATES GEOLOGICIAL SURVEY, MENLO PARK, CA
Report written by Robert Ellison, Turner Designs, Inc., 2002
7
FLUOROMETER COMPARISON REPORT