Journal of Student Research (2013)
Volume 2, Issue 1: pp.
48-51
Research Article
a.Department of Physics, Astronomy and Geophysics, Connecticut College, New London, CT06320
48
The Black Box Module for Low-Level Fluorescence
Detection Targeting Optogenetic Studies
Elizabeth Maret
a
and Mohamed Diagne
a
In optogenetics, genetic labeling by enhanced yellow fluorescent protein (EYFP) indicates transfected neurons expressing light-
gated channel proteins, Channelrhodopsin-2 (ChR2) and Halorhodopsin (NpHR) for millisecond timescale optical manipulation
of neurons.
However, poor spatial understanding of transfection relative to probing is responsible for blind stimulation in
complex mammalian systems and necessitates histological analysis of brain tissue.
Repeatable optogenetic studies on well-
trained and valuable subjects are therefore impossible.
We report here the Black Box Module as a novel fluorescence detection
system targeting EYFP emission and used in conjunction with a co-axial waveguide “optrode.
” Results of the Black Box
Module’s fluorescence detection performance in a bench-top environment simulating EYFP fluorescence with rhodamine 6g are
presented.
We show the Box’s filtration system to be sensitive to the EYFP-like fluorescence of rhodamine and its concentration
dependent emissions behaviors.
We anticipate these results to be the initial foundation in integrating the Box into optogenetic
studies as a tool for locating areas of ChR2 and NpHR transfection.
Keywords: Enhanced Yellow Fluorescent Protein, Black Box Module, Rhodamine 6G, fluorescence
1.0 Introduction
Optogenetics enables cell-type specific targeting and
millisecond timescale control of neuron action potential
events through viral vector transfection of light-responsive
channel proteins.
Light-gated channel proteins
Channelrhodopsin-2 (ChR2) and Halorhodopsin (NpHR) are
conjoined with enhanced yellow fluorescent proteins (EYFPs)
to visually label areas of neuron transfection
[1,2,4,6,8]
. Dual-
function optical stimulation and electrical recording of neuron
action potential events are accomplished through an
“optrode,” a waveguide-based neuroprobe
[10]
.
While the optrode has shown its value as a tool in precise
stimulation and collection of electrical action potentials when
located in an optogenetically transfected areas, locating areas
of transfection remains a challenge
[4]
. This leads to blind
probing, preventing repeatable studies and the possibility of
studies focused on brain layer targeting
[4,11]
. Collection of
fluorescence marking ChR2 and NpHR expressing cells has
initiated a new generation of low-light optical detection for
systems such as one-photon and two-photon fluorescence
microendoscopy and in vivo imaging at the single neuron
level
[3]
. Despite these advances, spatial understanding of
optical probing relative to areas of transfection is limited.
This compromises experimental success and requires
histological analysis of subjects to determine transfection
patterns
[4]
. It is thus both desirable and economic to perform
multiple optogenetic studies on valuable and well-trained test
subjects.
To address this issue, we present the Black Box Module
used in tandem with the optrode, as a novel system to collect
fluorescence from optogenetically modified neurons
exhibiting EYFP expression as is used to label ChR2 and
NpHR in optogenetics.
Using the optrode’s waveguide fiber
core, in addition to its simultaneous optical stimulation and
electrical recording dual-functionality, we believe the optrode
when used with the Black Box Module can also be used for
optical fluorescence collection.
Hence, the optrode-Box
combination can be used to indicate locations of transfection.
We substantiate this prediction by demonstrating how
light collected by the optrode and delivered through its optical
fiber into the Box isolates for EYFP fluorescence and in
addition is sensitive to concentration dependent properties of
rhodamine 6g.
This is achieved through a series of filters
within the light-tight environment of the Black Box.
Highly
filtered fluoresced light is detected by photomultiplier tube
(PMT).
Thus a signal detected by the PMT can verify the
optrode’s position in an optogenetically modified
environment while no signal indicates and non-optogenetic
environment.
Our Black Box system uses free space coupling in a
light-tight environment, opposed to the split fiber
fluorescence detection system of Diester et al (2011)
[4]
. We
believe this to be an improvement over the shared core of the
split waveguide fiber because it allows for increased
opportunities of optical filtration to isolate for fluorescent
light.
We present the results of a study using the Black Box
to detect concentration dependent emissions behavior in a
fabricated bench-top optogenetic environment using
rhodamine 6g fluorescent dye, which mimics EYFP
stimulation and emissions.
The results show the optrode-Box
set up to be sensitive to rhodamine 6g’s concentration
dependent emissions wavelength shift behavior while
maintaining the integrity of its filtration system.
2.0 Experimental Procedure
EYFPs have a quantum yield of 0.
61
[9]
. That means that
in optogenetic environments for every EYFP emission event
there are 64% more laser photons than fluorescence photons.
Hence, key to transfection labeling fluorescence detection is
creating an environment that can isolate detection for
fluorescence.
The Black Box Module (Figure 1) is an 18 in.
x
12 in.
x 7.5 in.
light-tight enclosure made from 3/8 in.
thick
black anodized aluminum plates, containing an optical
filtration system isolating for EYFP emission (525 nm peak)
(Figure 2).
The stimulating laser (Opto Engine), emitting at
473 nm, is fixed within the box and free-space couples to a
Journal of Student Research (2013)
Volume 2, Issue 1: pp.
48-51
Research Article
ISSN: 2167-1907
www.jofsr.
com
49
dichroic beamsplitter (Semrock Brightline FF520-Di02).
Initially, laser light is directed perpendicular from its source
by the beamsplitter and free-space coupled to an optical fiber,
which passes through the wall of the Box to butt-couple with
the optrode.
Laser light then exits the optrode to illuminate a
rhodamine solution and stimulate fluorescence.
Fluorescence,
scattered laser light, and ambient light are collected by the
optrode’s waveguide core to re-enter the Box.
Once inside,
collected light exits the fiber to strike the dichroic mirror at
normal incidence, allowing light above the 520 nm edge
wavelength to pass through.
The collected light then passes
through two identical optical filters (Semrock Brightline
FF01-542/27) with transmission bandwidth 528.
5-555.
5 nm
(Figure 2).
A pin-hole spatial filter controls beam divergence
before incidence on the photomultiplier tube (PMT)
(PerkinElmer MP 942).
The PMT is positioned within the
wall of the Box and an oscilloscope connects to the externally
protruding end.
Figure 1: Black Box Module stimulation and collection
schematic.
The Box’s method of EYFP fluorescence isolation using
optical filters is highlighted in Figure 2, showing the filter’s
transmission bandwidth to isolate for EYFP’s peak emissions
at 525 nm and blocking out laser light at 473 nm.
As
previously stated, initial fluorescence detection as measured
by the Box was performed using rhodamine 6g fluorescent
dye in an ethanol solution to mimic EYFP emissions.
Rhodamine’s stimulation and emissions spectra mimic that of
EYFP’s as shown by the close overlap of spectra in Figure 3.
Figure 2: EYFP absorption and emissions spectrum (blue).
The light green shading highlights the optical filter bandwidth
(542/27 (EM Filter)).
The solid blue line indicates the 473 nm
stimulating laser light (courtesy of Invitrogen Fluorescence
SpectraViewer).
Figure 3: EYFP (blue) and rhodamine 6g (green) absorption
and emissions spectra.
The stimulating laser light is
represented as the solid line at 473 nm.
(Courtesy of
Invitrogen Fluorescence SpectraViewer)
Literature reports rhodamine to exhibit concentration
dependent emissions qualities.
We used the Black Box to
measure fluorescence over a range of concentrations in
attempt to observe the Box’s sensitivity to the concentration
dependent behavior.
Fluorescence response was measured as
photon count events over 1x10
-7
M – 1x10
-2
M rhodamine
concentrations in an ethanol solution by increments of 1x10
-7
M, 5x10
-7
M, 1x10
-6
M, 5x10
-6
M, 1x10
-5
M, and so on until
1x10
-2
M. Thus, fluorescence photon counts were taken at a
total of 11 concentrations.
Fluorescence response was
measured as the frequency of photon count events per second
as recorded by the PMT.
A base noise count of photon events
on the PMT was measured in a 100% ethanol solution
between each fluorescence measurement.
Base noise counts
were directly related to the stimulating laser intensity.
Measurements for the 11 concentrations were taken at four
base count intensities: 1 kHz, 10 kHz, 100 kHz, and 1 MHz.
The relative fluorescence response N relative to the base
count was found by the equation:
N
c
m
c
b
c
b
(1)
where c
m
is the measured fluorescence count and c
b
is the base
count.
Rhodamine exhibits secondary emissions from the
approximately 475-525 nm overlap in its absorption and
emissions spectrum
[5]
. Measuring emissions at wavelengths
outside the area of overlap show low concentrations of
rhodamine to have a constant linear quantum yield in the 1x10
-6
M – 1.5x10
-4
M range
[5]
. Optical filters in the Box isolate for
fluorescence outside the overlap in the 528.
5-555.
5 nm
transmission bandwidth of the filter.
Thus, the Box’s measured
fluorescence response is expected to also exhibit a constant
linear trend until 1.
5x10
-4
M.
As rhodamine concentrations increase to 2x10
-4
M, the
monomer solution forms polymers.
The solution’s change in
composition alters the light interaction with the molecule, and
rhodamine’s emissions spectrum shifts towards infrared
wavelengths as concentration increases
[5,7]
. Kubin and Fletcher
(1982)
[7]
report that at 1.
5x10
-7
M, peak emission occurs at
approximately 525 nm compared to approximately 580 nm
emissions at 1x10
-3
M
[7]
. It must be noted that the Box’s filters
block out wavelengths above 555.
5 nm and Kubin and Fletcher
Journal of Student Research (2013)
Volume 2, Issue 1: pp.
48-51
Research Article
ISSN: 2167-1907
www.jofsr.
com
50
cite no upper limit to the concentration dependent red shift
behavior.
Therefore, as rhodamine concentrations increase and
the fluorescence emissions redshifts, the Box’s measured
response rate is expected to slow and show a decrease in
response as it reaches concentrations exceeding 1x10
-3
M.
Based on the properties of rhodamine described above there
are two key properties the Box’s sensitivity must accommodate
to be considered for optogenetic environments.
First, it must
pick up on the low concentration linear quantum yield to verify
its sensitivity to fluorescence detection.
Second, due to
rhodamine’s red-shift under increasing concentrations, the Box’s
measurements must show a decline in count rates at
concentrations beyond 2x10
-4
M to claim the validity of the
optical filters.
3.0 Results
3.1 Rhodamine Redshift
Data collectedover the 1 kHz, 10 kHz, 100 kHz and
1MHz base counts are shown in Figure 4.
Responses across
the 1 kHz, 10 kHz and 100 kHz base counts show the relative
response rate to notably slow between 1x10
-3
M and 5x10
-3
M.
Responses for the 1 MHz base count saturated the
sensitivity parameters of the PMT at concentrations above
1x10
-5
M and hence Figure 4 shows no fluorescence response
for concentrations above 1x10
-5
M.
Figure 4: Rhodamine’s relative fluorescence response for 1
kHz, 10 kHz, 100 kHz, and 1 MHz base counts of 1x10
-7
– 10
-
3
M concentrations.
3.2 Low-Concentration Constant Quantum Yield
Examining the 1 kHz base count behavior, Figure 5
shows the constant quantum yield behavior as measured by
the Box for the 1x10
-7
– 1x10
-4
M concentration range.
This
behavior is similarly observed at the 10 kHz, 100 kHz and 1
MHz base counts (not presented here).
Figure 5: Rhodamine’s low-concentration constant quantum
yield for 1 kHz base count.
4.0 Discussion
As expressed in Section 3.
1, Figure 4 presents
rhodamine’s relative response across all base counts.
Within
the 1x10
-3
M and 5x10
-3
M concentration range, the 1 kHz, 10
kHz and 100 kHz base counts show the response rate to slow.
This range corresponds to the region in which Kubin and
Fletcher (1982)
[7]
report a value of approximately 580 nm for
rhodamine peak emissions, which lies outside the
transmission bandwidth of the Box’s filters.
Moreover,
Figure 4 shows that within this range the fluorescent response
rate decreases between 5x10
-3
M and 1x10
-2
M. Since Kubin
and Fletcher do not cite an upper limit in rhodamine’s red
shift behavior, we believe the red shift behavior continues
such that the peak emissions wavelength shifts further outside
the filters’ transmission bandwidth.
Thus, as concentration
increases beyond 1x10
-3
M, more fluorescence is filtered out,
causing a decrease in detection by the PMT.
Section 3.
2 described the Box’s detection of rhodamine’s
low-concentration behavior.
The linear trend of the data
confirms rhodamine’s expected constant quantum yield at
concentrations below 2x10
-4
M, as expressed by Fischer and
Georges (1996)
[5]
.
The Box’s sensitivity to the concentration dependent
redshift and constant low-concentration linear behavior of
rhodamine confirms its ability to isolate for EYFP-like
fluorescence.
This presents the prospect of addressing the
explicit concern of Diester et al (2011)
[4]
in the need for
improved spatial understanding of optogenetic probing to
locate areas of transfection.
Considering that EYFP peak
emissions is understood to be constant compared to
rhodamine’s concentration emissions, we expect the Box’s
filtration and detection system to be able to separate and
detect EYFP fluorescence from scattering laser and noise light
signals.
To verify this, the next step is to use the Black Box
to measure actual EYFP fluorescence.
Provided this next
study is successful, we predict the Box-optrode system can
eventually be used to locate areas of Chr2- or NpHR-EYFP
expressing neurons indicated by collecting positive
fluorescence signals.
Journal of Student Research (2013)
Volume 2, Issue 1: pp.
48-51
Research Article
ISSN: 2167-1907
www.jofsr.
com
51
Before the Box-optrode system is regularly integrated
into optogenetic studies it is necessary to calibrate the system
for areas of transfection.
The potency of viral vector
transfection and how it spreads through a volume is still under
examination
[4]
. Thus it is important to examine the event
count signatures associated with differing EYFP potencies as
a further means of achieving spatial understanding when
probing.
5.0 Conclusion
By the ability of the Box’s low-level fluorescence
detection system used with the optrode to detect expected
behaviors in rhodamine’s concentration dependent
fluorescence response, we are confident in its function as a
tool for fluorescence detection.
This study also displays the
optrode-Box combination has the potential to jointly achieve
optical stimulation, optical and electrical recording.
We
anticipate the Box’s integration into in vivo optogenetic
studies to improve spatial understanding in optogenetic
probing and significantly reduce the need for terminal
histological analysis on expensive test subjects.
Acknowledgements
This work was supported by the Defense Advanced Research
Projects Agency (Repair Program) subcontract no.
25480040-
47135-B, and a startup fund from the Dean of Faculty Office
at Connecticut College.
The authors would like to extend their
sincere gratitude and appreciation to Professor Arto
Nurmikko of Brown University and his team for this
incredible undergraduate research opportunity.
Thanks to
Ilker Ozden and Jing Wang from Brown University for their
contribution in optimizing the module performance.
Role of the Funding Source
This work was supported by the Defense Advanced Research
Projects Agency (Repair Program) subcontract no.
25480040-
47135-B, and funds from the Dean of Faculty Office at
Connecticut College.
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