Journal of Neuroscience Methods 93 (1999) 163168

Two-dimensional photon counting imaging and spatiotemporal

characterization of ultraweak photon emission from a rats brain
in vivo
Masaki Kobayashi
a,
*, Motohiro Takeda
a
, Ken-Ichi Ito
b
, Hiroshi Kato
b
,
Humio Inaba
a,c
a
Biophotonics Information Laboratories, Yamagata Ad6anced Technology Research and De6elopment Center, 2-2-1 Matsuei,
Yamagata 990-2473, Japan
b
Department of Physiology, Yamagata Uni6ersity School of Medicine, 2-2-2 Iida-nishi, Yamagata 990-9585, Japan
c
Tohoku Institute of Technology, 35-1 Yagiyama-kasumi -cho, Taihaku-ku, Sendai 982-8577, Japan
Received 19 January 1999; received in revised form 27 August 1999; accepted 2 September 1999
Abstract
The process of metabolic reactions within living cells leads to spontaneous ultraweak light emission. The development of a
system for highly sensitive imaging and spatiotemporal analysis of ultraweak photon emission from a rats brain is reported in this
paper. The equipment used in this experiment consists of a two-dimensional photon-counting tube with a photocathode measuring
40 mm in diameter, a highly efcient lens system, and an electronic device to record time series of a photoelectron train with
spatial information. The sensitivity and ability to extract spatiotemporal information from sequential data of a single photoelec-
tron train were examined. The minimum detectable radiant ux density of the system was experimentally estimated to be
9.910
17
W/cm
2
with a 1-s observation time. Spontaneous photon emission was demonstrated from an exposed rats cortex in
vivo without adding any chemical agent or employing external excitation. An image of ultraweak photon emission was compared
with one obtained after cardiac arrest. The intensity after cardiac arrest was depressed to approximately 60% of before that. The
regional properties of time courses of emission intensity were also demonstrated, indicating the potential usefulness for
spatiotemporal characterization of photon emission with mapping of physiological information such as oxidative stress. This
technology constitutes a novel method, with the potential to extract pathophysiological information from the central nervous
system. 1999 Elsevier Science B.V. All rights reserved.
Keywords: Biophoton; Imaging; Photon counting; Reactive oxygen; Oxidative stress; Chemiluminescence
www.elsevier.com/locate/jneumeth
1. Introduction
During metabolic reaction processes, living organ-
isms spontaneously emit ultraweak light without any
external excitation or administration of chemilumines-
cent agents. This is referred to as biophoton emission
(Cilent, 1988; Inaba, 1988; Popp, 1988; Popp et al.,
1988; Slawinski, 1988; Van Wijk and Schamhart, 1988;
Usa et al., 1994; Amano et al., 1995; Devaraj et al.,
1997).
The intensity of emission is estimated to be less than
10
16
W/cm
2
on the surface. The biochemical mecha-
nism by which biophotons are emitted is not dependent
on specic enzymes or proteins for luminescence; so it
can be differentiated from bioluminescence phenomena
observed, for example, in reies or luminescent bacte-
ria, which have distinctive mechanisms for providing
luminescence with a high quantum yield that can be
recognized by the human eye.
Electronically excited states of constituents of living
cells are generally derived from oxidative metabolism
that accompanies the production of reactive oxygen
species (ROS; O

2
, H
2
O
2
, OH,
1
O
2
). In normal energy
metabolism, cellular respiration, a reaction in the elec-
tron transfer chain of inner mitochondrial membranes,
* Corresponding author. Present address: Yamagata Technopolis
Foundation, Yamagata Advanced Technology Research and Devel-
opment Center, 2-2-1 Matsuei, Yamagata 990-2473, Japan. Tel.:
+81-23-647-3130; fax: +81-23-647-3109.
E-mail address: kob@ckk.ymgt-techno.or.jp (M. Kobayashi)
0165-0270/99/$ - see front matter 1999 Elsevier Science B.V. All rights reserved.
PII: S0165- 0270( 99) 00140- 5
M. Kobayashi et al. / Journal of Neuroscience Methods 93 (1999) 163168 164
participates in ROS production, which is especially
facilitated under the highly reduced state of an electron
transfer chain. Biophoton emission reects the patho-
physiological state with respect to energy (ATP) pro-
duction by inner mitochondria and susceptibility to
oxidative stress, which is caused by excessive produc-
tion of ROS or a lack of activity for antioxidant
protection (Boveris et al., 1980; Cadenas et al., 1984).
Adamo et al. (1989) showed that triiodothyronine-in-
duced hypermetabolism in an in vivo rats brain leads
to increased ultraweak photon emission as a reection
of oxidative stress and suggested that ROS contributed
to premature aging in these animals.
We have described a highly sensitive imaging tech-
nique that is based on a two-dimensional photon detec-
tion and spatiotemporal characterization of ultraweak
light (Kobayashi et al., 1996, 1997a). To accomplish
this, we made the system based on a technique as
consecutive measurements of spatial and temporal in-
formation of individual photoelectrons with adequate
resolution of time. It also provides a photon correlation
function in two-dimensional eld with variety of time
resolution, and could contribute to the extraction of
non-random signals connected with physiological infor-
mation. This method has an advantage of capability on
analysis with varying time resolution and spatial resolu-
tion. However, it is not available in commercial equip-
ment of two-dimensional photon counting systems,
because of the restricted time resolution under the
time-lapse mode of imaging, and these systems also
have a disadvantage with a small area of photocathode
for macroscopic imaging such as our application.
The aim was to establish a technique whereby patho-
physiological information can be visualized in vivo
(Kobayashi et al., 1997b). In the current paper, this
system, by which biophoton images of a rats brain
could be characterized, was evaluated.
2. Materials and methods
2.1. Two-dimensional photon counting apparatus and
analytical procedures
The imaging system consisted of a two-dimensional
photon counting tube with a large active area, a highly
efcient lens system installed in a sample chamber, and
electronic equipment for identifying two-dimensional
spatial and temporal photoelectron data (Kobayashi et
al., 1996). A block diagram is shown in Fig. 1. The
photon counting tube (Model IPD 440, Photek, UK)
has a photocathode measuring 40 mm in diameter, with
a spectral sensitivity (S-20). It operates at a wavelength
that ranges from 350 to 900 nm and with a quantum
efciency of 9% at 500 nm, 5.5% at 600 nm, and 1.3%
at 800 nm. The active area of the photocathode for
imaging is a 2525 mm square.
The tube dark count was 76/s over the whole effec-
tive area, with a thermoelectronic device for cooling at
35C. Spatial resolution of the tube, which was deter-
mined by the readout precision of the resistive anode
incorporated into the photon counting tube, was ap-
proximately 200 mm.
Fig. 1. A block diagram of a highly sensitive imaging and spatiotemporal analysis system for ultraweak photon emission. Timing pulses in
photoelectron event from a position computer operate a four-cascaded counter to measure pulse-to-pulse interval based on an 80-MHz clock.
Time interval data and position data are transferred to a buffer memory, which is operated alternately for data storage and transfer, via FIFO
(fast-in-fast-out) memories, and nally are stored on a hard disk. IF represents an interface of each block of the circuit.
M. Kobayashi et al. / Journal of Neuroscience Methods 93 (1999) 163168 165
Fig. 2. Operational timing chart of four cascaded counters in a time interval counter.
A specially designed lens system (Fuji Optical Co.,
Tokyo, Japan) has a 0.45 NA (numerical aperture) and
is composed of six lenses and a built-in shutter mecha-
nism. Magnication of the lens system is 1.0, which
corresponds to an image size of 2525 mm. Relative
illumination of the lens system at an image height of
12.5 mm from the light axis is 88% on the axis. Spatial
resolution of the lens system was designed for approxi-
mately 5 line-pairs/mm to provide a superior NA.
Output pulses from the resistive anode are fed to a
position computer (IPD controller, Photek, UK) to
determine the XY position of each photoelectron
event. These data (nine bits, two channels) are consecu-
tively transferred to a specially designed pulse interval
counter. Simultaneously, it stores 27 bits of timing
data, represented as the time interval between two
successive photoelectron pulses, with two-dimensional
position data of each photoelectron event. The acquisi-
tion circuit for timing data is composed of a four-cas-
caded counter. Each stage of the counter is operated by
photoelectron pulses that serve as start and stop trig-
gers in turn, as illustrated on the timing chart of Fig. 2.
The cascaded structure of the counter potentially ac-
cepts successive pulses with a period of 37.5 ns without
a miss-count. However, the time resolution of data
acquisition is restricted by the dead time of the position
computer to a pulse-pair resolution of 10 ms. The time
resolution of the system can be improved if the calcula-
tion rate for each photoelectron on the position com-
puter can be increased. The maximum time interval for
photoelectrons, which is determined by the clock fre-
quency and data length of the counter (27 bits), is 1.7 s
with an 80-MHz clock. The miss-count probability
caused by a counter overow is theoretically negligible,
even in the case of dark counts assuming a random
time series. The total data record length depends on the
hard disk capacity of the computer. Here, using a 1
Gbyte hard disk, it is 1.2510
8
events.
After data acquisition, the data are transferred to a
workstation (Model SUN 3, Sun Microsystems, CA) to
reconstruct the photon counting images and analyze
spatiotemporal properties. This process demonstrates
the intensity kinetics in the regions of interest or the
timespace correlation of the photoelectrons.
2.2. The process of data collection for spatiotemporal
characterization and photon correlation analysis
Raw data accumulated in the pulse interval counter
are expressed by a set of sequences {(x
1
, x
1
), (x
2
, x
2
),,
(x
N
, x
N
)} where x
i
is the arrival time interval between
the (i 1)th and the i th photoelectron and x
i
is the
two-dimensional location of the i th photoelectron; and
N is the total number of photoelectron pulses. Initially,
a photon counting image, expressed as n(x, y), which is
the number of photoelectron pulses at position (x, y)
during a total measurement period, is constructed. Af-
ter setting regions of interest (r
j
={[x
j1
, x
j2
], [y
j1
, y
j2
]})
on the image plane, the number of photoelectron pulses
per unit time T (observation time) in each region are
calculated as n(r
j
, t, T) to extract the intensity time
course. The spatial and temporal correlations of emis-
sion intensity I(r
1
, t) I(r
2
, t +~) are calculated from
the photoelectron correlation that is derived from the
photoelectron detection probability at a space-time
point (r
1
, t) and detection probability after time ~ at r
2
,
represented by the conditional probability P
c
(r
2
, ~) of
two successive photoelectron pulses. The relationship is
expressed as
I(r
1
, t) I(r
2
, t +~)=an(r
1
, t, T)P
c
(r
2
, ~)
M. Kobayashi et al. / Journal of Neuroscience Methods 93 (1999) 163168 166
where the brackets denote ensemble averaging and
a, a constant.
The advantage of this system is the arbitrary selection
of spatial and temporal dimensions (r
j
and T, ~), which
can be achieved from a single measurement data point
instead of making a fresh measurement for each change
in the spatial or temporal dimensions (Kobayashi et al.,
1996).
2.3. General preparation of animals and measurement
procedure for spontaneous photon emissions from their
brains
Male Wistar rats (weighing 220300 g) were anes-
thetized in accordance with the NIH Guide for the Care
and Use of Laboratory Animals. The animals were
tracheotomized under anesthesia with pentobarbital (40
mg/kg body weight, i.p.) and articially ventilated after
cannulation of the external jugular vein for infusion
(rodent respirator Model 683, Harvard, MA) with a gas
mixture of 1% isourane in 45% oxygen and 55%
nitrogen. The rats were immobilized by pancuronium
bromide (1 mg/kg, i.v.) and an additional dose (1 mg/kg
per h) was subsequently infused with 1 ml/h of saline,
using a syringe pump (Model TOP-5200, TOP Co.,
Tokyo, Japan).
The rats were surgically prepared with an incision of
the skin to expose the skull. The parietal bone was
removed bilaterally while preserving the dura mater.
Rectal temperature was monitored throughout the ex-
periment and maintained at 38.290.5C by using a hot
water blanket. Each rat was mounted on a stereotaxic
frame and placed under the lens system in a completely
light-tight chamber. After focusing with weak light
illumination and dark adaptation to reduce phosphores-
cence (1 h), biophoton measurement was started.
3. System performance
3.1. Minimum detectable radiant ux density
Minimum detectable optical power is determined by
shot noise of the detector dark count. When minimum
detectable optical power is dened as unity of the
signal-to-noise ratio, the minimum detectable radiant
ux density on the sample surface (P
min
; W/cm
2
) is
expressed as
P
min
=
hc
u
|
d
p(V/2y)TA
with consideration of solid angle (d) for the light
collection efciency of the lens system. Here p is the
quantum efciency of the photocathode at wavelength u
and |
d
the standard deviation of the dark count within
the 1-s observation time; T is the observation time for
integration and A is the active area of the detector; c and
h represent the velocity of the light and Planks constant,
respectively.
|
d
was obtained experimentally to be 8.9 counts/s. P
min
at an observation time T=1 s, was estimated to be
8.010
17
(W/cm
2
) (=2.510
2
photons/s per cm
2
at
630 nm) using the parameters p=5.5% and d/2y=
10.2%,both of which were estimated from the numerical
aperture of the lens system. This means that a single
photoelectron corresponds to 1.810
2
photons on the
sample surface.
The above was evaluated experimentally by using a
uniform light source, incorporated with an electrolu-
minescent sheet (3030 mm) and neutral density lters.
The peak wavelength of the spectrum was 630 nm with
a 120 nm width. Intensity was calibrated by another
photon counting system (Kobayashi et al., 1998) to be
1.410
5
photons/s per cm
2
. When the light source was
installed in the focal plane, the averaged total photo-
count was 4.110
3
counts/s ( =6.510
2
counts/s per
cm
2
), which implied that a single photoelectron repre-
sents 2.210
2
photons. This is consistent with the
results of the calculation. However, a difference may
exist due to overestimation of the light collecting ef-
ciency of the lens system, without regard to the transmit-
tance property and the variation in relative illumination,
which depends on the image height. If these are consid-
ered, P
min
when T=1 s can be estimated: 9.910
17
W/cm
2
( =3.110
2
photon/s per cm
2
at 630 nm).
3.2. Analysis for spatiotemporal characteristics
To evaluate the performance of the spatiotemporal
characterization of ultraweak photon emission, the pro-
cess of obtaining timespace correlation of photoelec-
trons was examined. Optically attenuated light emitting
diodes (LEDs), which had an averaged intensity of 5.83
counts/s, were used for the evaluation. Using a 100-Hz
rectangular pulse, a pair of LEDs was directly operated
to blink alternately. Fig. 3(a) shows an image obtained
with 30 min integration. The image was displayed with
extraction of the area measuring 8.48.4 mm, and the
diameter of an LED was 2 mm. In Fig. 3(b), the intensity
time courses of both LEDs were obtained from the
square regions, as shown in Fig. 3(a), and the averaged
intensities on the two LEDs were comparable. The
correlation function, calculated with a time resolution of
100 ms from 56 k photoelectron data, which corresponds
to the measuring time of 3.010
3
s, is shown in Fig. 3(c),
in which the relevant phase delay between the two LEDs,
corresponding to the operation frequency, is indicated.
Fig. 3(d) is the same function calculated from 16 k data
(4.410
2
s measuring time). Although the correlation
function is relatively noisy, the phase delay is discernible.
The minimum number of data necessary to obtain
correlation depends on the dark count level. In this case,
the dark count for the selected regions was 0.27 counts/s.
M. Kobayashi et al. / Journal of Neuroscience Methods 93 (1999) 163168 167
3.3. Ultraweak photon emission images of a rats brain
and its spatiotemporal characterization
A typical example of biophoton emission images of a
normal rat brain, compared against the image obtained
after cardiac arrest which was led by excess dose of
pentobarbital, and extracted time courses of photon
emission, are shown in Fig. 4 with subtraction of the
dark counts. The images displayed in Fig. 4(a,b) were
obtained with 1-h integration before and after cardiac
arrest. The temporal variation of intensity of the whole
area of the brain is shown in Fig. 4(d) and time courses
of the four divided regions (Fig. 4c) in Fig. 4(e).
After cardiac arrest, the intensity of photon emission
was reduced to approximately 60% of the normal con-
dition, implying that the photon emission mechanism is
associated with cerebral blood ow.
Although temporal variations of intensity (Fig. 4e)
indicate similar patterns throughout the area (Fig. 4d),
the difference in intensity among the selected regions is
recognized. Localization of photon emission intensity is
also discernible from the image in Fig. 4(a), and the
spatial distribution is speculated to represent the local-
ization of ROS generation and could carry the informa-
tion of the regional susceptibility to oxidative stress.
4. Discussion
The present study introduced a system for highly
sensitive imaging and spatiotemporal analysis of ultra-
weak photon emission and evaluated its performance
through in vivo imaging of biophoton emission from a
rats brain. Spatiotemporal analysis of biophotons from
the cortical surface indicates potential for in vivo ex-
traction of physiological information from a brain.
The technique for spatiotemporal characterization of
ultraweak photon emission in a two-dimensional pho-
ton counting eld may be applicable to many other
optical measurements of brain function and physiology,
not only for biophoton emission, but for example, for
weak uorescence or chemiluminescence measurement
using infused chemical makers. In particular, for micro-
scopic measurements of dyed cells or transgenic cells
expressing luciferase, a new technique for data acquisi-
tion and processing that characterizes signal transduc-
tion and cellular communication will be better suited
for spatiotemporal correlation analysis of photo-
electrons.
In vivo visualization of biophoton emission from the
brain has characteristics with respect to ROS monitor-
ing and cellular injury. In general, brain tissue accounts
for approximately 20% of the whole body oxygen con-
sumption and contains a comparatively high concentra-
tion of polyunsaturated fatty acids. It is known to be
very susceptible to radical injuries subsequent to ROS
generation. Oxidative stress, for example under postis-
chemic reperfusion, leads to lipid peroxidation and
oxidative injury (Traystman et al., 1991), focally affect-
ing selectively vulnerable regions of the brain (Watson
et al., 1984; Bromont et al., 1988). Therefore in situ
identication of the spatiotemporal properties of oxida-
tive stress is most valuable and important for investiga-
tions of radical injuries.
Ultraweak biophoton emission observed under nor-
mal conditions suggests that intracellular respiration of
mitochondria for energy (ATP)-yielding metabolism
participates in photon emission through leakage of
ROS within the electron transport chain. It is suspected
that an excited species for photon emission is formed
through a radical reaction with ROS and intracellular
substances. Particularly in the case of unsaturated fatty
acids, excited species are generated as the result of a
lipid peroxidation process initiated by H
2
O
2
, metal-cat-
alyzed OH, or enzymatically produced ROS (Cadenas
et al., 1984).
Although intrinsic photon emission without the infu-
sion of chemical markers is extremely weak due to the
low efciency of the excitation mechanism, the mea-
surement of spontaneous photon emission has the ad-
vantage of being a native reection of physiological and
pathological conditions. A newly developed two-dimen-
sional photon counting imaging and analysis system of
Fig. 3. (a) An ultraweak photon emission image from two LEDs
measured to evaluate system performance. Using a 100-Hz rectangu-
lar pulse, two LEDs were switched to blink alternately. The image
size is 8.48.4 mm, and the diameter of an LED is 2 mm. (b) Time
courses of emission intensity from the two LEDs, with an observation
time of 50 s, were extracted from the square regions indicated in (a).
(c) Photoelectron correlation function extracted from two LED re-
gions with a time resolution of 100 ms. This was calculated from a
data number of 56 k, corresponding to the total measuring time of
3.010
3
s. (d) Photoelectron correlation function obtained under the
same condition of (c) except that the total number of data points in
the calculation was from 16 k data (4.410
2
s).
M. Kobayashi et al. / Journal of Neuroscience Methods 93 (1999) 163168 168
Fig. 4. (a) Ultraweak photon emission image of a normal rats brain, compared with (b) an image obtained after cardiac arrest. The integration
time is 1 h in both cases. The image size is 2525 mm. (c) A photograph of the sample, indicating the measurement eld. (d) Temporal changes
of the intensity of ultraweak photon emission extracted from the whole region of the brain, indicated by the outer square in (c). (e) Temporal
changes of the intensity of ultraweak photon emission extracted from the four regions ( c14) indicated in (c).
ultraweak photons has the potential to visualize the
pathophysiological brain function in vivo.
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