2D13

Towards spatially resolved magnetic field eﬀect
measurements in biological systems(Univ. of Tokyo, Graduate School of Arts and Sciences)
Jonathan R. Woodward◦ Joshua P. Beardmore and[Introduction] An important question addressed by spin chemists has been that of whether
magnetic fields can exert measurable influences in biology. Indeed the observation of such
eﬀects has proved quite controversial due to the diﬃculty of reproducing observed eﬀects acrossdiﬀerent laboratories. The most promising target for the observation of robust and significantmagnetic field eﬀects in biology has come from the attempt to understand the ability of many
species to navigate in the earths magnetic field. Much progress has been made since 2000,
when Ritz et al. reinvigorated Shulten’s original 1978 proposal of the radical pair as a magnetic
compass1 . This work identified the eye as the likely location of the sensor, and a group of
newly discovered proteins, called cryptochromes, as the likely source of the radical pair. From
one direction, spin chemists have tried to study both model reactions and simplified systems
involving cryptochromes and related photolyases in vitro 2 . From the other direction, biologists
have increasingly improved our understanding of the nature of the magneto-sensing ability andthe physical location and characteristics of the sensing apparatus. These two communities have
yet to meet in the middle and this research aims at trying to build a bridge between them
The presented research studies the eﬀects of external magnetic fields on biologically relevant,spin correlated radical pairs. A novel application of a continuous-wave laser detection system
has yielded improved resolution of the magnetic field eﬀects in a test system based on theinteraction between flavin mono-nucleotide (FMN) and hen egg white lysozyme (HEWL). The
detection system is being combined with a purpose built microscope that will directly observe
the species involved in spin-selective reactions within the sub-cellular structures in which they
are found in nature. Although we focus our eﬀorts on the study of flavoproteins in general andcryptochromes in particular, we aim at developing general experimental methods to observe
magnetic field eﬀects on reactions within microstructures in a spatially-resolved manner.
The origin of the magnetic eﬀects!"#*$%$&'()$
observed within spin correlated rad3456$'7$"!'$
ical pairs lies in the coupling of their<8!"#9!$$&'()%!:$;8!"#9!$$&'()%!:$
497$1=>=0?$electron spins to the nuclear spins
and also to external magnetic fields3 .
@$@$The radical pairs examined in this+!"8!"#9!$$&'()!:$
work are born from the electron ,-./012$
transfer between a lysozyme and aflavin based molecule in an excitedstate. The radical pair formed remains spin correlated. Application!"#$%$&'()$of an external magnetic field alters
the spin state of the radical pair andFigure 1: The proposed photochemical reaction scheme of
can ultimately aﬀect the products
the FMN-HEWL system.
formed upon re-encounter/reaction
between the two radicals. A schematic representation of the pathways involved in the FMNHEWL system are shown in Figure 1.
A$A$

[Experiment] In the experiments discussed FMN was excited from the ground state by theoutput of a Coherent CUBE laser at 449 nm. The output of this laser can either be continuouswave or modulated at up to 150 MHz by a TTL signal. The output of this laser is beam combinedwith that of a Coherent Sapphire LP 532. The 532 nm laser light is used to monitor changes in
the populations of the radical pair states via absorption spectroscopy. The absorption signal isdetected by a Nirvana auto-balanced photoreceiver (Model 2007) which can reduce noise fromlaser intensity fluctuations by over 50 dB. The output from this detector is then passed into aStanford Research Systems lock-in amplifier (Model SR830). Modulation of the absorption signal
is produced either through modulation of the ‘pump’ laser (449 nm) output or by application of
an AC magnetic field (provided by a custom built Helmholtz coil pair). The eﬀects of an appliedmagnetic field can then be observed.
Initial experiments have been performed utilising aqueous solutions in a standard cuvette. Theseexperiments have enabled the characteristics of the entire system to be studied before the addedcomplexity that arises from the implementation of microscopy. These experiments have also
yielded interesting results not previously observed in the FMN-HEWL system. A custom built
transmission mode, confocal microscope has also been constructed (see Figure 2) and absorption
imaging tests have been performed. The beam combined output of the two lasers can be diverted
from the cuvette experiment and launched into a single-mode fiber. This provides a beam withvery clean spatial mode, which then passes through a high NA, super apochromat objective lens.
The transmitted light is then collected through an identical lens and directed to the Nirvana
photoreceiver. A Thorlabs CMOS camera is also utilised for direct imaging (and navigation) ofthe sample. The sample is mounted to a Physik Instrumente P-545.3R7 3-axis peizo stage. This
stage provides 200 µm translation in all three axes with a maximum step resolution of 1 nm.
[Results and Discussion] By using both methods of signal modulation (via ‘pump’ light modulation or through an AC component in the applied magnetic field) we have been able to determine
the absolute change in the signal due to an external magnetic field and sensitively detect thechanges due to the field. The combined data from these complimentary methods provide thedata required to perform detailed simulations to determine many of the reaction kinetics of thescheme outlined in Figure 1.
The two methods of detecting the magnetic fieldeﬀects in this system also has important implications for imaging within the confocal microscope. Areas of interest can initially be determined by utilising the larger signals obtained using pump beam modulation. These areas canthen be scanned at higher resolution and using
the more sensitive technique. To date only the initial scanning method has been implemented. The
latest results will be discuss in detail during thepresentation.
[1] Ritz et al., Biophys. J. 2000, 78, 707-718.; Schultenet al., Z. Phys. Chem. 1978, NF111:1-5.
[2] See recently, Maeda et al., PNAS 2012, 109, 47744779.
[3] See Steiner and Ulrich, Chem. Rev. 1989, 89, 51147 and references therein.

Figure 2: A 3D design drawing of the confocal microscope apparatus.