Blab, Gerhard A.

Gerritsen, Hans C.

Knaus, Helene

Wösten, Han A.B.

Elsevier - Publisher Connector 

Nonlinear Spectral Imaging of Fungal Metabolism 

Sunday, February 16, 2014 205athe ToxR/TcpP/toxT protein-DNA complex important in early pathogenesis.
In addition to elucidating the regulatory pathway of V. cholerae, the impact
of this work will be to further provide a general model for outer-
membrane-bound transcription control in bacteria and nuclear-membrane-
bound transcription in eukaryotic cells.
Advances in UV-VIS-IR Spectroscopy
1033-Pos Board B788
Chemical Analysis Below the Diffraction Limit using Infrared-Coupled
Atomic Force Microscopy (AFM-IR)
Sara Heedy1, Michael Lo1, Eoghan Dillon1, Qichi Hu1, Craig Prater1,
Roshan Shetty1, Kevin Kjoller1, Curtis Marcott2, Alexandre Dazzi3,
Christopher Yip4.
1Anasys Instruments Corp., Santa Barbara, CA, USA, 2LightLight Solutions
LLC, Athens, GA, USA, 3Universite´ Paris-Sud XI - Campus d’Orsay, Paris,
France, 4Institute of Biomaterials and Biomedical Engineering, University of
Toronto, Toronto, ON, Canada.
High resolution infrared spectroscopy is a popular technique for investigating
biological structures. It is relatively simple to use, and in some cases con-
sidered to be a non-destructive technique. By combining atomic force
microscopy and infrared spectroscopy (AFM-IR) into a single bench-top in-
strument, it is possible to resolve chemical differences on the scale of ca. 100
to 200 nm, which often reveals information that could not have been obtained
with conventional infrared microspectroscopy. The AFM-IR technique is
based on observing the rapid thermal expansion and contraction of material
due to the absorption of nanoseconds-long IR radiation pulses, which is
collectively known as the photothermal induced resonance (PTIR) phenom-
enon. This rapid movement is captured by an AFM cantilever equipped with
a sharp tip that is in direct contact with the sample material. The resulting
amplitude of the ringdown response is directly related to the absorption char-
acteristics of the material across a given range of wavenumbers. Therefore,
AFM-IR spectral band shapes are similar to the bulk IR measurement and
the spectra are searchable against existing databases. By further modulating
the pulse frequency of the infrared laser radiation to coincide with the con-
tact resonance of the AFM cantilever, sensitivity is enhanced, enabling the
detection of ~ 20 nm-thick organic materials. In this presentation, we will
examine several biological systems using this AFM-IR technique. Spectral
changes in the IR spectra can be seen through the whole or cross-sections
of proteinaceous materials. Functional group IR images acquired using the
AFM-IR technique also reveal the spatial distribution of chemical species
in the form of absorption characteristics can be achieved at below the diffrac-
tion limit.
1034-Pos Board B789
Nonlinear Spectral Imaging of Fungal Metabolism
Helene Knaus1,2, Gerhard A. Blab1, Hans C. Gerritsen1, Han A.B. Wo¨sten2.
1Debye Institute, Utrecht University, Utrecht, Netherlands, 2Department of
Microbiology and Kluyver Centre for Genomics of Industrial Fermentation,
Utrecht University, Utrecht, Netherlands.
Nonlinear microscopy, which in combination with fluorescence spectroscopy is
called nonlinear spectral imaging (NLSI), affords access to specimen
morphology and (auto)fluorescence spectra at the same time. Thus, it measures
the biochemical composition, while also distinguishing different parts of the
tissue. Data interpretation is simplified by a new analysis method, the spectral
phasor [1], which results in a robust, quick, and semi-blind spectral unmixing of
fluorescent species.
NLSI and the spectral phasor are a very user-friendly technique and have the
potential to address a broad range of microbiological questions. We introduceRGB-representation of a non
linear spectral image of the whit
button mushroom (A. bisporus
left) and the corresponding pha
sor.them as a novel minimum-invasive tech-
nique to monitor the state of "fungal cells"
(hyphae). Fungi, both used as consumables
and organisms to produce industrial and
pharmaceutical compounds, require strin-
gent quality control during their growth.
To this end, and as one possible application
of NLSI and the spectral phasor, we present
their use in monitoring the quality and
freshness of white button mushrooms.
[1] F. Fereidouni, A.N. Bader, and H.C.
Gerritsen, Optics Express, 20,12729-
12741 (2012).-
e
,
-1035-Pos Board B790
A Micro-Perfusion System for the Fluorescence-Based Monitoring of
Physiological Responses to High Hydrostatic Pressures
Jeff Maltas, Zac Long, Alison Huff, Paul Urayama.
Physics, Miami University, Oxford, OH, USA.
Hydrostatic pressures of 102 to 103 atm affect a range of cellular processes,
including motility, cell division, nutrient uptake, fermentation, translation and
transcription, protein synthesis, and ultimately viability. In order to perform
the real timemonitoring of pressure effects, we present a micro-perfusion system
designed for spectroscopic measurements on cellular systems under high pres-
sure. The system consists of an optically-compatible pressure chamber and inter-
changeable fluid reservoirs. Perfusion is achieved using a dual pressure-
generator configuration, where one positive-displacement generator is com-
pressed while the other is retracted, thus maintaining a pressurized volume while
achieving fluid flow. Control over perfusion rates (typically in the 10 ml/s range)
and the ability to change between fluid reservoirs while under pressure (up to 600
atm) are demonstrated. Next, the system is used for the time-gated, spectral
monitoring of endogenous NADH fluorescence under pressure. Spectrofluori-
metric measurements utilize a nitrogen-discharge laser for sub-nanosecond pulse
width, 337-nmwavelength excitation, and an intensified CCD coupled to a spec-
trograph for nanosecond-gated spectral detection. Because emission from
NADH is routinely used at ambient pressure for monitoring mitochondrial func-
tion, we validate the system by observing the pressurized response of Saccharo-
myces cerevisiae (baker’s yeast) to mitochondrial functional modifiers (e.g.,
cyanide). Because the system is compatible with both spectroscopy and sub-
cellular resolution microscopy imaging, the system represents a robust tool for
investigating the biophysical effects of pressure on cellular systems.
1036-Pos Board B791
Fine-Grained Spatial and Temporal Resolution of Water and Protein
Contributions to Ultra-Fast and Slower Fluorescence Shifts from MD D
QM Simulations
Pedro L. Muı´n˜o1, J Nathan Scott1, Patrik R. Callis2.
1Chemistry, Saint Francis University, Loretto, PA, USA, 2Chemistry and
Biochemistry, Montana State University, Bozeman, MT, USA.
Vibrant discussion persists as to the origin of the time dependent fluorescence
spectral (Stokes) shift (TDFSS) in the range 100 fs to 100 ps for a number of
tryptophan (Trp)-containing proteins. TDFSS reports on the dynamic reorgani-
zation of the local environment around the large dipole of Trp following exci-
tation to the 1La state. Much of the discussion centers on the ubiquitous ‘‘slow’’
(10 ps-5 ns) TDFSS component found only in proteins. Details of what deter-
mines the fast (< 2ps) component in proteins are also of interest. Two questions
of interest are: (1) the relative contributions of protein and water; and (2) what
length scales characterize these contributions. To help answer these ongoing
questions we have performed molecular dynamics simulations in conjunction
with semiempirical quantum mechanics (MD þ QM) for the proteins STNase,
GB1, and monellin, each of which has been the subject of ultrafast experiments.
We have examined the spatial contributions to shifts at 1pm intervals of dis-
tance, which has revealed that only 5-10 waters less than 0.8nm from Trp
contribute to the TDFSS, while ~100 waters out to 1.5 nm often contribute
blue shifts that are the same in the ground and excited state. These simulations
raise a further question as to the mechanism of fast anti-correlated fluctuations
of protein and water contributions to the fluorescence shift.
1037-Pos Board B792
High throughput Time Resolved Fluorescence in a Microplate Reader
Karl J. Petersen1, Joseph M. Muretta1, Sutton E. Higgins2,
Kurt C. Peterson1, Gregory D. Gillispie1, David D. Thomas1.
1Biochemistry, Molecular Biology & Biophysics, University of Minnesota,
Minneapolis, MN, USA, 2Dartmouth College, Hanover, NH, USA.
We describe a high-throughput time-resolved fluorescence (TRF) spectrom-
eter, able to detect multiple fluorescence lifetimes across 384 wells with short
(< 5 min.) read times using direct waveform recording. The instrument com-
bines high-energy pulsed laser sources (5-10 kHz repetition rate, 1-3 ns pulse
width) with a photomultiplier and high-speed digitizer (1 GHz, effectively
5 GHz with interleaving) to record a complete fluorescence decay waveform
after each pulse. Single-well measurements of dyes with 200-fold signal
averaging (0.1 s acq. time) yield lifetimes comparable in accuracy and
precision to single photon counting (SPC.) Integrated software enables
immediate analysis by fitting exponential decays or by calculating a
model-independent truncated first moment. In a 384-well format changes
in quencher concentration are readily seen, with the first moment calculation
providing resolution comparable to exponential decay models. Further, we


Wosten, Han A. B.

Utrecht University Repository

Sunday, February 16, 2014 205athe ToxR/TcpP/toxT protein-DNA complex important in early pathogenesis.In addition to elucidating the regulatory pathway of V. cholerae, the impactof this work will be to further provide a general model for outer-membrane-bound transcription control in bacteria and nuclear-membrane-bound transcription in eukaryotic cells.Advances in UV-VIS-IR Spectroscopy1033-Pos Board B788Chemical Analysis Below the Diffraction Limit using Infrared-CoupledAtomic Force Microscopy (AFM-IR)Sara Heedy1, Michael Lo1, Eoghan Dillon1, Qichi Hu1, Craig Prater1,Roshan Shetty1, Kevin Kjoller1, Curtis Marcott2, Alexandre Dazzi3,Christopher Yip4.1Anasys Instruments Corp., Santa Barbara, CA, USA, 2LightLight SolutionsLLC, Athens, GA, USA, 3Université Paris-Sud XI - Campus d’Orsay, Paris,France, 4Institute of Biomaterials and Biomedical Engineering, University ofToronto, Toronto, ON, Canada.High resolution infrared spectroscopy is a popular technique for investigatingbiological structures. It is relatively simple to use, and in some cases con-sidered to be a non-destructive technique. By combining atomic forcemicroscopy and infrared spectroscopy (AFM-IR) into a single bench-top in-strument, it is possible to resolve chemical differences on the scale of ca. 100to 200 nm, which often reveals information that could not have been obtainedwith conventional infrared microspectroscopy. The AFM-IR technique isbased on observing the rapid thermal expansion and contraction of materialdue to the absorption of nanoseconds-long IR radiation pulses, which iscollectively known as the photothermal induced resonance (PTIR) phenom-enon. This rapid movement is captured by an AFM cantilever equipped witha sharp tip that is in direct contact with the sample material. The resultingamplitude of the ringdown response is directly related to the absorption char-acteristics of the material across a given range of wavenumbers. Therefore,AFM-IR spectral band shapes are similar to the bulk IR measurement andthe spectra are searchable against existing databases. By further modulatingthe pulse frequency of the infrared laser radiation to coincide with the con-tact resonance of the AFM cantilever, sensitivity is enhanced, enabling thedetection of ~ 20 nm-thick organic materials. In this presentation, we willexamine several biological systems using this AFM-IR technique. Spectralchanges in the IR spectra can be seen through the whole or cross-sectionsof proteinaceous materials. Functional group IR images acquired using theAFM-IR technique also reveal the spatial distribution of chemical speciesin the form of absorption characteristics can be achieved at below the diffrac-tion limit.1034-Pos Board B789Nonlinear Spectral Imaging of Fungal MetabolismHelene Knaus1,2, Gerhard A. Blab1, Hans C. Gerritsen1, Han A.B. Wösten2.1Debye Institute, Utrecht University, Utrecht, Netherlands, 2Department ofMicrobiology and Kluyver Centre for Genomics of Industrial Fermentation,Utrecht University, Utrecht, Netherlands.Nonlinear microscopy, which in combination with fluorescence spectroscopy iscalled nonlinear spectral imaging (NLSI), affords access to specimenmorphology and (auto)fluorescence spectra at the same time. Thus, it measuresthe biochemical composition, while also distinguishing different parts of thetissue. Data interpretation is simplified by a new analysis method, the spectralphasor [1], which results in a robust, quick, and semi-blind spectral unmixing offluorescent species.NLSI and the spectral phasor are a very user-friendly technique and have thepotential to address a broad range of microbiological questions. We introduceRGB-representation of a nonlinear spectral image of the whitbutton mushroom (A. bisporusleft) and the corresponding phasor.them as a novel minimum-invasive tech-nique to monitor the state of "fungal cells"(hyphae). Fungi, both used as consumablesand organisms to produce industrial andpharmaceutical compounds, require strin-gent quality control during their growth.To this end, and as one possible applicationof NLSI and the spectral phasor, we presenttheir use in monitoring the quality andfreshness of white button mushrooms.[1] F. Fereidouni, A.N. Bader, and H.C.Gerritsen, Optics Express, 20,12729-12741 (2012).-e,-1035-Pos Board B790A Micro-Perfusion System for the Fluorescence-Based Monitoring ofPhysiological Responses to High Hydrostatic PressuresJeff Maltas, Zac Long, Alison Huff, Paul Urayama.Physics, Miami University, Oxford, OH, USA.Hydrostatic pressures of 102 to 103 atm affect a range of cellular processes,including motility, cell division, nutrient uptake, fermentation, translation andtranscription, protein synthesis, and ultimately viability. In order to performthe real timemonitoring of pressure effects, we present a micro-perfusion systemdesigned for spectroscopic measurements on cellular systems under high pres-sure. The system consists of an optically-compatible pressure chamber and inter-changeable fluid reservoirs. Perfusion is achieved using a dual pressure-generator configuration, where one positive-displacement generator is com-pressed while the other is retracted, thus maintaining a pressurized volume whileachieving fluid flow. Control over perfusion rates (typically in the 10 ml/s range)and the ability to change between fluid reservoirs while under pressure (up to 600atm) are demonstrated. Next, the system is used for the time-gated, spectralmonitoring of endogenous NADH fluorescence under pressure. Spectrofluori-metric measurements utilize a nitrogen-discharge laser for sub-nanosecond pulsewidth, 337-nmwavelength excitation, and an intensified CCD coupled to a spec-trograph for nanosecond-gated spectral detection. Because emission fromNADH is routinely used at ambient pressure for monitoring mitochondrial func-tion, we validate the system by observing the pressurized response of Saccharo-myces cerevisiae (baker’s yeast) to mitochondrial functional modifiers (e.g.,cyanide). Because the system is compatible with both spectroscopy and sub-cellular resolution microscopy imaging, the system represents a robust tool forinvestigating the biophysical effects of pressure on cellular systems.1036-Pos Board B791Fine-Grained Spatial and Temporal Resolution of Water and ProteinContributions to Ultra-Fast and Slower Fluorescence Shifts from MD DQM SimulationsPedro L. Muı́ño1, J Nathan Scott1, Patrik R. Callis2.1Chemistry, Saint Francis University, Loretto, PA, USA, 2Chemistry andBiochemistry, Montana State University, Bozeman, MT, USA.Vibrant discussion persists as to the origin of the time dependent fluorescencespectral (Stokes) shift (TDFSS) in the range 100 fs to 100 ps for a number oftryptophan (Trp)-containing proteins. TDFSS reports on the dynamic reorgani-zation of the local environment around the large dipole of Trp following exci-tation to the 1La state. Much of the discussion centers on the ubiquitous ‘‘slow’’(10 ps-5 ns) TDFSS component found only in proteins. Details of what deter-mines the fast (< 2ps) component in proteins are also of interest. Two questionsof interest are: (1) the relative contributions of protein and water; and (2) whatlength scales characterize these contributions. To help answer these ongoingquestions we have performed molecular dynamics simulations in conjunctionwith semiempirical quantum mechanics (MD þ QM) for the proteins STNase,GB1, and monellin, each of which has been the subject of ultrafast experiments.We have examined the spatial contributions to shifts at 1pm intervals of dis-tance, which has revealed that only 5-10 waters less than 0.8nm from Trpcontribute to the TDFSS, while ~100 waters out to 1.5 nm often contributeblue shifts that are the same in the ground and excited state. These simulationsraise a further question as to the mechanism of fast anti-correlated fluctuationsof protein and water contributions to the fluorescence shift.1037-Pos Board B792High throughput Time Resolved Fluorescence in a Microplate ReaderKarl J. Petersen1, Joseph M. Muretta1, Sutton E. Higgins2,Kurt C. Peterson1, Gregory D. Gillispie1, David D. Thomas1.1Biochemistry, Molecular Biology & Biophysics, University of Minnesota,Minneapolis, MN, USA, 2Dartmouth College, Hanover, NH, USA.We describe a high-throughput time-resolved fluorescence (TRF) spectrom-eter, able to detect multiple fluorescence lifetimes across 384 wells with short(< 5 min.) read times using direct waveform recording. The instrument com-bines high-energy pulsed laser sources (5-10 kHz repetition rate, 1-3 ns pulsewidth) with a photomultiplier and high-speed digitizer (1 GHz, effectively5 GHz with interleaving) to record a complete fluorescence decay waveformafter each pulse. Single-well measurements of dyes with 200-fold signalaveraging (0.1 s acq. time) yield lifetimes comparable in accuracy andprecision to single photon counting (SPC.) Integrated software enablesimmediate analysis by fitting exponential decays or by calculating amodel-independent truncated first moment. In a 384-well format changesin quencher concentration are readily seen, with the first moment calculationproviding resolution comparable to exponential decay models. Further, we

Nonlinear Spectral Imaging of Fungal Metabolism

https://dspace.library.uu.nl/bitstream/1874/424616/1/1_s2.0_S0006349513024624_main.pdf

Nonlinear Spectral Imaging of Fungal Metabolism

Abstract

Similar works

Full text

Available Versions

Elsevier - Publisher Connector

Elsevier - Publisher Connector

Utrecht University Repository