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Holographic Tracking of Archaea and Bacteria Over Millimeter Length Scales
582a
Wednesday, February 15, 2017
2863-Pos Board B470 Holographic Tracking of Archaea and Bacteria Over Millimeter Length Scales Katie L. Thornton, Laurence G. Wilson. Physics, University of York, York, United Kingdom. Swimming microorganisms usually move in three dimensions, but camera sensors are two-dimensional. This can restrict the scope of experiments using standard techniques, which typically rely on tracking objects close to a twodimensional image plane. This approach is the same used in the macroscopic domain, where ‘machine vision’ approaches to object recognition and tracking have been very successful. The work in our lab takes a different approach by using aspects of classical optics and signal processing to design new image processing algorithms, to ‘mine’ more information out of digital images. This has two advantages: (i) We can extract three dimensional imaging data from two-dimensional images; (ii) by moving away from traditional ‘machine-vision’ ideas, we can redesign imaging systems that are cheaper and more flexible. I will present a few examples from recent work in holographic tracking of microorganisms that use image-processing algorithms to obtain three-dimensional data on swimming trajectory and cell shape. In particular, the ability to follow hundreds or thousands of individual swimming bacteria in volumes of up to the scale of cubic millimetres allows us to address questions on the statistics and variability of cell swimming trajectories. We have demonstrated this technique to characterise the swimming behaviour of several motile, co-habiting halophilic archaea, as well as standard laboratory strains of bacteria such as E. coli. 2864-Pos Board B471 Differential Interference Contrast Microscopes with Switchable Shear Direction and Quadrilateral Shear Michael Shribak, Elena Iourieva. Marine Biological Laboratory, Woods Hole, MA, USA. A conventional differential interference contrast (DIC) microscope is based on the interference of two beams, which optic axes are sheared by a sub-resolution distance. When the beams are traveling through the specimen the interference creates image contrast, which depends on the gradient of optical path difference (OPD) encountered along the shear. The contrast disappears if the OPD gradient is perpendicular to the shear direction. The image contrast also depends on the bias OPD introduced by microscope and specimen’s absorption and/or scattering. The DIC image is not quantitative. The orientation-independent (OI-) DIC microscope overcomes these limitations. The OI-DIC rapidly switches the shear direction by 90 and controls the bias. The microscope captures two complementary sets of DIC images with orthogonal shear directions. The images are processed to compute OPD map, which displays the specimen’s morphology and can serve as a landmark for fluorescent staining. The OPD map can be used to measure the sold content (DNA, protein) of the cells and to reconstruct the refractive index. New 4-beam DIC microscope employs interference of four beams, which optic axes are sheared quadrilateral. The 4-beam DIC creates orientationindependent OPD gradient magnitude image, which can be seen by an eye through the ocular in real-time. In order to obtain an OPD map, the microscope captures a set of images with different biases. The bias control unit can be placed anywhere between the polarizers. Therefore the 4-beam DIC is simpler to implement, in comparison to the OI-DIC. Other interference and phase microscopy techniques use modified or restricted numerical apertures (NA) of the condenser and/or objective lens. Often times the contrast of their raw images is low and is strongly affected by wavefront aberrations. The OI-DIC and the 4-beam DIC employ: (1) the full NAs of condenser and objective lenses, (2) high-contrast raw images, (3) the optical image subtraction, and (4) the computation image subtraction. Thus, the OI-DIC and the 4-beam DIC can provide the best resolution images. 2865-Pos Board B472 One-Nanometer Steps in the Motion of a Linear Molecular Motor Serratia marcescens Chitinase A Resolved by Gold Nanoprobe Ryota Iino1,2, Akihiko Nakamura2. 1 Institute for Molecular Science, Okazaki, Japan, 2Okazaki Institute for Integrative Bioscience, Okazaki, Japan. Serratia marcescens chitinase A (SmChiA) is a linear molecular motor moving on and hydrolyzing crystalline chitin processively. Here, we directly visualized steps and pauses in the motion of SmChiA. By using gold nanoparticle (40- or 20-nm in diameter) as a low-load probe, movement of SmChiA was observed with total internal reflection dark-field microscopy at 1000 fps or 2000 fps. Step sizes were 1.1 nm and 1.2 nm for forward
and backward steps, respectively, consistent with the length of the product, chitobiose (1 nm). The ratio of forward to backward steps was 18, corresponding to the energy difference of 2.9 kBT. Interestingly, 2.4 nm forward steps were also observed, and chitotetraose without one acetyl group was detected with MALDI-TOF Mass as a by-product. These results indicate that SmChiA skips one chitobiose unit if deacetylated, because SmChiA cannot hydrolyze deacetylated chitin. Furthermore, distribution of pause was fitted by single exponential decay with time constant of 22 ms, indicating single rate-limiting step. 2866-Pos Board B473 Micro-Spectroscopy of Bio-Assemblies at the Single Cell Level Jeslin Kera, Debopam Chakrabarti, Alfons Schulte. University of Central Florida, Orlando, FL, USA. Confocal absorption microscopy has the benefits of requiring no labels and low light intensity for excitation while providing a signal from the contrast generated by the attenuation of propagating light due to absorption. This enables spatially resolved measurements of single live cells and bio-molecules in nano-liter solutions. We present experiments on model systems over the spectral range from the near-infrared to the ultraviolet. The spectral identification of biomolecules with characteristic absorption bands in the ultraviolet at spatial resolution in the micron range will be discussed. 2867-Pos Board B474 High Precision Indirect Optical Manipulation of Live Cells with Functionalised Microtools Gaszton Vizsnyiczai1, Badri Aekbote2, Andra´s Bu´za´s3, Istva´n Grexa3, Pal Ormos3, Lo´ra´nd Kelemen3. 1 Dipartimento di Fisica, Universita` di Roma ‘‘La Sapienza’’, Rome, Italy, 2 Center for Soft and Living Matter, Ulsan National Institute of Science and Technology, Ulsan, Korea, Republic of, 3Institute of Biophysics, Biological Research Centre, Szeged, Hungary. Mechanical manipulation of live cells is crucial to numerous biological experiments. Optical trapping and manipulation is a popular and widely used approach, however, direct optical trapping suffers from serious limitations: The intensive laser illumination damages the cells, in addition, since trapping occurs due to interaction of light with high index of refraction regions in the cells, the trapping is not defined sufficiently well for high precision manipulation. To overcome these problems microbeads have already been used for trapping cells indirectly thereby reducing the irradiation damage and increasing trapping efficiency with their high refractive index contrast. The drawbacks can be completely eliminated by the use of tailor made microtools designed specifically for this task, so that indirect trapping and manipulation is applied. The trapping light interacts only with the tools, and the shape of the tool can be optimized for accurate and efficient manipulation around all spatial coordinates as well as for optimal cell handling. The microtools of complex shape are fabricated by two-photon excitation photopolymerization. Appropriate chemical surface functionalization establishes controlled and strong attachment of the cells. Holographic optical traps are used to precisely control the position of the cells in 6D, the positional accuracy is in the order of 10 nm. The power of the approach is demonstrated by greatly improved imaging of live cells. 2868-Pos Board B475 Fibrin Network Formation and Thrombolysis using a Birefringence Measuring Naoyuki Yokoyama1, Hayata Machida1, Yuito Tsukamoto1, Shinya Ohkubo2. 1 Department of Artificial Organs, National Institute of Technology, Numazu College, Numazu-City, Shizuoka, Japan, 2Department of Optical information engineering, National Institute of Technology, Numazu College, Numazu-City, Shizuoka, Japan. Background: Fibrin is a fibrous plasma protein, which is known as precursor of clot. The polymerized fibrin network together with platelets forms a thrombus which causes fatal thromboembolism especially in the extracorporeal circulation using heart-lung machine or dialyzer. Because the length of fibrin is shorten than visible ray wavelength, we cannot detect fibrin using an optical microscope. Objective: To develop early phase thrombus detection system, we proposed fibrin imaging method using the birefringence properties. In this study, the relationship between fibrin network formation/resolution and optical birefringence properties was evaluated. Method: Birefringence imaging system was consisted of halogen light source, red light interference filter, polarizer, quarter wave plate, optical condenser, sample flow pass, 10 objective lens, and CCD image sensor. Experimental plasma, which was kept at temperature of 36 C and coagulated gradually,
Wednesday, February 15, 2017
2863-Pos Board B470 Holographic Tracking of Archaea and Bacteria Over Millimeter Length Scales Katie L. Thornton, Laurence G. Wilson. Physics, University of York, York, United Kingdom. Swimming microorganisms usually move in three dimensions, but camera sensors are two-dimensional. This can restrict the scope of experiments using standard techniques, which typically rely on tracking objects close to a twodimensional image plane. This approach is the same used in the macroscopic domain, where ‘machine vision’ approaches to object recognition and tracking have been very successful. The work in our lab takes a different approach by using aspects of classical optics and signal processing to design new image processing algorithms, to ‘mine’ more information out of digital images. This has two advantages: (i) We can extract three dimensional imaging data from two-dimensional images; (ii) by moving away from traditional ‘machine-vision’ ideas, we can redesign imaging systems that are cheaper and more flexible. I will present a few examples from recent work in holographic tracking of microorganisms that use image-processing algorithms to obtain three-dimensional data on swimming trajectory and cell shape. In particular, the ability to follow hundreds or thousands of individual swimming bacteria in volumes of up to the scale of cubic millimetres allows us to address questions on the statistics and variability of cell swimming trajectories. We have demonstrated this technique to characterise the swimming behaviour of several motile, co-habiting halophilic archaea, as well as standard laboratory strains of bacteria such as E. coli. 2864-Pos Board B471 Differential Interference Contrast Microscopes with Switchable Shear Direction and Quadrilateral Shear Michael Shribak, Elena Iourieva. Marine Biological Laboratory, Woods Hole, MA, USA. A conventional differential interference contrast (DIC) microscope is based on the interference of two beams, which optic axes are sheared by a sub-resolution distance. When the beams are traveling through the specimen the interference creates image contrast, which depends on the gradient of optical path difference (OPD) encountered along the shear. The contrast disappears if the OPD gradient is perpendicular to the shear direction. The image contrast also depends on the bias OPD introduced by microscope and specimen’s absorption and/or scattering. The DIC image is not quantitative. The orientation-independent (OI-) DIC microscope overcomes these limitations. The OI-DIC rapidly switches the shear direction by 90 and controls the bias. The microscope captures two complementary sets of DIC images with orthogonal shear directions. The images are processed to compute OPD map, which displays the specimen’s morphology and can serve as a landmark for fluorescent staining. The OPD map can be used to measure the sold content (DNA, protein) of the cells and to reconstruct the refractive index. New 4-beam DIC microscope employs interference of four beams, which optic axes are sheared quadrilateral. The 4-beam DIC creates orientationindependent OPD gradient magnitude image, which can be seen by an eye through the ocular in real-time. In order to obtain an OPD map, the microscope captures a set of images with different biases. The bias control unit can be placed anywhere between the polarizers. Therefore the 4-beam DIC is simpler to implement, in comparison to the OI-DIC. Other interference and phase microscopy techniques use modified or restricted numerical apertures (NA) of the condenser and/or objective lens. Often times the contrast of their raw images is low and is strongly affected by wavefront aberrations. The OI-DIC and the 4-beam DIC employ: (1) the full NAs of condenser and objective lenses, (2) high-contrast raw images, (3) the optical image subtraction, and (4) the computation image subtraction. Thus, the OI-DIC and the 4-beam DIC can provide the best resolution images. 2865-Pos Board B472 One-Nanometer Steps in the Motion of a Linear Molecular Motor Serratia marcescens Chitinase A Resolved by Gold Nanoprobe Ryota Iino1,2, Akihiko Nakamura2. 1 Institute for Molecular Science, Okazaki, Japan, 2Okazaki Institute for Integrative Bioscience, Okazaki, Japan. Serratia marcescens chitinase A (SmChiA) is a linear molecular motor moving on and hydrolyzing crystalline chitin processively. Here, we directly visualized steps and pauses in the motion of SmChiA. By using gold nanoparticle (40- or 20-nm in diameter) as a low-load probe, movement of SmChiA was observed with total internal reflection dark-field microscopy at 1000 fps or 2000 fps. Step sizes were 1.1 nm and 1.2 nm for forward
and backward steps, respectively, consistent with the length of the product, chitobiose (1 nm). The ratio of forward to backward steps was 18, corresponding to the energy difference of 2.9 kBT. Interestingly, 2.4 nm forward steps were also observed, and chitotetraose without one acetyl group was detected with MALDI-TOF Mass as a by-product. These results indicate that SmChiA skips one chitobiose unit if deacetylated, because SmChiA cannot hydrolyze deacetylated chitin. Furthermore, distribution of pause was fitted by single exponential decay with time constant of 22 ms, indicating single rate-limiting step. 2866-Pos Board B473 Micro-Spectroscopy of Bio-Assemblies at the Single Cell Level Jeslin Kera, Debopam Chakrabarti, Alfons Schulte. University of Central Florida, Orlando, FL, USA. Confocal absorption microscopy has the benefits of requiring no labels and low light intensity for excitation while providing a signal from the contrast generated by the attenuation of propagating light due to absorption. This enables spatially resolved measurements of single live cells and bio-molecules in nano-liter solutions. We present experiments on model systems over the spectral range from the near-infrared to the ultraviolet. The spectral identification of biomolecules with characteristic absorption bands in the ultraviolet at spatial resolution in the micron range will be discussed. 2867-Pos Board B474 High Precision Indirect Optical Manipulation of Live Cells with Functionalised Microtools Gaszton Vizsnyiczai1, Badri Aekbote2, Andra´s Bu´za´s3, Istva´n Grexa3, Pal Ormos3, Lo´ra´nd Kelemen3. 1 Dipartimento di Fisica, Universita` di Roma ‘‘La Sapienza’’, Rome, Italy, 2 Center for Soft and Living Matter, Ulsan National Institute of Science and Technology, Ulsan, Korea, Republic of, 3Institute of Biophysics, Biological Research Centre, Szeged, Hungary. Mechanical manipulation of live cells is crucial to numerous biological experiments. Optical trapping and manipulation is a popular and widely used approach, however, direct optical trapping suffers from serious limitations: The intensive laser illumination damages the cells, in addition, since trapping occurs due to interaction of light with high index of refraction regions in the cells, the trapping is not defined sufficiently well for high precision manipulation. To overcome these problems microbeads have already been used for trapping cells indirectly thereby reducing the irradiation damage and increasing trapping efficiency with their high refractive index contrast. The drawbacks can be completely eliminated by the use of tailor made microtools designed specifically for this task, so that indirect trapping and manipulation is applied. The trapping light interacts only with the tools, and the shape of the tool can be optimized for accurate and efficient manipulation around all spatial coordinates as well as for optimal cell handling. The microtools of complex shape are fabricated by two-photon excitation photopolymerization. Appropriate chemical surface functionalization establishes controlled and strong attachment of the cells. Holographic optical traps are used to precisely control the position of the cells in 6D, the positional accuracy is in the order of 10 nm. The power of the approach is demonstrated by greatly improved imaging of live cells. 2868-Pos Board B475 Fibrin Network Formation and Thrombolysis using a Birefringence Measuring Naoyuki Yokoyama1, Hayata Machida1, Yuito Tsukamoto1, Shinya Ohkubo2. 1 Department of Artificial Organs, National Institute of Technology, Numazu College, Numazu-City, Shizuoka, Japan, 2Department of Optical information engineering, National Institute of Technology, Numazu College, Numazu-City, Shizuoka, Japan. Background: Fibrin is a fibrous plasma protein, which is known as precursor of clot. The polymerized fibrin network together with platelets forms a thrombus which causes fatal thromboembolism especially in the extracorporeal circulation using heart-lung machine or dialyzer. Because the length of fibrin is shorten than visible ray wavelength, we cannot detect fibrin using an optical microscope. Objective: To develop early phase thrombus detection system, we proposed fibrin imaging method using the birefringence properties. In this study, the relationship between fibrin network formation/resolution and optical birefringence properties was evaluated. Method: Birefringence imaging system was consisted of halogen light source, red light interference filter, polarizer, quarter wave plate, optical condenser, sample flow pass, 10 objective lens, and CCD image sensor. Experimental plasma, which was kept at temperature of 36 C and coagulated gradually,