As a platform for high resolution bioimaging, biochips with a refractive index very close to that of a culture medium (water) are highly demanded. CYTOP (AGC Corporation) is a promising material, since its refractive index (1.34) is almost similar to that of water (1.33). However, high transparency in also the ultraviolet region and high chemical resistance of CYTOP make it difficult to fabricate 3D microstructures for biochips. In this study we present fabrication method of 3D biochips based on CYTOP for cell observation by the molding process using 3D micro/nanostructures fabricated by two-photon polymerization with femtosecond laser.
We fabricated hierarchical glass biochips with interior characteristics that can be tailored by adjusting process parameters. By a new derived processing method, termed Femtosecond Laser Assisted chemical Etching Nanoscale Glass Deformation (FLAE-NGD), we have developed graded and hierarchical configurations with dimensions from several hundred micrometers to several hundred nanometers as relevant glass model platforms that mimic cancer cell intravasation-extravasation processes. We were able to control the dimensions of both the widths and lengths of the channels as well as shape and curvatures of interior glass pillars. Various curvatures were successfully prepared for the study of the migration and invasion processes of cancer. We have further evaluated the effect of x-ray exposure on melanoma cells grown in glass biochips and determined the increase of intracellular reactive oxygen species production and cellular DNA breaks with the applied irradiation dose.
KEYWORDS: Super resolution, Polymers, Microfluidics, 3D microstructuring, Two photon polymerization, Fabrication, Water, Femtosecond phenomena, Transparency, Refractive index
Development of biochips enabling distortion-free imaging in the microchannel filled with a culture medium (water) is required to investigate behavior of living cells in micro and nano environments. Fluoropolymer CYTOP is a promising material as a platform of biochips for the distortion-free imaging, because the refractive index of CYTOP (1.34) is almost same as 1.33 of water. In this study, we have developed a new 3D fabrication method for CYTOP by two-photon polymerized structures using a femtosecond laser as molds, which will be used for fabrication of micro and nano environment platforms for super-resolution bioimaging.
We report the ability of three-dimensional (3D) printing from pure fluorescent protein in the colors red, blue and green via femtosecond laser direct write (fs-LDW) technology. We utilize the protein variants mScarlet, enhanced blue fluorescent protein (EBFP2) and enhanced green fluorescent protein (EGFP) for red, blue and green colors, respectively. The family of fluorescent protein is a well-established biological tool used for fluorescence labelling and pH indication. We demonstrate that the feature of fluorescence is retained after fabrication. We further provide details regarding the fluorescence retention, in particular which fabrication parameters achieve the best outcomes.
Fs-LDW is a promising technique for fine 3D printing of biomaterials such as protein due to nonlinear multiphoton absorption processes facilitating localized microfabrication along a designated laser light path. The use of protein as a precursor material for fs-LDW is attractive because the fabricated structures retain their native functions as demonstrated by several reports. These reports range over various combination of protein and photoactivators, but pure protein can also be utilized as a precursor. The resulting proteinaceous microstructures with native function retained and submicron feature sizes might offer diverse biomedical or biochip applications.
This work highlights the capabilities of fs-LDW from pure protein with a biologically highly regarded protein, broadening the scope of the 3D printing technology as well as providing a new use for the fluorescent protein family.
Cancer metastasis is the process in which cancer cells developed in the primary tumor start to spread in the body through bloodstream or lymphatic systems and home in on secondary sites, where they may generate new tumors. At the first stage, individual cancer cells migrate through narrow confined nanometric spaces or channels of micrometric interstitial spaces. It is thus challenging to create synthetic environments that mimic in vivo characteristics, fabricating relevant biosystems along with imaging techniques for sub-cellular visualization in order to understand mechanism of cancer cell migration, in particular in confining environments. Femtosecond laser assisted chemical etching (FLAE) is a technology performing subtractive processing of glass in order to create 3D microfluidic structures embedded in a microchip with the micrometric feature size. We evaluate herein relevant glass platforms capable to offer both observation of collective cancer cells migration over long periods and individual visualization at unicellular and subcellular levels on the target cell. Glass microfluidic biochips with micrometric characteristics are first fabricated by FLAE, hosting in vivo like microenvironments. Then, by applying two photon polymerization one may generate biomimetic polymeric architectures with confining channels inside microchannels. The fabricated 3D glass nanofluidics is applied to observe behavior of cancer cell deformation and migration in narrow spaces, providing new findings.
Recent advances in three-dimensional (3D) printing of pure proteinaceous microstructures by femtosecond laser direct write (fs-LDW) are presented. Fs-LDW utilizes light-matter interaction to fabricate micro- and nanostructures along the designated path of the focused laser light. Extremely short pulses suppress heat effects while enhancing the likelihood of non-linear light absorption processes. Fs-LDW thereby allows the fabrication with spatially well-confined features and nearly arbitrary shapes in 3D with a high resolution for diverse materials including protein. Mixtures of protein and photoactivator were usually used so far as precursors to fabricate proteinaceous 3D microstructures. Here, we show that proteinaceous 3D microstructure can be fabricated using the precursor without photoactivator. Such omission of photoactivator might be relevant for biomedical and microfluidic applications because the use of photoactivator is accompanied with the undesired side-effects of photoactivator molecules leaching from created structures and cause physical alteration in the device or allergic reactions for medical use. Raman spectroscopy reveals the absence of photoactivator in the created structures and acid-catalyzed hydrolysis verifies covalent cross-linking as the printing mechanism. We further demonstrate our recent findings in terms of function retention by antibody staining. Due to the diverse abundance of proteins with different native protein functions, we believe fs-LDW of pure proteinaceous microstructures offers many applications in biological studies and medical applications.
Lab-on-a-chip devices have been intensively developed during the last decade when emerging technologies offered possibilities to manufacture reliable devices with increased spatial resolution. These biochips allowed testing chemical reactions in nanoliter volumes with enhanced sensitivity and lower consumption of reagents. There is space to further consolidate biochip assembling processing since the new technologies attempt direct fabrication in view of reducing costs and time by increasing efficiency and functionalities.
Rapid prototyping by ultrafast lasers which induces local modifications inside transparent materials of both glass and polymers with high precision at micro- and nanoscale is a promising tool for fabrication of such biochips. We have developed a new technology by combining subtractive ultrafast laser assisted chemical etching of glasses and additive two-photon polymerization to integrate 3D glass microfluidics and polymer microcomponents in a single biochip. The innovative hybrid "ship-in-a-bottle" approach is not only an instrument that can tailor 3D environments but also a tool to fabricate biomimetic in vivo structures inside a glass microfluidic chip. It was possible to create appropriate environment for cell culturing and to offer robustness and transparency for optical interrogation. Cancer cells were cultivated inside biochips and monitored over short and long periods. With the view of understanding cancer cells specific behavior such as migration or invasiveness inside human body, introduction of different geometrical configurations and chemical conditions were proposed. The cells were found responsive to a gradient of nutrient concentration through the microchannels of a 3D polymeric scaffold integrated inside glass biochip.
We demonstrate three-dimensional (3D) microstructuring inside glass by ultrafast laser to fabricate microfluidic chips
integrated with some functional microcomponents such as optical attenuators and optical waveguides. The fabricated
microchips are applied to understand phenomena and functions of microorganisms and cyanobacteria. Ultrafast laser
irradiation followed by thermal treatment and wet etching in dilute hydrofluoric acid solution resulted in fabrication of
3D microfludic structures embedded in a photosensitive glass. The embedded microfludic structures enabled us to easily
and efficiently observe Phormidium gliding to the seedling root, which accelerates growth of the vegetable. In addition,
integration of optical attenuators and optical waveguides into the microfluidic structures clarified the mechanism of the
gliding movement of Phormidium. We termed such integrated microchips nanoaquariums, realizing the highly efficient
and functional observation and analysis of various microorganisms.
We demonstrate to fabricate microfluidic chips integrated with some functional microcomponents such as optical
attenuators and optical waveguides by femtosecond laser direct writing for understanding phenomena and functions of
microorganisms. Femtosecond laser irradiation followed by annealing and wet etching in dilute hydrofluoric acid
solution resulted in fabrication of three-dimensional microfludic structures embedded in a photosensitive glass. The
embedded microfludic structures enabled us to easily and efficiently observe Phormidium gliding to the seedling root,
which accelerates growth of the vegetable. In addition, integration of optical attenuators and optical waveguides into the
microfluidic structures clarified the mechanism of the gliding movement of Phormidium. We termed such integrated
microchips nanoaquariums, realizing the highly efficient and functional observation and analysis of various
microorganisms.
We demonstrate fabrication of microfluidic chips integrated with different functional elements such as optical filters and
optical waveguide for mechanism study of gliding movement of Phormidium to a seedling root using a femtosecond (fs)
laser. Fs laser direct writing followed by annealing and successive wet etching in dilute hydrofluoric (HF) acid solution
resulted in formation of three dimensional (3D) hollow microstructures embedded in a photostructurable glass. The
embedded microfludic structures enabled us to easily and efficiently observe Phormidium gliding to the seedling root,
which accelerates growth of the seedling. In addition, fabrication of optical filter and optical waveguide integrated with
the microfluidic structures in the microchip clarified the mechanism of the gliding movement. Such microchips, referred
to as a nano-aquarium, realize the efficient and highly functional observation and analysis of the gliding movement of
Phormidium.
A microchip made of UV transparent polymer (CYTOP) that can perform selective cell culture has been fabricated by F2
laser surface modification. The refractive index of CYTOP is almost the same as that of culture medium, which is
essential for three dimensional (3D) observation of cells. The F2 laser modification of CYTOP achieves hydrophilicity
only on the laser irradiated area with little deterioration of the optical properties and surface smoothness. After the laser
modification, HeLa cells were successfully cultured and strongly adhered only on the modified area of CYTOP. The
cells patterned on CYTOP were applied for clear 3D observation using an optical microscope in phase contrast mode.
We are developing an ultra-high-sensitivity and ultra-high-speed imaging system for bioscience, mainly for imaging of microbes with visible light and cells with fluorescence emission. Scarcity of photons is the most serious problem in applications of high-speed imaging to the scientific field. To overcome the problem, the system integrates new technologies consisting of (1) an ultra-high-speed video camera with sub-ten-photon sensitivity with the frame rate of more than 1 mega frames per second, (2) a microscope with highly efficient use of light applicable to various unstained and fluorescence cell observations, and (3) very powerful long-pulse-strobe Xenon lights and lasers for microscopes. Various auxiliary technologies to support utilization of the system are also being developed. One example of them is an efficient video trigger system, which detects a weak signal of a sudden change in a frame under ultra-high-speed imaging by canceling high-frequency fluctuation of illumination light. This paper outlines the system with its preliminary evaluation results.
We demonstrate the fabrication of three-dimensional (3-D) hollow microstructures embedded in photostructurable glass
by a femtosecond (fs) laser direct writing. Fs laser direct writing followed by annealing and successive wet etching in
dilute hydrofluoric (HF) acid solution resulted in the rapid manufacturing of microchips with 3-D hollow microstructures
for the dynamic observation of living microorganisms in fresh water. The embedded microchannel structure enables us
to analyze the continuous motion of Euglena gracilis. A microchamber with a movable microneedle demonstrates its
ability for the elucidation of the information transmission process in Pleurosira laevis. Such microchips, referred to as
nano-aquariums realize the efficient and highly functional observation of microorganisms.
We demonstrate the fabrication of three-dimensional (3D) hollow microstructures embedded in photostructurable glass
by a nonlinear multiphoton absorption process using a femtosecond (fs) laser. Fs laser direct writing followed by
annealing and successive wet etching in dilute hydrofluoric (HF) acid solution resulted in the rapid manufacturing of
microchips with 3-D hollow microstructures for the dynamic observation of living microorganisms in fresh water. The
embedded microchannel structure enables us to analyze the continuous motion of Euglena gracilis and Dinoflagellate.
Such microchips, referred to as nano-aquariums realize the efficient and highly functional observation of
microorganisms.
An ultrafast video microscope (UVM), the frame rate of which reaches one million per second has been developed. Our
UVM system provides pictures with high-contrast and high-resolution for differential interference contrast (DIC), phase
contrast, or dark field imaging. It allows us to observe fast events that occur in live cells when irradiated by ultrashort
laser pulses. Femtosecond laser pulses can be used to manipulate, stimulate, and destroy specific cells and organelles
under the microscope. The irradiation of such an intense laser immediately results in some physical events, such as
microbubble generation, plasma formation, and photoporation. We investigate biophysical mechanisms underlying the
ultrafast processes. Our data will contribute to development of new bio-imaging modalities, which implement laser cell
transfection. We also present a new method to observe side views of live cells on a substrate. We used a polymer
material CYTOP as the substrate for HeLa cells. CYTOP has a refractive index of 1.34, which is close to 1.33 of water.
We investigate generation of microbubbles beneath the plasma membranes with a time resolution of one microsecond for
the purpose of improving the efficiency of photoporation.
The two-photon excitation fluorescence (TPEF) process of an enhanced green fluorescent protein (EGFP) for fluorescence signals was adaptively controlled by the phase-modulation of femtosecond pulses. After the iteration of pulse shaping, a twofold increase in the ratio of the fluorescence signal to the laser peak power was achieved. Compared with conventional pulses optimized for peak power, phase-optimized laser pulses reduced the bleaching rate of EGFP by a factor of 4 while maintaining the same intensity of the fluorescence signal. The ratio of two-photon (2P) fluorescence from EGFP and three-photon (3P) fluorescence from the essential amino acid L-Tryptophan was also enhanced by using the adaptive pulse shaping technique. To achieve a trade-off between the 2P/3P fluorescence ratio and the 2P fluorescence intensity, we then engineered the cost function in the self-learning algorithm. These methods have much potential for immediate application to various important biological and medical studies.
Two-photon excited fluorescence process of green fluorescent protein was adaptively controlled by shaped femtosecond excitation laser pulses. The phase control was given by a fused-silica spatial light modulator. We could completely intentionally increase and decrease of fluorescence efficiency against the excitation intensity. This fully-controllable method can become a robust solution for various practical problems such as photobleaching of the samples on a two-photon fluorescence microscopy.
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