Thermal Lens Microscope Research Articles

Thermal diffusivity imaging with the thermal lens microscope

A coaxial thermal lens microscope was used to generate images based on both the absorbance and thermal diffusivity of histological samples. A pump beam was modulated at frequencies ranging from 50 kHz to 5 MHz using an acousto-optic modulator. The pump and a CW probe beam were combined with a dichroic mirror, directed into an inverted microscope, and focused onto the specimen. The change in the transmitted probe beam's center intensity was detected with a photodiode. The photodiode's signal and a reference signal from the modulator were sent to a high-speed lock-in amplifier. The in-phase and quadrature signals were recorded as a sample was translated through the focused beams and used to generate images based on the amplitude and phase of the lock-in amplifier's signal. The amplitude is related to the absorbance and the phase is related to the thermal diffusivity of the sample. Thin sections of stained liver and bone tissues were imaged; the contrast and signal-to-noise ratio of the phase image was highest at frequencies from 0.1-1 MHz and dropped at higher frequencies. The spatial resolution was 2.5 μm for both amplitude and phase images, limited by the pump beam spot size.

Detection of nonfluorescent molecules using differential interference contrast thermal lens microscope for extended nanochannel chromatography

An ultrasensitive absorbance detector, the differential interference contrast thermal lens microscope (DIC-TLM), was employed for a chromatography system using silica nanochannel. Recently, separation of ultrasmall volume sample has been strongly required for single-cell biological and chemical analysis. Previously, we have developed a chromatography system using nanochannels of ~100 nm scale (extended nanochannels) fabricated on a silica substrate. The extended nanochromatography realized highly efficient separation of samples <1 fL without packing materials. However, its detection method was limited to fluorescence method due to the small volume, and a new detector based on absorbance has been required. On the contrary, we have also developed DIC-TLM, a photothermal spectrometer based on absorption and thermal relaxation of sample for determination of concentration of nonfluorescent molecules in extended nanochannel. In this paper, we combined the extended nanochromatography and the DIC-TLM for separation and detection of nonfluorescent dyes. Particularly, basic performances of the DIC-TLM including quantitative performance and sensitivity were deliberated for injected samples of ~fL volume.

Development of Reflection Objective-Employed Collinear Mode-mismatched Thermal Lens Microscope

A reflection objective-employed collinear thermal lens microscope (roco-TLM) is developed in which a reflection objective lens is used for collinearly focusing, in a mode-mismatched configuration, both an excitation laser beam at 260 nm and a probe laser beam at 632 nm. With the microscope, unsymmetrical distortions of a thermal lens image obtained with a reflection objective were reduced. The cause of the diffraction pattern in a thermal lens image observed with the roco-TLM is discussed as well.

Development of Reflection Objective-Employed Collinear Mode-mismatched Thermal Lens Microscope

A reflection objective-employed collinear thermal lens microscope (roco-TLM) is developed in which a reflection objective lens is used for collinearly focusing, in a mode-mismatched configuration, both an excitation laser beam at 260 nm and a probe laser beam at 632 nm. With the microscope, unsymmetrical distortions of a thermal lens image obtained with a reflection objective were reduced. The cause of the diffraction pattern in a thermal lens image observed with the roco-TLM is discussed as well.

Polarization modulation thermal lens microscopy for imaging the orientation of non-spherical nanoparticles

In this paper a far field optical technique we call polarization modulation thermal lens microscopy (PM-TLM) is used for imaging the orientation and dichroism of non-spherical nanoparticles. In PM-TLM, the polarization state of a pump beam is periodically modulated which in turn causes morphology related intensity fluctuations in a continuous probe beam, thus allowing high signal to noise ratio detection with using lock-in amplification. Since PM-TLM uses nanoparticle absorption as the contrast mechanism, it may be used to detect and image nanoparticles of far smaller dimensions than can be observed by conventional dark field optical microscopy. The technique, its implementation and experiment results are presented.

Sensitive Determination of Concentration of Nonfluorescent Species in an Extended-Nano Channel by Differential Interference Contrast Thermal Lens Microscope

A photothermal detector, named differential interference contrast thermal lens microscope (DIC-TLM), was used to determine the concentration of a nonfluorescent solution in a nanochannel. This method exploits a local change in the refractive index of a solution caused by light absorption. A solution was introduced into a 100-nm scale channel by a pressure-driven nanofluidic control system and its concentration was determined by DIC-TLM. The limit of detection (LOD) was 2.4 microM in a nanochannel that was 21 microm wide and 500 nm deep. The LOD was 3 orders of magnitude smaller than that of conventional method. Moreover, the detection volume was accurately determined to be merely 0.25 fL by using a nanochannel with an optical path length of 500 nm. Based on these results, the number of detected molecules was calculated to be 390. In addition, the concentration of a solution in a nanochannel that was 790 nm wide and 500 nm deep could be determined. Finally, the relationship between sensitivity and channel size was investigated and the sensitivity was found to decrease with decreasing nanochannel size, which indicates that the changes in the refractive indices of water and silica cancel each other out. The DIC-TLM realizes sensitive detection of nonfluorescent species in nanochannels without requiring any special fabrication techniques. Therefore, DIC-TLM is expected to be a highly useful analytical technique in nanofluidics.

Micro OS-ELISA: Rapid noncompetitive detection of a small biomarkerpeptide by open-sandwich enzyme-linked immunosorbent assay (OS-ELISA) integrated into microfluidic device

A novel detection system that combines the merits of open-sandwich (OS) enzyme-linked immunoadsorbent assay (ELISA) and a microfluidic sensor chip system, and which enables rapid and noncompetitive immunodetection of small antigens of less than 1000 in molecular weight, has been proposed. Equipped with a sensitive thermal lens microscope, a minute amount of the carboxyl-terminal peptide of human osteocalcin (BGP), a biomarker for bone metabolism, was quantified utilizing antigen-dependent stabilization of an antibody variable region (OS principle). In a short analysis time (approximately 12 min), we could attain a detection limit comparable to that of the microplate-based OS ELISA (1 microg L(-1)). In addition, the effects of several pretreatments for serum-derived samples were investigated: an albumin absorption resin, addition of a protease inhibitor cocktail and heat treatment. Each pretreatment was found to be effective. Consequently, intrinsic BGP and its fragments could be detected in healthy human serum with a superior detection limit and working range compared to those of the conventional competitive ELISA method.

Development of a Differential Interference Contrast Thermal Lens Microscope for Sensitive Individual Nanoparticle Detection in Liquid

A thermal lens microscope (TLM) with a new principle was developed to improve the detection limit of conventional TLM. The detection limit was decreased by introducing a differential interference contrast (DIC) method which realizes background-free photodetection. The new differential interference contrast thermal lens microscope (DIC-TLM) exploits phase contrast resulting from a photothermal effect instead of refraction used in conventional TLM. In order to produce high phase contrast, we fabricated a pair of DIC prisms with a large shear value of 5 microm which is in accordance with the thermal diffusion length. First, we verified the principle of DIC-TLM. The background of TLM measurement was reduced to 1/100 by differential interference, and the signal-to-background (S/B) ratio was improved by 1 order of magnitude. The signal was confirmed to originate from phase contrast, and the expansion of the shear value was effective. Furthermore, we demonstrated counting of individual gold nanoparticles (5 nm) using DIC-TLM. The particles were counted with high signal-to-noise (S/N) ratio, and the S/N ratio was improved by 1 order of magnitude. Finally, we discuss the possibility of single molecule counting in a liquid.

Lateral spatial resolution of thermal lens microscopy during continuous scanning for nonstaining biofilm imaging

The possible application of continuous scanning thermal lens microscopy (TLM) as alternative online biofilm observation method is studied. As biofilm is a heterogeneous sample, the influence of spatially limited thermal flow at the sample heterogeneities and the biofilm-environment border has to be considered. The influence of the edges on the lateral resolution with respect to scanning velocity during continuous scanning TLM was therefore evaluated. Lateral scanning experiments on 100 nm thin gold stripes showed that the maximum scan speed can be predicted from a time constant of a lock-in amplifier and the beamwidth. Since three-dimensional mapping is needed to fully characterize the biofilm structure, depth scanning experiments with stained 4 μm thick polystyrene samples with the coaxial TLM setup were evaluated for signal width at full width at half maximum. Thus, a minimum step width for depth scanning of 10 μm for observation has been acquired. A three-dimensional image of unstained biofilm grown in a flow chamber was acquired using continuous scanning TLM.

Individual Nanoparticle Detection in Liquids by Thermal Lens Microscopy and Improvement of Detection Efficiency Using a 1-μm Microfluidic Channel

Nanoparticles are a key material in nanoscience and nanotechnology due to their unique physicochemical properties, so an analytical method is increasingly required. In the present research, we developed a method for individual nanoparticle detection by thermal lens microscopy and microfluidic chips. Pulsed signals were clearly observed, as nanoparticles were passing through the detection volume. The scale of the microfluidic channel was reduced from 100 to 1 microm to improve the detection efficiency. As a result, a detection efficiency of 100% was demonstrated.

Label-free detection of amino acids using gold nanoparticles in electrokinetic chromatography–thermal lens microscopy

To achieve label-free detection of amino acids in capillary-based electrokinetic chromatography–thermal lens microscopy (EKC–TLM), gold nanoparticles (GNPs), which possess the absorption around 500 nm attributed to surface plasmon resonance (SPR), were added to the background solution (BGS). Since the SPR absorption of the GNPs exhibits a sensitive response toward environmental changes and the degree of aggregation, the sensitive detection of non-absorbing species is expected by using the GNPs in EKC–TLM (GNP–EKC–TLM). In the GNP–EKC–TLM analysis of glutamic acid (Glu), a sharp peak was observed when the GNPs were added to the BGS. The plot of the peak area of Glu against its concentration gave a good linear relationship and the limit of detection was estimated to be 25 μg/mL. Furthermore, a baseline separation of lysine and Glu was successfully achieved. Thus, the EKC separation and label-free TLM detection of the amino acids can be realized only by adding the GNPs into the BGSs.

Thermal Lens Spectrometric Detection in Flow Injection Analysis and Separation Techniques

Recent applications of thermal lens spectrometry in liquid chromatography (HPLC, ion chromatography), capillary electrophoresis, and flow injection analysis are reviewed. Possible effects of performing TLS measurements in flows are discussed initially for different TLS configurations. The emphasis is given to the analysis of real samples, and the performances of various detection schemes in combination with chromatographic techniques and FIA are discussed particularly in terms of sensitivity, sample throughput, and eventual interferences from complex matrices. Coaxial TLS detection with continuous wave excitation is most suitable for detection in HPLC, ion chromatography, and FIA using long‐path length cells, when large volume samples are available. On the other hand, transverse or crossed beam TLS was found most suitable for detection in CE where it provides low absolute LODs, which are lowered even further by thermal lens microscopy on microchips, where the capability of detecting a single molecule in the detection volume was demonstrated.

Thermooptical detection in microchips: From macro‐ to micro‐scale with enhanced analytical parameters

In this paper, we compared the methods of photothermal spectroscopy used in different spatial scales, namely thermal-lens spectrometry (TLS) and thermal-lens microscopy (TLM) to enhance the performance parameters in analytical procedures. All of the experimental results were confirmed by theoretical calculation. It was proven that the design for both TLM and TLS, despite a different scale for the effect, is governed by the same signal-generating and probing conditions (probe beam diameter at the sample should be equal to the diameter of the blooming thermal lens), and almost does not depend on the nature of the solvent. Theoretical and experimental instrumental error curves for thermal lensing were coincident. TLM obeys the same law of instrumental error as TLS and shows better repeatability for the same levels of thermal-lens signals or absorbances. TLS is more advantageous for studying low concentrations in bulk, while TLM shows much lower absolute LODs due to better repeatability for low amounts. The behavior of the thermal-lens signal with different flow rates was studied and optimum conditions, with the minimum contribution to total error, were found. These conditions are reproducible, are in agreement with the existing theory of the thermal response in thermal lensing, and do not significantly affect the design of the optimum scheme for setups. TLM showed low LODs in solvent extraction (down to 10(-8) M) and electrokinetic separation (10(-7) M), which were shown to be governed by discussed instrumental regularities, instead of by microchemistry.

Numerical analysis of thermal lens effect for sensitive detection on microchips

Thermal lens microscope (TLM) is a sensitive detection method for nonfluorescent molecules and widely applied to detection in a capillary or on a microchip. In this paper, we developed a flexible design tool for TLM systems to meet various applications utilizing a microspace. The TL effect was modeled, including signal processing, and calculated by combining fluidic dynamics and wave optics software. The coincidence of the calculations and experiments was investigated by measuring the effects of optical path length or focus positions of the excitation beams on TL signals which are quite difficult to calculate by a conventional method. Good agreement was shown and the applicability of the TLM design tool was verified.

Circular dichroism thermal lens microscope in the UV wavelength region (UV-CD-TLM) for chiral analysis on a microchip

We have developed a circular-dichroism thermal lens microscope for UV wavelengths (UV-CD-TLM), for the first time, to realize sensitive chiral analysis on a microchip. Quasi-continuous-wave phase modulation of a pulsed UV laser was used to generate left-circularly polarized light and right-circularly polarized light and to detect the generated TL signal amplitude and phase with a lock-in amplifier. The amplitude and phase were used to determine the concentration and chirality, respectively, of a sample. The basic principle of UV-CD-TLM for chiral analysis on a microchip was verified by measuring aqueous solutions of optically active camphorsulfonic acids (CSA). Lower limits of detection (LOD) were calculated at S/N = 2 and were 8.7 x 10(-4) mol L(-1) (DeltaA = 5.2 x 10(-6) Abs.) for (+)-CSA and 8.4 x 10(-4) mol L(-1) (DeltaA = 5.0 x 10(-6) Abs.) for (-)-CSA. In terms of number of molecules, LODs for UV-CD-TLM were calculated to be 8.7 fmol and 8.4 fmol, respectively. This is at least three orders of magnitude lower than previously obtained. The applicability of UV-CD-TLM for chiral analysis on a microchip was verified.

Pesticide analysis by MEKC on a microchip with hydrodynamic injection from organic extract

An integrated microchip for monitoring carbamate pesticides in environmental water using continuous flow chemical processes is under development, i. e., the integration of hydrolysis, azo-derivatization, liquid-liquid extraction, electrophoretic separation, and quantification. The separation of the derivatives of four carbamate pesticides (carbaryl, carbofuran, propoxur, and bendiocarb) extracted in the continuous flow of a 1-butanol phase was studied in a silica microchip using micellar EKC. A baseline separation of four pesticide derivatives was achieved on a silica chip using hydrodynamic injection with electroosmotic gating. Detection using a thermal lens microscope showed good linearity in the concentration range of 10(-6 )-10(-5 )M with an LOD of 5 x 10(-7) M, which is superior to that of conventional CE with UV absorption detection at a level of 10(-4) M.

Multiple enzyme linked immunosorbent assay system on a capillary-assembled microchip integrating valving and immuno-reaction functions

Multiple enzyme linked immunosorbent assay (ELISA) chip is developed by using capillary-assembled microchip (CAs-CHIP) technique, which involves simple embedding of 2–3 mm length of square capillaries possessing valving and immuno-reaction functions into the microchannels fabricated on a PDMS substrate. In contrast to the previously reported ELISA chips, our system enables not only the flexible design of the multi-ELISA chip required for many different diagnostic purposes, but also the valving operation required for a reliable analysis. Here, a thermo-responsive polymer-immobilized capillary was used together with a small Peltier device, as a valving part, and different antibody-immobilized capillaries were used as immuno-reaction part. Sample solution and detecting reagent solutions were sequentially introduced through the valving capillary, and the valve is closed to completely stop the solution flow inside the immuno-reaction capillaries and detected using thermal lens microscope (TLM). Different anti-IgGs (human, goat, chicken) were immobilized and used as ELISA parts of CAs-CHIP. Sequential introductions of the mixed IgG solution, mixed enzyme–antibody solution and substrate solution facilitated the multiple determinations of 0.1 ng mL −1 IgGs (human, goat, chicken) with total analysis time of about 30 min. The valve-integrated multi-ELISA chip developed here can be applied for many different diagnostic purposes by using different immuno-reaction capillaries necessary for a specific clinical diagnostic application.

Application of a Micro Multiphase Laminar Flow on a Microchip for Extraction and Determination of Derivatized Carbamate Pesticides

Determination of carbamate pesticides such as carbaryl, carbofuran, propoxur and bendiocarb was demonstrated on a microchip with newly designed microchannels developed for efficient solvent extraction. The pesticides were hydrolyzed to corresponding naphthols, coupled with p-nitrobenzenediazonium fluoroborate reagent, and then extracted into 1-butanol as colored azo derivatives and detected with a thermal lens microscope. Optimum flow rates for the aqueous and organic phases were evaluated in the continuous-flow chemical process established in the microchip. The calibration lines showed good linearity in the range of concentrations of 0.03 - 3 ppm (10(-7) - 10(-5) M) and a mass detection limit down to a nanogram level was achieved that is at least two orders of magnitude lower than the LODs for conventional spectrophotometric methods. Azo derivatives of the pesticides were successfully separated and identified by micellar electrokinetic chromatography (MEKC) using a sample prepared on a bulk scale.

Development of the microchip-based repeatable immunoassay system for clinical diagnosis

A microchip-based semi-automated heterogeneous immunoassay was developed wherein a flow injection-like system and repeatable-use gel were employed. Microfluidics used a pump with highly accurate control of flow rate, two injector valves and a microchip. The results of a system suitability test based on dye injection for microfluidics including a thermal lens microscope as a detector were good. A regeneration buffer for antigen–antibody interaction of brain natriuretic peptide (BNP) was found which did not lose binding activity of the antibody on the gels and integration of such an immunoassay scheme into a semi-automated microfluidics system would facilitate repeated-use heterogeneous immunoassay. Good reproducibility of BNP assay was obtained with the selected regeneration buffer. The concentration of BNP in the range of 0.1–100 pg mL−1 (n = 3) was successfully determined using the system and protocol developed here.

Confocal Dual-Beam Thermal-Lens Microscope: Model and Experimental Results

The use of a confocal detection scheme in a dual-beam thermal-lens microscope is presented. It allows the measurement of absorption factors smaller than 10-6 using a heating laser power of 100 mW incident on the sample. A simple mathematical model based on gaussian beam propagation is presented that takes into account the main features of the instrument: the confocal detection scheme and the presence of chromatic aberrations in the objective lenses, and that allows to estimate the sensitivity and resolution of the instrument. This model is validated by experimental measurements on different test samples. Results are presented that prove a 450 nm axial resolution when using a NA=1.2 water immersion objective lens.