Quantitative Phase Imaging of Live Cells
The company developed a digital holographic photothermal microscope system which allows acquisition of quantitative phase image of live cell. Also since photothermal imaging utilizes the principle of temperature induced phase change for imaging, our system allows one to see minute objects that couldn’t normally be seen using microscope. The extracted phase values can be used to determine the temperature distribution of the photothermal images thereby allows live cells monitoring.
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Photothermal Imaging
Photothermal microscopy (PTM) is a noninvasive technique that has been applied to many biomedical studies. PTM utilizes two pulsed lasers as pump–probe lasers. The significant merit of this technique is that it is able to detect a sample smaller than the diffraction limit. The sample is irradiated with a pump pulse, which causes a temperature excursion. The absorbed thermal energy diffuses radially outward, forming a thermal field. When the thermal field expands beyond the diffraction limit of the microscope, a probe pulse is used to probe the sample plane. The probe pulse would pick up the temperature-induced changes in the refractive index. To detect the phase changes, a phase contrast module is employed to convert the modulated phase into amplitude before detection by a CCD camera.
(a) Phase contrast image of a lymphocyte cell. (b) Photothermal image of the same lymphocyte cell (60x, N.A. 0.65)
Digital Holographic Photothermal Microscope
Unlike PTM using a phase contrast module, the phase is picked up using digital holography technique. The acquired phase is recorded in hologram, and through digital reconstruction 3D phase image can be obtained. In addition, from the acquired phase one can work backward to determine the temperature change and then the absorption contrast of the sample. Since absorption contrast of normal cells is very different from malignant cells, the system can be used as a diagnostic or monitoring tool.
The schematic setup of the system is shown below. In this setup, the probe laser, also the imaging laser, is a frequency-doubled Nd:YAG pulsed laser with a wavelength of 532 nm. The laser output is split into a reference beam and an object beam using a nonpolarized beam splitter (BS1). The sample or biological specimen is placed on the X –Y translational microscope sample stage.
The pump laser is a Nd:YAG laser pumped optical parametric oscillator working at 540 nm wavelength. The laser pulse passes through BS2 and is focused by the microscope objective onto the sample. Upon absorption of the energy of the pump pulse, a temperature excursion is created on the sample. As heat is diffusing outward, this will induce a phase change on the surrounding medium. A probe pulse is sent at a time delay of the pump pulse to pick up the induced phase.
Schematic of digital holographic photothermal microscopy system
Reconstructed Red Blood Cell phase images taken (a) before pumping and (b) 350 ns after pumping of RBCs (pump energy, 0.4 μJ; probe energy, 0.12 μJ; scale bar, 5 μm; beam spot size, 15 μm; color bar in radians)
Phase profiles of RBCs before pumping and 350 ns after pumping (X axis, distance in micrometers, Y axis, phase in radians).
Interested party wants to monitor live cells assay using our digital holography photothermal microscope is welcomed to discuss with us on the sample’s requirement.