Cytometry provides statistical analysis of a large population of cells. While flow cytometry enables characterizing gene and protein expression with throughput up to 100,000 cell/sec, it lacks morphological information. Image flow cytometry addresses this need by providing 3D structural information of the cell population, and has found numerous applications in biology and pharmacology. The LBRC has also been at the forefront in developing 3D-resolved image cytometry for cells and biological tissues. Today, most image cytometers are based on fluorescent contrast; however, developing label-free approaches has several advantages. For instance, interferometric microscopy based cytometers are virtually free from photodamage and allow for long-term, time-lapse imaging. Furthermore, without exogenous labels, sample preparation is easy and has minimal cytotoxicity. Finally, many biotechnology applications such as mesenchymal stem cell sorting or embryo selection for in vitro fertilization are not compatible with exogenous labels. Figure 1: Schematic system setup. L1-L8, lenses (f1, 5, 8 = 200 mm, f2 = 125 mm, f3 = 150 mm, f4 = 350 mm, f6 = 75 mm, f7 = 300 mm); M1-M2, mirrors; D1-D2, digital micromirror devices (DMDs); OL1-OL2, objective lenses (Zeiss, 40X, oil immersion, NA = 1.3); SMFC, single-mode fiber coupler; BS, beam splitter. Inset shows the mask pattern displayed by D2.
DMD based high-speed optical diffraction tomography
Optical diffraction tomography (ODT) is an emerging microscopy technique for label-free three-dimensional (3D) cellular imaging based on refractive index (RI) mapping [1-3]. The imaging throughput of typical angle-scan ODT is limited since multiple interferograms need to be measured for different illumination angles. The imaging speed of angle-scan ODT can be improved using a digital micromirror device (DMD) high-speed angle scanning [4]. When using a DMD, undesired diffraction can lead to limited contrast ratio of the interference fringe and hence inaccurate RI mapping. LBRC has developed a novel method to dynamically filter the diffraction noise present in DMD based OCT systems [5]. Figure 1 shows the system design of the DMD-based ODT system, which is based on the Mach-Zehnder interferometer combined with an beam illumination part using two DMDs. A 532-nm laser beam is split into sample and reference beams using a 1×2 single-mode fiber coupler. The sample beam, collimated by lens L1, is reflected by the mirror M1 and the first DMD D1 (DLP LightCrafter 9000) before illuminating the sample. D1 is programmed to display binary amplitude grating patterns using the Lee hologram scheme that generates multiple diffraction orders, among which the three primary ones, 0th and ±1 orders, are shown in Fig. 1.
The reflected beams from D1 form a series of diffraction spots at the Fourier plane of lens L2, where a second DMD D2 (DLP LightCrafter 3000) is placed for filtering. Since only one diffraction order is needed to illuminate the sample, the presence of multiple orders can significantly deteriorate the image quality of calculated phase images needed for tomographic reconstruction. However, the proposed dynamic spatial filtering method can perfectly get rid of all the diffraction noise. Specifically, a mask pattern illustrated in the inset 1 of Fig. 1 is displayed onto D2 so that only the 1st order diffraction order is allowed through (filtering the remaining orders). The sample beam, free from diffraction noise, is then used to illumintate the sample at different illumination angles. The 3D RI distribution of a human red blood cell (RBC) was measured to demonstrate the imaging applicability of this method. Figure 2 shows the cross-sectional slices of a reconstructed refractive index tomogram of a red blood cell using the DMD-based ODT system. The cross-sections clearly show the characteristic biconcave shape of a RBC and the uniform RI distribution in the x-y plane cross-section slice indicates a uniform distribution of the hemoglobin (Hb) protein in RBC cytoplasm. The blood sample was collected from Massachusetts General Hospital, MA.
The imaging throughput of our current system is ~26 tomograms/sec, which is mainly limited by the number of interferograms acquired, camera frame rate, and data processing speed. LBRC is further developing new technology to increase the throughput beyond 1kHz tomograms/sec.