Measurement of subcellular texture by optical Gabor-like filtering with a digital micromirror device

Abstract

We demonstrate an optical Fourier processing method to quantify object texture arising from subcellular feature orientation within unstained living cells. Using a digital micromirror device as a Fourier spatial filter, we measured cellular responses to two-dimensional optical Gabor-like filters optimized to sense orientation of nonspherical particles, such as mitochondria, with a width around 0.45 μm. Our method showed significantly rounder structures within apoptosis-defective cells lacking the proapoptotic mitochondrial effectors Bax and Bak, when compared with Bax/Bak expressing cells functional for apoptosis, consistent with reported differences in mitochondrial shape in these cells. By decoupling spatial frequency resolution from image resolution, this method enables rapid analysis of nonspherical submicrometer scatterers in an under-sampled large field of view and yields spatially localized morphometric parameters that improve the quantitative assessment of biological function.Fundamental biological processes, such as programmed cell death (apoptosis), involve timedependent dynamic alterations in the morphology and subcellular organization of submicrometer scale organelles. As in any structure-function relationship, these morphological changes are controlled by important molecular pathways and must be quantified objectively to gain a more complete understanding of cellular function. Electron microscopy (EM) can directly image these structures but suffers from low throughput and cannot track dynamic structure changes. Such limitations motivate the development of high throughput optical methods that quantify time-dependent changes in morphology on very small-length scales and over a large field of view.To assess morphological changes with subwave-length sensitivity in unstained living cells, we developed an optical scatter imaging (OSI) technique based on Fourier spatial filtering using an iris with a variable diameter as a Fourier filter in a dark-field microscope [1]. In this Letter, we demonstrate how the assessment of sample morphology can be greatly extended by utilizing a spatial light modulator (SLM) as a Fourier filter. As a proof of concept, we used the SLM to implement 2D Gabor-like filters that can characterize particle orientation and roundness. Gabor filters have been used extensively in texture analysis of digital images. In the space domain, a Gabor filter corresponds to a sinusoidal wavelet with Light from a ~5 mW He-Ne laser (λ o =632.8 nm) was passed through a spinning diffuser and coupled into a multimode fiber whose output was collimated and launched into the microscope's condenser aligned in central Köhler illumination (NA < 0.05) to provide a spatially coherent plane wave. Image acquisition consisted of collecting on the CCD a stack of spatially filtered dark-field images using a spatial filter bank generated by the DMD. The DMD is a 1024×768 array of individually addressable 13.7 μm×13.7 μm mirrors, which can be programmed to deflect the light toward or away from the CCD detect...

Detection of mitochondrial fission with orientation‐dependent optical Fourier filters

“…For each time point, the degree of particle orientation, defined as ''orientedness,'' is extracted at each pixel from the Gabor-filtered images by taking the ratio of maximum to average signal response as a function of Gabor filter orientation, u, as described in (33), thereby generating ''orientedness'' response maps of the object (Fig. 1, insets 2 and 3).…”

“…All samples are studied on the optical Fourier processing microscope described in (33) fitted and aligned with a 633 oil immersion objective with numerical aperture (NA) 5 1.4, or a 203 dry objective with NA 5 0.75 (Carl Zeiss, Gottingen, Germany). For Fourier filtering, light from a $5 mW HeliumNeon laser (k o 5 632.8 nm) was passed through a spinning diffuser and coupled into a multimode fiber whose output was collimated and launched into the microscope's condenser aligned in central Köhler illumination (NA \ 0.05) to provide a spatially coherent plane wave illumination of the sample.…”

“…Gabor filtering is implemented on the DMD as described in (33). An orientation-sensitive filter bank is used, consisting of Gabor-like filters with orientations u 5 08 to u 5 1808 in 208 increments (Fig.…”

“…In this article, we present a proof-of-concept for detecting mitochondrial fission using an optical Fourier filtering modality based on Gabor filters that we developed recently and that is sensitive to size (32) and orientation (33,34) of sub-micron and micron-sized structures. The method is implemented by analyzing the output of a conventional microscope with a spatial light modulator to infer the scattering properties of the sample object (see also Supp.…”

Quantifying subcellular dynamics in apoptotic cells with two-dimensional Gabor filters

“…Here, we present a wide-field optical scatter imaging modality with sensitivity to dynamic alterations in size, shape, and orientation of sub-micron structures to investigate early apoptotic changes. The method utilizes optical Fourier filtering based on tunable Gabor-like filters for their ability to directly characterize object structure in situ and without relying on a predictive scatter model such as Mie theory [8–10]. Using an experimental model consisting of endothelial cells treated with the apoptosis inducer, staurosporine (STS), we apply this method to analyze mitochondrial and nuclear dynamics during the first three hours of apoptosis.…”