This work was supported by grants from the National Institutes of Health (U54CA143906 to K.G.P., O.J.T.M and R01HL101972 to O.J.T.M.) and an Oregon Medical Research Foundation Early Clinical Investigator Award (K.G.P.). O.J.T.M. is an American Heart Association Established Investigator (13EIA12630000). We thank Dr. Eric Anderson of the Knight Cancer Institute for preparing cell samples used in this work.

1. Microscope Specifications
To carry out imaging in the correct fashion the microscope should have the following specifications:
<ol>
	<li>Possess both differential interference contrast (DIC) and brightfield (BF) contrast.</li>
	<li>Have computer-controlled z-axis movement.</li>
	<li>Have an adjustable aperture stop to vary the condenser lens numerical aperture. An aperture with a graded rule or electronic readout is required to know the value of the numerical aperture. The numerical apert...

<ol>
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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Microscopic interferometry, Research. 8, 385-395 (1955).</li><li>Nomarski, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microinterf&#233;rom&#232;tre+diff&#233;rentiel+&#224;+ondes+polaris&#233;es.">Microinterf&#233;rom&#232;tre diff&#233;rentiel &#224; ondes polaris&#233;es.</a> J. Phys. Radium. 16, (1955).</li><li>Lang, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nomarski+differential-interference+contrast+microscopy.">Nomarski differential-interference contrast microscopy.</a> Zeiss Inform. 70, 114-120 (1968).</li><li>Allen, R. D., Allen, N. S., Travis, J. 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A., Paganin, D., Roberts, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+optical+phase+microscopy.">Quantitative optical phase microscopy.</a> Opt. Lett. 23, 817-819 (1998).</li><li>Paganin, D., Nugent, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-interferometric+phase+imaging+with+partially+coherent+light.">Non-interferometric phase imaging with partially coherent light.</a> Phys. Rev. Lett. 80, 2586-2589 (1998).</li><li>Frank, J., Altmeyer, S., Wernicke, G. N. o. n. -. i. n. t. e. r. f. e. r. o. m. e. t. r. i. c. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=non-iterative+phase+retrieval+by+Green&#8217;s+functions.">non-iterative phase retrieval by Green&#8217;s functions.</a> J. Opt. Soc. Am. A. 27, 2244-2251 (2010).</li><li>Phillips, K. G., Velasco, C. R., Li, J., Kolatkar, A., Luttgen, M., Bethel, K., Duggan, B., Kuhn, P., McCarty, O. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+quantification+of+cellular+mass,+volume,+and+density+of+circulating+tumor+cells+identified+in+an+ovarian+cancer+patient.">Optical quantification of cellular mass, volume, and density of circulating tumor cells identified in an ovarian cancer patient.</a> Front. Oncol. 2, 1-8 (2012).</li><li>Phillips, K. G., Jacques, S. L., McCarty, O. 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In general, NIQPM is a diffraction-limited technique validated on optical path-lengths ranging from 0.25-44.7. Validation was performed on n = 1.596 polystyrene spheres ranging in diameter from 0.11-9.8 &#956;m suspended in Fluoromount&#160;G (data not shown). Cells possess optical path-lengths that range from nearly 0-7.
When measuring the density distribution of a specimen one may find that the pseudo DIC image looks fine while the density map possesses unwanted background contributions. Thi...

The use of optical microscopy is now ubiquitous in the investigation of cellular organisms. Due to their low endogenous absorbance and weak scattering properties over the visible optical spectrum, cells do not strongly affect the amplitude of optical waves traversing them and thus appear semitransparent when imaged with standard bright field microscopes. Cellular specimens do, however, slow down optical waves traveling through them in a manner that is linearly related to the amount of local mass density in a particular region of space through which the light travels. Utilization of this heterogeneous time lag or &#8220;phase&#8221; profile of optical waves transmitted through microscopic specimens was first described in 1935 by Frits Zernike1 and experimentally realized by Zernikein 19422. Zernike was awarded the Nobel prize in 1953 for this achievement. Zeiss commercialized this modality in 19453.&#160;In 1955, Smith and Nomarski would present their initial work on the use4 and theory5 of differential interference contrast (DIC) microscopy, a modality that uses the spatial gradient of phase as a contrast mechanism. DIC was commercialized by Zeiss in 1965in close collaboration with Nomarski6. In 1981, two laboratories demonstrated the first recorded live cell DIC imagery with the incorporation of video cameras into the optics train of the DIC microscope7,8. The era of live cell imaging was born.
Since this time, the execution of both phase contrast and DIC on commercial microscopes has largely been unchanged. These methods are principally utilized by biologists to produce images of cells for qualitative purposes: monitoring of morphology, tracking of subcellular structures, and investigations of membrane dynamics9. These techniques are qualitative in their &#8220;off-the-shelf&#8221; configuration as both phase and DIC images are arbitrary functions of the light source intensity, the illumination optics settings, and CCD camera gain, gamma, and exposure settings.
A small legion of physicists and optical engineers has endeavored to make commercial imaging modalities quantitative. Among the first efforts were two letters to Nature in 1952 and 1953 in which the physician-turned-biophysicist Robert Barer demonstrated the use of phase microscopy to determine cellular dry mass of cells by estimating phase shifts through these cell types using a commercially available phase microscope10,11.&#160;The field has developed a multitude of techniques over the ensuing years based around three basic label-free contrast mechanisms: phase microscopy10,11, DIC microscopy12-17, and bright field18-22 to determine optical path-length, phase, mass density, refractive index, and cellular volume.
In parallel, a large collection of custom optical instruments have also been developed since the 1950s, and have made far-reaching optical measurements ranging from applications in parasite growth23, to documenting the cell cycle24 to investigating membrane dynamics of red blood cells25.&#160;In particular, the past ten years has seen a wealth of label-free quantitative microscopy in the form of diffraction phase microscopy26, tomographic phase microscopy27, digital holographic microscopy28, phase sensitive optical coherence microscopy29, spatial light interference microscopy30, Hilbert phase microscopy31, and quantitative phase microscopy32. Despite their collective successes, these instruments have not been disseminated to the larger field of biological researchers owing, mostly, to their complex instrumentation and computational requirements.
Herein we describe the use of a standard optical microscope to perform quantitative measurements of mass, volume, and density on cellular specimens through a combination of bright field and DIC imagery. Two primary approaches are presented: noninterferometric quantitative phase microscopy (NIQPM), to perform measurements of total cell mass and subcellular density distribution, and Hilbert transform differential interference contrast microscopy (HTDIC), to determine volume. The primary advantages of NIQPM and HTDIC lay in their technological accessibility. The imaging conditions required for their successful execution are within the scope of normal operation of most commercially available microscopes. Additionally, the post-processing algorithms are stable, quick, and robust &#8211; having been implemented in MATLAB using fast Fourier transform (FFT) based algorithms whenever possible.
NIQPM is a method to reconstruct phase and the axially integrated mass density of cellular specimens from bright field imagery. Summation of this axially integrated mass density over the area of the specimen gives the total dry mass content of the specimen. The NIQPM protocol is based on the experimental foundations laid by Paganin and Nugent18,19 &#8211; in which it was demonstrated that the phase profile of a cell could be reconstructed from through-focus bright field imagery of the sample &#8211; and the theoretical work of Frank, Altmeyer, and Wernicke20 &#8211; on solving the paraxial wave models in an efficient FFT-based manner. The connection of phase to the dry mass density is based upon the work by Barer10,11 and Popescu33.
Volumetric information can be obtained from through-focus DIC imagery under high NA illumination conditions that enable optical sectioning of the specimen along the optical axis. Hilbert transform processing on the DIC image stacks greatly enhances edge detection algorithms for localization of the specimen borders in three dimensions by separating the gray values of the specimen intensity from those of the background. This work originates with Arinson&#160;et al.34 although we have introduced both Fourier filtering methods to enhance contrast and a Sobel-based edge detection method for automated volumetric analysis of the sample. We have also validated HTDIC previously on polystyrene spheres ranging in size from the diffraction limit up to 20 &#956;m in diameter36.
While both NIQPM and HTDIC are technologically accessible owing to their development on commercial microscopes, the methods are fundamentally limited by the hardware configuration of the microscopes themselves. The primary limitations of these techniques are two-fold: slow z-stack acquisition time on commercial scopes, due to translation of the entire sample stage as opposed to just the objective lens, currently limits the investigation of phenomena faster than roughly 1 frame/minute, and secondly, diffraction effects restrict the utility of NIQPM and HTDIC to objects ranging in size from 0.2 up to 10 and 20 &#956;m in diameter, respectively. Hence, the specimen and its associated time dynamics of interest must meet certain size and temporal constraints to enable the use of these methods on typical &#8220;off-the-shelf&#8221; instruments. Excitingly, most fixed cellular specimens are readily investigated with these methods.
An overview of the NIQPM and HTDIC protocols are given in Figure 1. In Figure 2 we illustrate optimal and suboptimal through-focus imaging under both low and high NA illumination conditions for both bright field and DIC imagery. Figures 3 and 4 demonstrate the parameter dependence of the NIQPM algorithm highlighting successful and unsuccessful implementations. Figure 5 illustrates the steps involved in the HTDIC image-processing algorithm and demonstrates the optimal implementation of the algorithm to determine cellular volume.

<table border="0" cellpadding="0" cellspacing="0" width="675">
	<tbody>
		<tr>
			<td>Zeiss Axio Imager 2 microscope</td>
			<td>Carl Zeiss MicroImaging GmbH, Germany</td>
			<td>Axio Imager D2</td>
			<td>Microscope</td>
		</tr>
		<tr>
			<td>Green filter (&#955; = 540 &#177; 25 nm)</td>
			<td>Chroma Technology Corp., Bellows Falls, Vermont</td>
			<td>D540/25x</td>
			<td>Green filter</td>
		</tr>
		<tr>
			<td>SlideBook 5.5 software</td>
			<td>Intelligent Imaging Innovations, Denver, Colorado</td>
			<td></td>
			<td>Image acquistion software</td>
		</tr>
		<tr>
			<td>Polystyrene microspheres</td>
			<td>Bangs Laboratory, Inc., Fishers, IN</td>
			<td>PS06N</td>
			<td>Polystyrene spheres</td>
		</tr>
		<tr>
			<td>Fluoromount-G</td>
			<td>SouthernBiotech, Birmingham, Alabama</td>
			<td>0100-01</td>
			<td>Mounting media</td>
		</tr>
	</tbody>
</table>

quantitative optical microscopy: measurement of cellular biophysical features with a standard optical microscope

We describe the use of a standard optical microscope to perform quantitative measurements of mass, volume, and density on cellular specimens through a combination of bright field and differential interference contrast imagery. Two primary approaches are presented: noninterferometric quantitative phase microscopy (NIQPM), to perform measurements of total cell mass and subcellular density distribution, and Hilbert transform differential interference contrast microscopy (HTDIC)&#160;to determine volume. NIQPM is based on a simplified model of wave propagation, termed the paraxial approximation, with three underlying assumptions: low numerical aperture (NA) illumination, weak scattering, and weak absorption of light by the specimen. Fortunately, unstained cellular specimens satisfy these assumptions and low NA illumination is easily achieved on commercial microscopes. HTDIC is used to obtain volumetric information from through-focus DIC imagery under high NA illumination conditions. High NA illumination enables enhanced sectioning of the specimen along the optical axis. Hilbert transform processing on the DIC image stacks greatly enhances edge detection algorithms for localization of the specimen borders in three dimensions by separating the gray values of the specimen intensity from those of the background. The primary advantages of NIQPM and HTDIC lay in their technological accessibility using &#8220;off-the-shelf&#8221; microscopes. There are two basic limitations of these methods: slow z-stack acquisition time on commercial scopes currently abrogates the investigation of phenomena faster than 1 frame/minute, and secondly, diffraction effects restrict the utility of NIQPM and HTDIC to objects from 0.2 up to 10 (NIQPM) and 20 (HTDIC) &#956;m in diameter, respectively. Hence, the specimen and its associated time dynamics of interest must meet certain size and temporal constraints to enable the use of these methods. Excitingly, most fixed cellular specimens are readily investigated with these methods.

Correct sample illumination during through-focus image acquisition is critical to the successful implementation of the NIQPM and HTDIC algorithms. In Figure 2 we illustrate low and high NA illumination under both DIC and bright field contrast for a polystyrene sphere and the human colorectal adenocarcinoma cell line SW620. Figures 2A, 2C, 2I, and 2K demonstrate optimal imaging for NIQPM. Figures 2F, 2H, 2N, and 2P demons...

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Quantitative Optical Microscopy: Measurement of Cellular Biophysical Features with a Standard Optical Microscope

We describe the use of a standard optical microscope to perform quantitative measurements of cellular mass, volume, and density through a combination of bright field and differential interference contrast imagery.

We describe the use of a standard optical microscope to perform quantitative measurements of mass, volume, and density on cellular specimens through a ...

quantitative-optical-microscopy-measurement-cellular-biophysical

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15.3K ビュー.  Oregon Health & Science University, School of Medicine. We describe the use of a standard optical microscope to perform quantitative measurements of cellular mass, volume, and density through a combination of bright field and differential interference contrast imagery.