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

Summary

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.

Abstract

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) 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 “off-the-shelf” 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) μm 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.

Introduction

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 “phase” profile of optical waves transmitted....

Protocol

1. Microscope Specifications

To carry out imaging in the correct fashion the microscope should have the following specifications:

1. Possess both differential interference contrast (DIC) and brightfield (BF) contrast.
2. Have computer-controlled z-axis movement.
3. 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 aperture should range from 0.1 for NIQPM up to 0.9 (or higher) for HTDIC.
4. The microscope should have a narrow band color filter to be utilized fo....

Results

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.......

Discussion

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 μm suspended in Fluoromount 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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Zeiss Axio Imager 2 microscope	Carl Zeiss MicroImaging GmbH, Germany	Axio Imager D2	Microscope
Green filter (λ = 540 ± 25 nm)	Chroma Technology Corp., Bellows Falls, Vermont	D540/25x	Green filter
SlideBook 5.5 software	Intelligent Imaging Innovations, Denver, Colorado		Image acquistion software
Polystyrene microspheres	Bangs Laboratory, Inc., Fishers, IN	PS06N	Polystyrene spheres
Fluoromount-G	SouthernBiotech, Birmingham, Alabama	0100-01	Mounting media