Modulated Polarization Microscopy: A Promising New Approach to Visualizing Cytoskeletal Dynamics in Living Cells

Abstract

In an effort to visualize cytoskeletal filaments in living cells, we have developed modulated polarization microscopy. Modulated polarization microscopy visualizes cytoskeletal filaments based on their birefringence but differs from the standard polarization microscopy by exploiting the angle dependence of birefringence. A prototype instrument has been developed using two Faraday rotators under computer control to change the angle of plane polarized light at a known rate. By placing one Faraday rotator before and one after the specimen, rotation produced by the first Faraday rotator is cancelled by the second. This allows the use of fixed polarizer and analyzer in a crossed configuration and continuous imaging of the specimen between crossed polarizers. The variation in polarization angle of light illuminating the specimen causes birefringent elements to oscillate in brightness. Images acquired as polarization angle is varied are then processed by a Fourier filter image-processing algorithm. The Fourier filtering algorithm isolates those signals that vary at the proper rate, whereas static or random signals are removed. Here we show that the modulated polarization microscope can reveal cytoskeletal elements including stress fibers and microtubules in living cells.

New, simple theory-based, accurate polarization microscope for birefringence imaging of biological cells

“…Therefore, the movement of our sample cells within the measurement time of 20 s needs to be less than the spatial resolution of our system, which is about 300 nm. Table 1 shows a comparison of our microscopes with two other microscopes reported by Oldenbourg et al 7,9,10 and Kuhn et al, 8 respectively. As briefly explained in Sec.…”

“…Specifically for biological applications, two different types of polarization microscopes have been reported. 7,8 One type uses two liquid crystal variable phase retarders for generating four discrete input polarization states, and the other type uses two Faraday rotators for continuous rotation of linearly polarized light with respect to a sample by controlling an external magnetic field.…”

“…Due to the white beam source and the bandpass filter employed in this microscope, however, the beam coming into the LC variable phase retarders is not monochromatic. 7,8 For example, the birefringence ⌬n of a commercially available, nematic, LC phase retarder for wavelengths of 610 and 630 nm are 0.138036 and 0.137028, respectively. 11 For ⌬ of 20 nm at = 630 nm, a spread of 3% in wavelength, each LC retarder could yield an error of 0.001 in birefringence.…”

“…8 The two Faraday rotators, one positioned before a specimen and the other after the specimen, are used to modulate polarization angles. Since the high current in the solenoid of Faraday rotators generates a lot of heat, cooling systems were added to the Faraday rotators of the microscope.…”

“…It is well known that only discrete data points such as five-or four-phase retardation values are good enough to obtain the complete birefringence information of a sample. [7][8][9][10][11] Even though the measurement speed of these previous methods are very fast, the accuracy in a measured phase retardation image is limited in these methods. The major advantage of our method is that more accurate phase retardation can be obtained compared to previous methods by having 30 or even more phase scanning data points and fitting them with a sine function.…”