Leo G. Krzewina

Single-exposure optical sectioning by color structured illumination microscopy.

Structured illumination microscopy (SIM) is a wide-field technique that rivals confocal microscopy in optical sectioning ability at a small fraction of the acquisition time. For standard detectors such as a CCD camera, SIM requires a minimum of three sequential frame captures, limiting its usefulness to static objects. By using a color grid and camera, we surpass this limit and achieve optical sectioning with just a single image acquisition. The extended method is now applicable to moving objects and improves the speed of three-dimensional imaging of static objects by at least a factor of three.

Quantitative imaging of cellular adhesion by total internal reflection holographic microscopy

Total internal reflection (TIR) holographic microscopy uses a prism in TIR as a near-field imager to perform quantitative phase microscopy of cell-substrate interfaces. The presence of microscopic organisms, cell-substrate interfaces, adhesions, and tissue structures on the prisms TIR face causes relative index of refraction and frustrated TIR to modulate the object beams evanescent wave phase front. We present quantitative phase images of test specimens such as Amoeba proteus and cells such as SKOV-3 and 3T3 fibroblasts.

Quantitative Analysis by Digital Holography of the Effect of Optical Pressure on a Biological Cell

Digital Holographic Microscopy produces quantitative phase analysis of a specimen with nanometric (sub-wavelength) precision. The deformation caused by optical pressure can be observed and used to calculate physical properties of a biological cell.

Three-Dimensional Tracking of Optically Trapped Particles by Digital Gabor Holography

A new technique for 3-D position detection of optically trapped particle by digital Gabor holography is demonstrated with accuracy of ~100 nm. The particle complex optical field is reconstructed via the angular spectrum method.

Structured Illumination Imaging

A structured illumination microscopy (SIM) setup is the one in which the specimen is illuminated with a specific spatial pattern rather than with uniform illumination. The most common use of structured illumination is in optical sectioning microscopy. Linearly sinusoidal SIM is a fast optical sectioning tool that requires only simple modification to the conventional light microscope followed by a bit of image processing. A mechanical actuator attached to a linear sinusoidal grating is used in place of the grid with a coherent light source. The sectioned image should be computed with high numerical precision (e.g., double-precision floating point). Then one can apply further image processing if necessary. If so, high precision should be used there as well. The values resulting from the floating-point calculations must be scaled to the integer range of the final output file. For example, for 8-bit precision, the overall range would be scaled from 0 to 255. But this is not completely straightforward. Usually a series of optical sections is gathered at regular intervals along the z -axis to cover a three-dimensional specimen fully. The entire set of images must be considered when scaling gray-level values, since all sections should be scaled consistently.

Optical sectioning by selective illumination feedback microscopy

Selective illumination feedback microscopy is presented as a form of optical sectioning microscopy, or the ability to optically extract focused slices from axially extended objects. A liquid crystal spatial light modulator (SLM) is used to project structured light onto an object, whose image is captured by a CCD camera and processed by computer to discern the in-focus areas of the image. The processed image is fed back to the SLM to illuminate only the in-focus areas of the object. The final image captured by the CCD exhibits optical sectioning. The selective illumination principle is demonstrated both experimentally and with computer simulations, implying a range of potential three-dimensional microscopy applications.

Dynamic structured illumination microscopy: focused imaging and optical sectioning for moving objects

Structured illumination microscopy (SIM) is a valuable tool for three-dimensional microscopy and has numerous applications in bioscience. Its success has been limited to static objects, though, as three sequential image acquisitions are required per final processed, focused image. To overcome this problem we have developed a multicolored grid which when used in tandem with a color camera is capable of performing SIM with just a single exposure. Images and movies demonstrating optical sectioning of three-dimensional objects are presented, and results of applying color SIM for wide-field focused imaging are compared to those of SIM. From computer modeling and analytical calculations a theoretical estimate of the maximum observable object velocity in both the lateral and axial directions is available, implying that the new method will be capable of imaging a variety of live objects. Sample images of the technique applied to lens paper and a pigeon feather are included to show both advantages and disadvantages of CSIM.

Three-Dimensional Microscopy by Selective Illumination with Feedback

A novel method of 3D microscopy is demonstrated based on illumination of an object by a processed image of itself in a feedback loop. Using a simple apparatus, we obtain 8 mum optical sectioning resolution

3D optical trapping calibration and optical micromanipulation using 808-nm diode-laser bar

It has recently been demonstrated that diode laser bars can be used to not only optically trap red blood cells in flowing microfluidic systems but also, stretch, bend, and rotate them. To predict the complex cell behavior at different locations along a linear trap, 3D optical force characterization is required. The driving force for cells or colloidal particles within an optical trap is the thermal Brownian force where particle fluctuations can be considered a stochastic process. For optical force quantification, we combine diode laser bar optical trapping with Gabor digital holography imaging to perform subpixel resolution measurements of micron-sized particles positions along the laser bar. Here, diffraction patterns produced by trapped particles illuminated by a He-Ne laser are recorded with a CMOS sensor at 1000 fps where particle beam position reconstruction is performed using the angular spectrum method and centroid position detection. 3D optical forces are then calculated by three calibration methods: the equipartition theorem, Boltzmann probability distribution, and power spectral density analysis for each particle in the trap. This simple approach for 3D tracking and optical control can be implemented on any transmission microscope by adding a laser beam as the illumination source instead of a white light source.

High-precision three-dimensional position measurement of particles by digital Gabor holography

A single exposure of digital Gabor holography (DGH) is used for simultaneous three-dimensional measurement of particle position. The particle sample is set up such that its position can be electro-mechanically manipulated using calibrated piezoelectric transducers in both the lateral and axial directions. The central position of the reconstructed image of the particle is determined by low-pass filtering, thresholding, and center-of-mass calculation. We have obtained less than 20 nm resolution in both the lateral and axial directions in a direct and unambiguous manner. The method is applied to calibration of optical trap strength.