Visualization of fast-moving cells in vivo using digital holographic video microscopy

Abstract

Digital in-line holography offers some significant advantages over conventional optical holography and microscopy to image biological specimens. By combining holography with digital video microscopy, an in-line holographic video microscope is developed and is capable of recording spatial 3D holographic images of biological specimens, while preserving the time dimension. The system enables high-speed video recording of fast cell movement, such as the rapid movement of blood cells in the blood stream in vivo. This capability is demonstrated with observations of fast 3-D movement of live cells in suspension cultures in response to a gentle shake to the Petri dish. The experimental and numerical procedures are incorporated with a fast reconstruction algorithm for reconstruction of holographic video frames at various planes (z axis) from the hologram and along the time axis. The current system enables both lateral and longitudinal resolutions down to a few micrometers. Postreconstruction processing of background subtraction is utilized to eliminate noise caused by scattered light, thereby enabling visualization of, for example, blood streams of live Xenopos tadpoles. The combination of digital holography and microscopy offers unique advantages for imaging of fast moving cells and other biological particles in three dimensions in vivo with high spatial and temporal resolution.

Digital Holography and Cell Studies

“…Suspension cells are difficult to track using traditional microscopy as they quickly move out of focus. DHM has been used to follow a population of human endothelial cells in their 3-D trajectory through a solution (Sun et al, 2008). The cells were followed for 189 μm along the Z axis.…”

“…Sun and colleagues showed that they could follow the movement of blood cells in vivo in a tadpole blood vessel (Sun et al, 2008). This shows that DHM can be a useful tool for in vivo studies, enabling researchers to directly follow the effect of a treatment on e.g.…”

Accurate single-shot quantitative phase imaging of biological specimens with telecentric digital holographic microscopy

“…This unique feature has been determinant to find applications of DHM in living cell screening, [1][2][3][4] particle tracking, [5][6][7][8][9][10][11] and MEMS evaluation, [12][13][14] among many others. [15][16][17][18][19][20][21][22][23][24] The ability of performing QPI has been extended to other microscopy methods like spatial light interference microscopy or spatial light interference tomography; 25,26 hence similar applications have been reported by the use of these imaging approaches.…”

“…Cell volume, [15][16][17] osmotic membrane water permeability coefficient, 18 and the integral intracellular refractive index 17,19 are some of the quantitative biological parameters that can be evaluated with DHM via the phase retrieving of the sample. DHM combined with digital video microscopy is also an effective tool to fully localize in three-dimensions living cells in controlled or natural environments [9][10][11] as a way to derive insight about the cell behavior. Its unique feature of label-free imaging of cells has turned DHM into a suitable tool for cell sorting and counting.…”

Segmentation and visualization of digital in-line holographic microscopy of three-dimensional scenes using reconstructed intensity images

“…[5][6][7][8][9][10][11][12][13] Previous 2D segmentation of digital holographic microscopy intensity reconstructions used active contours.…”