Contrast-enhanced digital holographic imaging of cellular structures by manipulating the intracellular refractive index

Abstract

The understanding of biological reactions and evaluation of the significance for living cells strongly depends on the ability to visualize and quantify these processes. Digital holographic microscopy (DHM) enables quantitative phase contrast imaging for high resolution and minimal invasive live cell analysis without the need of labeling or complex sample preparation. However, due to the rather homogeneous intracellular refractive index, the phase contrast of subcellular structures is limited and often low. We analyze the impact of the specific manipulation of the intracellular refractive index by microinjection on the DHM phase contrast. Glycerol is chosen as osmolyte, which combines high solubility in aqueous solutions and biological compatibility. We show that the intracellular injection of glycerol causes a contrast enhancement that can be explained by a decrease of the cytosolic refractive index due to a water influx. The underlying principle is proven by experiments inducing cell shrinkage and with fixated cells. The integrity of the cell membrane is considered as a prerequisite and allows a reversible cell swelling and shrinking within a certain limit. The presented approach to control the intracellular phase contrast demonstrated for the example of DHM opens prospects for applications with other quantitative phase contrast imaging methods.

Morphological Measurement of Living Cells in Methanol with Digital Holographic Microscopy

“…However, a change of the cellular refractive index may happen when seeding the Hela cells in different culture medium [13, 28]; meanwhile, as seen from (4), ${\stackrel{-}{n}}_{c}false(x,yfalse)$ is the function of the spatial coordinate, and the intracellular refractive such as the nucleoli and cytoplasm also possesses different refractive index. In view of the refractive index change caused by the intracellular refractive and the addition of different amounts of methanol in our experiment, we only give the optical thickness of Hela cells to describe the morphological feature.…”

Optical tweezers-assisted measurements of elastic light scattering

“…One approach that allows verifying scattering models and helps to understand cell physiology is the so-called immersion optical clearing technique, where the medium surrounding a cell is immersed by an optical clearing agent (OCA) with a higher refractive index and often with hyperosmotic properties 1,[18][19][20][21] . An increase in the refractive index of the cell's surroundings reduces the amount of light scattering occurring within the cell.…”

Quantitative phase microscopy via optimized inversion of the phase optical transfer function

“…A key advantage of these modifications is that they often lead to modular compatibility with commercial microscopes [7,8,[12][13][14][15][16][17][18], the results of which have been transformative for the biomedical application of QPI [2][3][4][19][20][21][22][23][24][25][26][27][28][29][30][31][32][33][34][35][36]. Quantitative phase microscopy (QPM or QPI via microscopy) has been enabling for reasons such as the ability to perform multimodal investigations correlating QPI data with images from other microscopy modalities: for example, fluorescence [37].…”