Diffraction phase and fluorescence microscopy

Yongkeun Park,Gabriel Popescu,Michael S Feld,Ramachandra R Dasari,Kamran Badizadegan

doi:10.1364/oe.14.008263

Abstract

We have developed diffraction phase and fluorescence (DPF) microscopy as a new technique for simultaneous quantitative phase imaging and epi-fluorescence investigation of live cells. The DPF instrument consists of an interference microscope, which is incorporated into a conventional inverted fluorescence microscope. The quantitative phase images are characterized by sub-nanometer optical path-length stability over periods from milliseconds to a cell lifetime. The potential of the technique for quantifying rapid nanoscale motions in live cells is demonstrated by experiments on red blood cells, while the composite phase-fluorescence imaging mode is exemplified with mitotic kidney cells.

Highlights

During recent years optical phase-based and fluorescence techniques have advanced considerably [1]
We have developed diffraction phase and fluorescence (DPF) microscopy as a new technique for simultaneous quantitative phase imaging and epi-fluorescence investigation of live cells
The DPF instrument consists of an interference microscope, which is incorporated into a conventional inverted fluorescence microscope

Summary

Introduction

During recent years optical phase-based and fluorescence techniques have advanced considerably [1]. Phase contrast (PC) and differential interference contrast (DIC) microscopy have been used extensively to infer morphometric features of cells without the need for exogenous contrast agents [1] Both PC and DIC are qualitative in terms of optical path-length measurement, i.e. the relationship between the irradiance and phase of the image field is generally nonlinear. Full-filed phase measurement techniques, on the other hand, provide simultaneous information from a large number of points on the sample. Several such methods have been proposed in the literature [916]. DPM allows for fast imaging rates without compromising phase stability This type of measurement lacks specificity, i.e. the optical path-length detected is not characteristic to a particular molecular structure. We present for the first time, to our knowledge, an investigation that combines optical path-length maps of live cells with images of specific structures tagged by fluorophores

Diffraction phase and fluorescence microscope

DPF microscopy of live cells

Summary