Long-Term Recordings of Live Human Cells Using Phase Contrast Microscopy - Biomedical Optical Phase Microscopy and Nanoscopy - page 41

CHAPTER 3
Long-Term Recordings of Live Human Cells
Using Phase Contrast Microscopy

Yumi Uetake, Greenfield Sluder
Department of Cell Biology, University of Massachusetts Medical School, Worcester, MA

Editor: Lisa L. Satterwhite

3.1 Introduction

In this chapter, we describe a method based on phase contrast microscopy to conduct long-
term ( B 5 6 days) continuous time-lapse observations of individual untransformed human cells

and later conduct correlative immunofluorescence characterization of cells previously followed
in vivo. From an instrumentation standpoint, there are multiple ways to prepare and observe
cells; here, we describe what has worked well for us in a convenient and cost-effective
fashion. We start with some general considerations of the optical systems suitable for live cell
time-lapse analysis, and then we will cover the specifics of our methodology.

Phase contrast and differential interference contrast (DIC) microscopy, described in details
in Chapters 1 and 2 respectively, are the most effective and commonly used methods for
long-term live cell time-lapse observations. Fluorescence microscopy, used with fluorescent
protein tagged cellular structures, has also been used to good effect for specific and
generally shorter-term applications. However, this contrast mode suffers from the greatly
increased likelihood of photodamage to the live cells. Cells are stressed by blue light used
to excite green fluorescent protein (GFP) and we have found that green excitation light for
the red fluorescent proteins is only slightly less damaging (Galdeen and Sluder,
unpublished). Over time, photodamage leads to cell cycle arrest and eventually cell death.
Short of cell death, a pernicious consequence of photodamage is compromised cell viability
that can influence experimental outcome and lead to false conclusions.

We use phase contrast microscopy for long-term observations because it provides good
contrast in a direct independent fashion and minimizes the amount of light that impinges upon
the cells. DIC is a polarized light-based contrast mode and when used in a high extinction