Single Cell Analysis: Flourescence Microscopy

Since their advent, proteomics and genomics have developed in ways that underline the rate and volume of data acquisition and analysis. They have, by necessity, worked on large populations of cells and thereby reported on population averages rather than their distributions.In this process they have missed rare, but important events and been unable to analyse cells that are only produced in small numbers. This is of course not by choice but owing to a paucity of techniques that allow experimentalists to measure protein levels at the single-cell level.

In addition, genome sequence information provides powerful insights into cellular complexity but limited information pertaining to how individual parts of a cell are work together in time and space to form dynamic cellular processes and how cellular interactions consequently translate to create higher order functions. This currently hampers an understanding of the mechanisms behind the morphological design of organisms that depends on programmes of cellular division, apoptosis, immune response etc. that are closely linked to these spatially and temporally dependent signals.

Parameters based upon averages of large populations are often misleading. Cellular heterogeneity is widespread in bacteria and increasingly apparent in eukaryotic cells. The complex and highly interconnected network of signalling pathways, their spatially dependent nature and reliance upon low-abundance molecules produces stochastic behaviour that subsequently underpins heterogeneity in cellular systems. The noise in biological function subsequently expresses itself in many different forms, from noise-driven divergence of individual cell fates through to noise-induced amplification of signals.

Similarly, the study of rare cells such as stem cells and progenitor cells does not lend itself to high throughput population-based protocols. In these cases, the development of single-cell analysis techniques that allow multiple measurements to be conducted on the same cell as a function of time is vital if we are to unravel the inner workings of these extraordinary systems.

Focusing on the characteristics of a group can obscure the differences between the individuals in it. Yet when it comes to biological cells, scientists typically derive information about their behaviour, status, and health from the collective activity of thousands or millions of them. Analyzing individual cells allows researchers to distinguish between a uniform population of cells and a group of cells with members having, say, different protein content.

A more precise understanding of differences between individual cells could lead to better treatments for diseases such as cancer, diabetes etc. Detecting minute differences between individual cells could improve medical tests and treatments. An understanding of the relationship between biological heterogeneity and signalling pathway regulation that may result in disease states is therefore critical and offers the potential to drive novel therapeutic interventions developed in response to single-cell behaviours.

Thus, Single-cell studies are crucial in order to productively study the complexity of intracellular processes.

However, tools that are capable of harvesting large amounts of proteomic data from single cells remain rather limited, largely owing to the difficulty involved in dealing with the small volumes and quantities of analytes concerned. Despite the limitations, over the last decade or two, there has been significant progress in developing assays capable of determining levels of specific proteins and interrogating enzyme activity in single cells .Fluorescence Microscopy is one such method which can be sucessfully used for Single cell analysis.

Lots of agents are used for the fluorescence, one such widely used protein is EGFP (Enhanced Green Fluorescent Protein). In the 1960s and 1970s, GFP, was first purified from Aequorea victoria and its properties studied by Osamu Shimomura. In A. victoria, GFP fluorescence occurs when aequorin interacts with Ca2+ ions, inducing a blue glow. Some of this luminescent energy is transferred to the GFP, shifting the overall color towards green. However, it’s utility as a tool for molecular biologists did not begin to be realized until 1992 when Douglas Prasher reported the cloning and nucleotide sequence of wtGFP in Gene. A 37 °C folding efficiency (F64L) point mutant to this scaffold yields enhanced GFP (EGFP) which was discovered in 1995 by the lab of Ole Thastrup. EGFP allowed the practical use of GFPs in mammalian cells. EGFP has an extinction coefficient (denoted ε) of 55,000 M−1cm−1. The fluorescence quantum yield (QY) of EGFP is 0.60. The relative brightness, expressed as ε•QY, is 33,000 M−1cm−1.

EGFP expressing cells under Flourescence Microscope

EGFP can also be expressed in different structures enabling morphological distinction. In our cases, the gene for the production of EGFP is spliced into the genome of the organism in the region of the DNA that codes for the target proteins and that is controlled by the same regulatory sequence; that is, the gene's regulatory sequence now controls the production of EGFP, in addition to the tagged protein. In cells where the gene is expressed, and the tagged proteins are produced, EGFP is produced at the same time. Thus, only those cells in which the tagged gene is expressed, or the target proteins are produced, will fluoresce when observed under fluorescence microscopy. The fluorescence can be used to even identify position of the target protein and the its amount produced at a specific time point.

Analysis of such time lapse movies has redefined the understanding of many biological processes in our case helped us to understand protein transport, and RNA dynamics, which in the past had been studied using fixed (i.e., dead) material.