Thursday, 12 May 2016

Lipid Staining: Explore your own detection tool….


Lipid droplets are found in almost every level of biological kingdom. Also, they are being characterized in almost every cell, which play an important role in energy storage, cell membrane repair, and substrate synthesis etc. The method of lipid staining to visualize lipid droplets under the microscope, that we are using today is a step up from the conventional methods but it will take little more advancement to optimize the protocol and use it in different fields of study.
Lipid staining using different type’s dyes is a very efficient and cost effective tool to screen 1000s of species for neutral lipids. Fluorescent stains enable the researchers to visualize the lipids and oil droplets under the fluorescence microscope. However, even with today high tech microscopy, it`s not so simple to take high-resolution photographs of lipids. There are many problems such as an accurate sample binding and sample leakage when viewing sample under a microscope.
It is likely to become a favorite tool for observing neutral lipids at preliminary steps for large-scale production various industrial and medical applications. Therefore, optimized protocol should be designed for large-scale sample study. It requires modification of the available protocols in the given literature to obtain the common calibration curve for efficient binding and fluorescence when studied for cellular imaging. These dyes are the one of the most vital methods for detecting cytoplasmic lipids.
The increase in technological advancements in staining and microscopy is helping the researchers to quantify lipids under various conditions and less time

Lipid Staining and fluorescent microscope

It provides us an inexpensive tool to measure the neutral lipid content by excluding the requirement of expensive gravimetric analysis. Considering this fact, it is very essential requirement to conduct an easy and repeatable measurement of lipid.  It is estimated that lipid measurement using GC-MS, HPLC, or TLC will cost you around $50 to $100 per sample (including technical team and equipment requirement).  On the contrary, in terms of signal fluorescence and my finite experience, there are no significant achievements in lipid staining methods.
The different staining system provides us with different signal intensity that varies with factors associated with intracellular environment. To ensure the best lipid staining method, the researcher needs to optimize the conditions according to species requirement. In this article, I will explain the use of fluorescent dyes (Nile Red and BODIPY) for lipid detection and measurement. In addition to this, the article will also discuss the insights about various factors (concentration, incubation temperature, staining temperature etc) associated with the protocol.   
Indeed, there are some disadvantages of using dyes to estimate the exact quantity of neutral lipid as it has potential to bind to other proteins (non-lipid parts) hydrophobic domain of which might interfere in fluorescence. 

There are two major fluorochromes which are used to study lipid droplets are Nile red and    BODIPY 

Concentration (ug/ml)
Staining Temp
Incubation Temp (min)
Target Molecule
Wavelength  (Ex/Em)
Nile Red
2 -7
Lipid Droplets

Table 1: Various specific parameters needed to keep in mind while using dyes to quantify lipids.
Our idea is to identify the conditions where dye is strongly colored, completely soluble, and unable to interact with unwanted domains. All the above-mentioned parameters (Table-1) can be changed accordingly, which can effectively target your sample molecule. 

Why lipid Staining is the future?

It provides a rapid and cost effective method to calculate neutral lipid content avoiding time-consuming gravimetric method.
They give quick response and require less number of equipment.

How to improvise the protocol?

As of now, there is no standard protocol defined for staining, which is necessary to be optimized for replicable results. There are two major steps in this procedure (i) dye enter the cells (ii) dye incorporates with cytoplasmic lipids.
To improve the lipid estimation using fluorescent dyes, there are a number of techniques to optimize the staining conditions, which can be applied to increase the efficacy. For ex: We can use solvents as stain carrier such as ethanol, DMSO, chloroform, acetone etc. to improve permeation issues. But these solvents have the potential to bring variations in fluorescent measurement. Literature studies have also mentioned that repeated use of Nile red in acetone changes the solute concentration. In my lab, I am using DMSO as a solvent carrier as it is efficient for diffusion across the cell membrane.
I will advise you to use solvent concentration 1% to 2%, and all experiments with dye are strictly conducted in dark room. Some researchers advised to use BODIPY but it varies from organism to organism.
To improve the permeation of stain inside the cell wall and poor fluorescence signal, several physical and chemical methods have been advised such as liquid nitrogen.
The maximum fluorescence has been observed in DMSO concentration between 20-25% (v/v). In another approach, lipid staining was increased by multifold times by adding glycerol (0.05g/ml) and keeping the sample in dark shakers for 5 min at 42°C can further increase it. We can also vary the temperature range from 37°C to 50°C
Varied electric field and microwave irradiation have also been exemplified to increase the efficiency, which have shown high fluorescence and low variability. 

Fixation for lipid droplets
Fixation is used to preserve the cell component exactly.  Different methods can be used for fixation like incubating the cell mixture with cold methanol and acetone for 5-10 mins.  Personally, I have used 0.1% osmium tetroxide for 24-48 hours (depends on tissue thickness) after fixing the sample with 10% formalin for 24 hours. Also, the fixation methods will have different results in different cell types.

Things that can be done
·         Optimize the physical and chemical conditions to increase the permeation of dye inside the cell without destroying the host machinery.
·         For ex: optimization of staining and incubation temperature for separate dye in relation to different algae species.
·         Optimization of dyes concentration.
·         Conditions that will only lead to binding of dye only to target molecule i.e., TAG (Triacylglycerol)
The important finding of this article is that lipid staining is the most effective tool for studying the structure of lipid droplets in cells. There is, however, a requirement to optimize the various factors, which could induce certain, changes such fixation strategy, dye concentration and incubation time etc. Thus small chemical and physical changes could indicate the onset of new technology which will aimed to study lipid droplets. 

Judith Rumin, Hubert Bonnefond, Bruno Saint-Jean, Catherine Rouxel, Antoine Sciandra, Olivier Bernard, Jean-Paul Cadoret and Gael Bourgaran.The use of fluorescent Nile red and BODIPY for lipid measurement in microalgae. Biotechnology for biofuels, 8:42,2015

Philip Green Span, Eugene P Mayer, and Stanley D Fowler, Nile red: A selective fluorescent stain for intracellular lipid droplets. The journal of cell biology, Vol 100, March 1985

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