Overview
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
DYE
|
Concentration (ug/ml)
|
Staining Temp
|
Incubation Temp (min)
|
Target Molecule
|
Wavelength (Ex/Em)
|
Nile Red
|
0.01-100
|
37C-40C
|
0.5-14
|
TAG
|
450-550/
575-580
|
BODIPY
|
0.067-0.1
|
25C-30C
|
2 -7
|
Lipid Droplets
|
488/510
|
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.
References
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
