Tag Archives: resolution

Malaria parasites send each other genes

Communication between cells takes many forms. There could be communication by direct contact, by sending out free molecules (like hormones) or by special structures (e.g. synapses).

But how can parasites, that dwell inside their host cell, communicate with one another?

A very elegant mechanism used by Malaria parasites was found, and is described in a recent Cell paper (actually, it was published in the same issue as my paper).

Malaria parasites (Plasmodium falciparum) are transferred from mosquitoes to humans, where they infect red blood cells (RBCs). Once inside the RBCs, the parasite need to sexually differentiate into sexual forms that are competent for transmission by the next mosquito.

When trying to understand which signals are transferred between the parasites that dwell in different RBCs, they mixed two cultures, each expresses a different drug resistant gene and a different fluorescent protein. Surprisingly, the mixed cultures survived when both drugs were added, and parasite cells exhibited both colors.

Further analysis showed that the parasites send out tiny vesicles (their size is ~70 nm). These vesicles are similar to endosome-derived “exosomes” ,and therefor are referred in the paper as “exosome-like” vesicles. In most papers that study exosomes, they are visualized by electron microscopy. However, in this paper, Atomic Force Microscopy (AFM) was employed.  AFM resolution is ~1000-fold better than light microscopy (yet lower than electron microscopy).  In essence, AFM uses a tiny cantilever with a very sharp tip that travels over the sample. The tip is deflected from the sample based on forces exerted from the surface (e.g. mechanical contact force, electrostatic forces, magnetic forces, Van der Waals force).  The deflection is registered by a laser light.

Principle of AFM. Source: Wikipedia

These vesicles supposedly contain the plasmid DNA that enables the lateral inheritance of the new characteristic (drug resistance & fluorescent protein).

Malaria parasites-derived

Malaria parasites-derived “exosome-like” vesicles as seen by Atomic Force Microscopy. Upper row: fraction that does not contain the vesicles. Lower row: fraction that does contain vesicles. Source: Regev-Rudzki et al. Cell 153(5): 1120-1133

The authors put a lot of effort in proving that the information – DNA plasmid – is transferred via these vesicles.  They perform DNA FISH and PCR to show that the “acceptor” parasites contain these genes. Alas, they never show that the vesicles that they isolated also contain this DNA. This should have been simple to do: they already have the isolated exosomes and just need to do PCR on them. I do not know why this was not requested by the reviewers.

Their last figure, which is intended to give a broader biological meaning to their findings, suggests that this form of communication is required for production of gametocytes (a sexual differentiation step required for intake by the mosquito). They show that the gametocytes contain both fluorescent markers, and are produced at greater numbers when parasites are co-cultured.

Malaria gametocytes from co-cultures express both fluorescent proteins. Source: Regev-Rudzki et al. Cell 153(5): 1120-1133.

Malaria gametocytes from co-cultures express both fluorescent proteins. Source: Regev-Rudzki et al. Cell 153(5): 1120-1133.

However, they do not show that application of the isolated vesicles can induce this sexual differentiation. More so, the exosomes may contain other factors (proteins, RNAs) that can induce sexual differentiation, and be unrelated to the DNA transfer observed.

In any case, an interesting paper, that may have major clinical applications in the future.

ResearchBlogging.orgRegev-Rudzki N, Wilson DW, Carvalho TG, Sisquella X, Coleman BM, Rug M, Bursac D, Angrisano F, Gee M, Hill AF, Baum J, & Cowman AF (2013). Cell-Cell Communication between Malaria-Infected Red Blood Cells via Exosome-like Vesicles. Cell, 153 (5), 1120-33 PMID: 23683579

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Calculating the pixel sizes on images

In most microscopy images that are published in research papers, there appears a scale bar.  The scale bar is like a ruler that allows you to compare sizes and distances in images from different sources.  Although a scale bar is helpful for assessing by eye, many image processing programs allows you to measure distances in the image. The problem is that these measurements are in pixels. That is what I encountered when I wanted to measure certain objects in my images. How to convert from pixels to nanometers (or microns) requires a simple formula and some prior data as follows:

  1. Objective magnification
  2. Lens magnification (in some microscopes, it is possible to get extra mag of 1.25x, 1.6x or 2x.
  3. C mount (is usually 1x)
  4. Pixel size – is the actual pixel size of the camera that is attached to the microscope.
  5. Binning – i.e. combining a cluster of pixels to a single pixel. The common options are 1X1, 2X2 and 4X4. Binning is usually used to reduce noise, but at the expense of resolution.

The formula:

Image pixel size = camera pixel size x binning / (obj. mag x lens mag x C mount)

Example:

For Cascade 512 camera (16µm/pixel on CCD), at 60x mag and 1×1 binning:

Pixel size = 16×1/(60x1x1) = 0.2667µm = 266.7nm.

Obviously, the smaller the pixel size of the camera, the better the resolution (i.e. actual pixel size in the image).

Here is a helpful table of pixel sizes (in nanometers) for some common cameras:

pixel sizes

Note that all cameras listed here have a square pixel size (e.g. 9300×9300). Some cameras have rectangular pixel sizes (e.g. ExwaveHAD 3CCD with 6350×7400).  In such cases, the length and width of the pixel sizes should be calculated separately. However, I am told that microscope-intended cameras today have only square pixels, not rectangular.

For an explanation how to add a scale bar in ImageJ, click here.

For an explanation how to add a scale bar in photoshop, click here.

Numerical aperture and resolution

While reading about microscopy, I came across mentions of a parameter called numerical Aperture (NA).

NA is a measure of the ability of the objective lens to gather light and resolve it, at a fixed distance from the specimen, through a specific media (e.g. air, water, immersion oil).

As shown in the figure, light from the specimen enter the objective in an inverted cone as seen in this figure:

The angle µ is half the angular aperture A.  NA is calculated by the following equation:

NA = n(sin µ)

n is the value of the refractive index of the medium between the specimen (the cover glass) and the objective. The value of n is between 1.0 (for air) up to 1.51 (for special immersion oil).

When imaging through air (usually with low magnifications), then n=1 and so NA depends solely on the sin of µ which theoretically can be a maximum of 1 (sin(90°)=1). In practice, it is difficult to achieve NA>0.95 with “dry” microscopy (i.e. through air).

Therefore, many microscope objectives (mostly for high magnifications) are designed to be used with water (n=1.33), glycerin (n=1.47) or immersion oil (n=1.51).

So how does the NA relate to resolution?

The resolution of an optical microscope is defined as the shortest distance between two points that can still be distinguished by the observer.  This can be perceived by the concept of Airy diffraction patterns (named after physicist George Biddell Airy).

I will not go into the physics of it now (since I need to learn it first). It is enough to say that point sources of light in the specimen (e.g. fluorescent proteins) appear as bright disks, surrounded by bright concentric rings.

The limit of resolution is determined by the ability to distinguish between two closely spaced Airy disks. The 3-dimentional representation of the Airy disks is called the point spread function. The resolution of the microscope is determined (in part) by the NA of the objective. You can explore it yourself using the Java tutorial of NA & resolution at MicroscopyU. You can see that the larger the NA, the better the resolution.

The resolution is determined not only by the NA of the objective, but also of the sub-stage condenser. The alignment of the two, to create an accurate light cone, is also important.

Another parameter that greatly affects the resolution is the wavelength (λ) of light that is used.  Generally, the shorter the wavelength, the better the resolution. There are three equations developed to describe the resolution (r):

r = 0.5λ/NA

r = 0.61λ/NA

r = 1.22λ/(NA(obj)+NA(cond))

NA in the first two equations refers to the general NA of the microscope.

These equations were calculated based on multiple optical factors and, in some instances, the actual resolution may be even better than the limit calculated by the equations.

However, other factors, like background, contrast, illumination and more can also affect the resolution.

The practical limit (for green light) is ~250nm.

Just to put things in perspective, the size of an epithelial cells is ~30µm, that of a yeast cell is ~4-5µm. The size of the yeast nucleus is ~1µm. A chromosomally packed DNA length of 100kb (100,000 bases) is ~500nm, when stretched. Viruses are usually ~30-50nm. A single ribosome is also ~30nm. A single average protein will be ~5nm in size. Therefore, viewing single molecules in cells is a real challenge.