Category Archives: Confocal

The wild ride of the exosomes

Exosomes are extracellular vesicles that are thought to mediate cell-to-cell communication in eukaryotes. Briefly, exosomes are 50-100 nanometer (nm) sized vesicles produced by the endosomal system. They are exported out of the cell and can be found in every bodily fluid: plasma, saliva, milk, urine and more. These vesicles then enter recipient cells, and the cargo they carry (proteins, RNA molecules and lipids) modulate the physiology and/or gene expression of the recipient cell. Exosomes catch a lot of attention lately because of their clinical significance. First, exosomes might be used as biomarkers for some diseases (most importantly tumors). Second, they are being considered for therapeutics as a delivery system.

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New data on SmartFlare – do they detect mRNA?

Almost exactly a year ago, I wrote a post regarding my concerns with SmartFlare, supposedly a novel method for live imaging of RNA in cells.

In a nutshell, SmartFlare are gold nanoparticles covered in oligos specific to a certain mRNA of interest. Supposedly, cells internalize these particles and, once the mRNA hybridize to the oligo, a complementary fluorecently labeled oligo is being unquenchhed and “flares”, indicating the present of said mRNA.

You can read about my concerns in that older post, but apparently I wasn’t the only one concerned about their validity.

Raphaël Lévy from U. of Liverpool (UK) was concerned as well. He endeavored into an open science project to try and answer his concerns (which is why I allow myself to openly review his paper).

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Transcription caught on camera part 2: Fab-ulous Histones

In eukaryotes, the DNA is packages tightly in nucleosomes, which are composed primarily out of histone proteins. There are four major types of histones (1,2,3 & 4). Extensive work has been done on how histones facilitate and regulate transcription. It turns out that there are multiple post-translational modifications on histones, such as methylation and acetylation that are linked to transcription regulation. The majority of the studies use a method called chromatin immunoprecipitation (ChIP) to study these modifications. In essence, an antibody specific for a certain modification is used to affinity-purify only modified histones, along with any DNA region they are associated with. Thus, one could get a map of the specific modified histone along the chromosomes and correlate this locations with transcription activity, ChIP maps of other transcription related proteins etc…

There are two problems with this approach. The first, since the cells are fixed, the time resolution is limited to several minutes, at best. Second, the results are an average of the entire cell population, and therefore factors considered linked may not actually be present in the same cell, same genomic location at the same time.

So, Timothy Stasevich et al. tried a different approach by using a novel method to image histone modifications in live cells.

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Imaging gene expression – methods & protocols

A new book in the “Methods in molecular biology” series, recently published, contains 23 imaging protocols in three major research areas: gene expression & RNA dynamics, genome & chromatin dynamics, and nuclear process & structures.

book cover: Imaging Gene Expression

This is a fairly good overview of the field and can help both beginners and researchers looking for new ideas.

The book can be freely downloaded.

Here’s the contents of the book:

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Basics in Confocal microscopy and image analysis

Nowadays, confocal microscopy is possibly the most widely used optical method in biological research. This methods creates better (and prettier) images than widefield microscopy (whether transmitted light or epifluorescence). The main advantages of confocal vs. widefield microscopy is the elimination of out-of-focus glare (thus increasing resolution and increasing signal-to-noise ratio) and the ability to collect serial optical sections of the specimen (z-sections).

The basic configuration of the optics is similar to that of the epifluorescnece microscope. The addition that created the confocal microscope, invented by Marvin Minsky in 1955, was to add two pinholes. The light produced by lamp (or laser) passes through the first pinhole on the way to the specimen. The light that is reflected (bright light) or emitted (fluorescent light) from the specimen passes through a second pinhole on the way to the detector (eyepiece, camera or any recording device). The two pinholes have the same focus – thus they are confocal. The light from other focal planes cannot go through the second pinhole, and this reduces the background “glare” of out-of-focus fluorescence seen in epifluorescence widefield microscopes.

Since biological samples usually have thickness of a few microns at least, one can get an image of a thin slice of the sample (e.g. 0.1 µm) without physically slicing the sample (optical section). We can then move the focus along the Z axis to get clear images of up or down sections. Thus, for a cell 3µm thick, we can have 30 hi-resolution images 0.1 µm thick from bottom to top (z-sections). These images can then be stacked one on top of the other (z-stacking) to create a single 2D image or to reconstruct a 3D image of the sample.

Here’s an example from an experiment I did last week (note that this is a widefield, not confocal microscope):

Above is a composite image of 31 Z sections of U2OS cells, create by the ImageJ program. The”pseudo-blue” represents the blue fluorescnce of a dye called DAPI (4′,6-diamidino-2-phenylindole)  which intercalates into DNA, and is therefore a popular nuclear dye. When bound to DNA, it is excited by UV light (peak at 358nm) and emits blue/cyan light (peak at 461nm). The “pseudo-red” color represents the fluorescence of mCherry-ZBP1 fusion protein. mCherry is an RFP.

You can see in the image that the first and last few images in the series are out of focus. You can therefore choose the best or sharpest Z-section according to your needs.

However, most people do not show a single Z section since then we miss a lot of information that is found in other sections. The available programs today allow “stacking” the section to create a projection of all the sections into a single image.

Here is the maximum projection of the Z sections shown above:

Maximum projection means that the algorithm chooses, for each pixel, the highest value found in any of the 31 Z sections. However, since we chose all 31 sections, we can still see a “glare” or halo. This is a result of the “halos” from the out of focus sections.

I therefore choose only a few sections to create the next image:

This image is now sharper and better looking.

The program allows you other options besides maximu projection: you can choose minimum, average, median, and even standrad deviation, seen in the next image (DAPI channel only):

It looks very cool. I stacked the entire 31 sections (of a differnt field), so you can see the halo from the out-of-focus sections sorounding the “black” rim of the nucleus (black since it has the minimal standrd deviation value for all the images). The blue zones, with high SD, suggest a larger differnce in fluorescence between the differnt sections.

Above, I mentions that the blue and red are pseudo colors. What actually happened was that the images I took with the microscope at each channel (range of wavelengths) is actually maintained as a greyscale image.

Using the image analysis program you can then merge the images of the differnt channels (up to 4 in ImageJ) to create a color image. When creating the merged image, you determine what color to assign to each channel. Here is the same image, but with the colors reversed:

You should take that into account when you see pretty pictures in sceintific journals.

The program also allows to creade 3D representations of your Z stack. but I haven’t learned how to do that.

There are many other tools that one can use with the image analysis program besides creating the image. One important feture is the ability to measure the intensity of the fluorescent signal (actually, the pixels) in certain areas within the cell. You can measure distances and angles between objects and probably many moer that I still have to learn.