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As the labeled cell passes through the interrogation point, it is illuminated by the excitation lasers. The fluorochromes, fluoresce; emitting photons of a higher wavelength than the excitation source. This is typically modeled using spectral viewers such as in the figure below, which shows the excitation (dashed lines) and emission (filled curves) for Brilliant Violet 421TM (purple) and Alexa Fluor 488Ⓡ (green).  Figure 1: Excitation and emission profiles of BV421TM and AF488Ⓡ  In traditional fluorescent flow cytometry (TFF), the instrument measures each fluorochrome off an individual detector. Since the detectors we use — photomultiplier tubes (PMT) and avalanche photodiodes (APD)…

Extracting specific cells still remains an important aspect of several emerging genomic techniques. Prior knowledge about the input cells helps to put the downstream results in context. The most common isolation technique is cell sorting, but it requires a single cell suspension and eliminates any spatial information about the microenvironment. Spatial transcriptomics is an emerging technique that can address some of these issues, but that is a topic for another blog.  So what does a researcher who needs to isolate a specific type of cell do? The answer lies in the technique of laser capture microdissection (LCM). Developed at the National…

Incorporating quality control as a part of the optimization process in  your flow cytometry protocol is important. Take a step back and consider how to build quality control tracking into the experimental protocol.  When researchers hear about quality control, they immediately shift their attention to those operating and maintaining the instrument, as if the whole weight of QC should fall on their shoulders.   It is true that core facilities work hard to provide high-quality instruments and monitor performance over time so that the researchers can enjoy uniformity in their experiments. That, however, is just one level of QC.  As the experimental…

Clinical trials are studies designed to test the novel methods of diagnosing and treating health conditions – by observing the outcomes of human subjects under experimental conditions.  These are interventional studies that are performed under stringent clinical laboratory settings. Contrariwise, non-interventional studies are performed outside the clinical trial settings that provide researchers an opportunity to monitor the effect of drugs in real-life situations. Non-interventional trials are also termed observational studies as they include post-marketing surveillance studies (PMS) and post-authorization safety studies (PASS). Clinical trials are preferred for testing newly developed drugs since interventional studies are conducted in a highly monitored…

Fluorophore selection is important. I have often been asked by my facility users which fluorophore is best suited for their experiments. The answer to this is mostly dependent on whether they are using a widefield microscope with set excitation/emission cubes or a laser based system that lets you select the laser and the emission window. Once you have narrowed down which fluorophores you can excite and collect the correct emission, you can further refine the specific fluorophore that is best for your experiment.  In this blog  we will discuss how to determine what can work with your microscope, and how…

As we continue to explore the steps involved in optimizing a flow cytometry experiment, we turn our attention to the detectors and optimizing sensitivity: instrument voltage optimization.  This is important as we want to ensure that we can make as sensitive a measurement as possible.  This requires us to know the optimal sensitivity of our instrument, and how our stained cells are resolved based on that voltage.  Let’s start by asking the question what makes a good voltage?  Joe Trotter, from the BD Biosciences Advanced Technology Group, once suggested the following:  Electronic noise effects resolution sensitivity   A good minimal PMT…

In the first blog of this series, we explored the power of sequencing the genome at various levels. We also dealt with how the characterization of the RNA expression levels helps us to understand the changes at the genome level. These changes impact the downstream expression of the target genes. In this blog, we will explore how NGS sequencing can help us comprehend DNA modification that affect the expression pattern of the given genes (epigenetic profiling) as well as characterizing the DNA-protein interactions that allow for the identification of genes that may be regulated by a given protein.  DNA Methylation Profiling…

Image quality is critical for accurate and reproducible data. Many people get stuck on the magnification of the objective or on using a confocal instead of a widefield microscope. There are several other factors that affect the image quality such as the numerical aperture of the objective, the signal-to-noise ratio of the system, or the brightness of the sample.  Numerical aperture is the ability of an objective to collect light from a sample, but it contributes to two key formulas that will affect your image quality. The first is the theoretical resolution of the objective. It is expressed with the…

Why is Next Generation Sequencing so powerful to explore and answer both clinical and research questions. With the ability to sequence whole genomes, identifying novel changes between individuals, to exploring what RNA sequences are being expressed, or to examine DNA modifications and protein-DNA interactions occurring that can help researchers better understand the complex regulation of transcription. This, in turn, allows them to characterize changes during different disease states, which can suggest a way to treat said disease.  Over the next two blogs, I will highlight these different methods along with illustrating how these can help clinical diagnostics as well as…

In my previous blog on  experimental optimization, we discussed the idea of identifying the best antibody concentration for staining the cells. We did this through a process called titration, which  focuses on finding the best signal-to-noise ratio at the lowest antibody concentration. In this blog we will deal with sample blocking As a reminder, there are two other major binding concerns with antibodies. The first is the specific binding of the Fc fragment of the antibody to the Fc Receptor expressed on some cells. This protein is critical for the process of destroying microbes or other cells that have been…

TIRF is not as common as other microscopy based techniques due to certain restrictions. We will discuss these restrictions, then analyze why it might be perfect for your experiment.  TIRF relies on an evanescent wave, created through a critical angle of coherent light (i.e. laser) that reaches a refractive index mismatch.  What does it mean in practice?  A high angle laser reflects off the interface of the coverslip and the sample. Although the depth that this wave penetrates is dependent on the wavelength of the light, in practice it is approximately 50-300nm from the coverslip. Therefore, the cell membrane is…

NGS methodologies have been used to produce high-throughput sequence data. These data with appropriate computational analyses facilitate variant identification and prove to be extremely valuable in pharmaceutical industries and clinical practice for developing drug molecules inhibiting disease progression. Thus, by providing a comprehensive profile of an individual’s variome — particularly that of clinical relevance consisting of pathogenic variants — NGS helps in determining new disease genes. The information thus obtained on genetic variations and the target disease genes can be used by the Pharma companies to develop drugs impeding these variants and their disease-causing effect. However simple this may allude…